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Transcriptional regulation mechanism mediated by miRNA-DNA•DNA triplex structure stabilized by Argonaute
... In this model, purine or pyrimidine rich (> 75%) miR-NAs form triple-helical structures with purine-rich duplex DNA via Hoogsteen or reverse Hoogsteen interaction in the major groove of the duplex DNA. This interaction may alter the DNA topography and allow binding of transcription factors, resulting in transcriptional activation or suppression [68,69]. Paugh et al. developed an algorithm (Trident) to search for potential triplex-formation sites. ...
... Moreover, in primary leukemia cells, largely, the expression of miRNAs that are predicted by Trident are positively correlated with the expression of their target [68]. However, as few evidence exists for triplex formation of miRNAs in vivo [69,70], this model still needs to be validated upon further study. ...
... MiRNA can also directly target promoter facilitated by transcription preinitiation complex to alter histone modification [56]. In the RNA-DNA triplex model, miRNA forms triple-helical structure with DNA to activate or suppress transcription via altering DNA topography [68,69]. Recently, enhancer associated mechanism has also been discovered [71]. ...
MicroRNAs (miRNAs) are endogenous non-coding RNAs that contain approximately 22 nucleotides. They serve as key regulators in various biological processes and their dysregulation is implicated in many diseases including cancer and autoimmune disorders. It has been well established that the maturation of miRNAs occurs in the cytoplasm and miRNAs exert post-transcriptional gene silencing (PTGS) via RNA-induced silencing complex (RISC) pathway in the cytoplasm. However, numerous studies reaffirm the existence of mature miRNA in the nucleus, and nucleus-cytoplasm transport mechanism has also been illustrated. Moreover, active regulatory functions of nuclear miRNAs were found including PTGS, transcriptional gene silencing (TGS), and transcriptional gene activation (TGA), in which miRNAs bind nascent RNA transcripts, gene promoter regions or enhancer regions and exert further effects via epigenetic pathways. Based on existing interaction rules, some miRNA binding sites prediction software tools are developed, which are evaluated in this article. In addition, we attempt to explore and review the nuclear functions of miRNA in immunity, tumorigenesis and invasiveness of tumor. As a non-canonical aspect of miRNA action, nuclear miRNAs supplement miRNA regulatory networks and could be applied in miRNA based therapies.
... In this setting, it must be underlined that there is considerable evidence demonstrating that RNA molecules can direct chromatin and DNA modifications at specific loci [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57] ...
... 47,[53][54][55][56] Also, some miRNAs seem to target specific gene promoters and to modulate gene transcription activity and local chromatin modifications, even though more experiments are needed to ascertain whether these effects are direct. 57 have been shown to bind mRNAs in vivo. [94][95][96] Second, it has been reported in yeast that some mRNAs are bound by proteins that interact co-translationally with the mRNA-encoded proteins. ...
... 177 It has also been proposed that miRNAs act on targeted promoters by forming RNA:dsDNA triplexes. 57,175 Fourth, RNAs can theoretically target specific loci and induce the BOX 3: piRNA biogenesis ...
Understanding the molecular mechanisms behind the capacity of cancer cells to adapt to the tumor microenvironment and to anticancer therapies is a major challenge. In this context, cancer is believed to be an evolutionary process where random mutations and the selection process shape the mutational pattern and phenotype of cancer cells. This article challenges the notion of randomness of some cancer-associated mutations by describing molecular mechanisms involving stress-mediated biogenesis of mRNA-derived small RNAs able to target and increase the local mutation rate of the genomic loci they originate from. It is proposed that the probability of some mutations at specific loci could be increased in a stress-specific and RNA-depending manner. This would increase the probability of generating mutations that could alleviate stress situations, such as those triggered by anticancer drugs. Such a mechanism is made possible because tumor- and anticancer drug-associated stress situations trigger both cellular reprogramming and inflammation, which leads cancer cells to express molecular tools allowing them to “attack” and mutate their own genome in an RNA-directed manner.
... Interestingly, AGO proteins are known to play important roles in the establishment of heterochromatin as well as the silencing of repetitive sequences (18). There are several examples in mammals, in which small RNAs function in the nucleus to modulate gene expression, including silencing and activation at the epigenetic level, and in an AGOdependent manner (18)(19)(20)(21)(22)(23)(24). In this regard, miR-223 binds to complementary binding sites within the promoter of its target gene Nfia, repressing transcription by influencing epigenetic events (20). ...
... Given that RNA can also form duplexes with DNA, it is reasonable to think that miRNAs require direct hybridization with genomic DNA to specifically find regulatory regions. Often, a search for consensus sequences for miRNA-binding sites in promoter regions is conducted in order to explain potential instances of miRNA mediated-TGS (24). There is no common mechanism to control transcriptional processes; thus, it is difficult to predict whether non-coding RNAs targeting a specific sequence in the promoter will have a repressive or activating regulatory effect, and it is likely that the presence of specific transcription factors and other DNA-or RNA-binding proteins in the relevant tissue or cell line are important determinants. ...
... Previously, we proposed a miRNA-DNA·DNA triplehelical structure formation mediated by a miRNA:AGO complex, as a plausible non-canonical gene regulation strategy targeting important nuclear events, such as transcription (24). In this sense, miRNA-directed TGS may occur by means of a parsimonious mechanism through TATA-box motif is one of the most prevalent core promoters, located immediately upstream the transcription start site (TSS), wherein the pre-initiation complexes (PICs) are assembled. ...
Emerging evidence suggests that components of the small RNAs pathway interact with chromatin to regulate nuclear events, such as gene transcription. However, it has recently been reported that in some cases, gene transcription regulation by cellular miRNAs can occur via targeting the TATA-box motif without altering epigenetic modifications. This observation supports the notion that multiple mechanisms of miRNA-based transcriptional regulation exist, enhancing our understanding of the complexity of small RNA-mediated gene regulatory pathways. Here, we remark that miRNA-mediated transcriptional modulation, through the TATA-box motif, may be a synergistic approach for transcriptional control.
... Understanding the mechanism behind the precise regulation of follicle growth and ovulation, compared to atresia, is highly essential to improve fertility. Although the functions of some nuclear miRNA have been found in cancerous and other cell lines [13,14], the role of nuclear miRNA in regulating ovarian cell gene expression remains unknown. ...
... The final model is the miR-DNA-DNA triplex, which is stabilized by the AGO protein. This complex can be formed when the promoter is in an open conformation, as it grants the miRNA access [13]. Transcription is regulated by disrupting the pre-initiation complex through altering the topography of the chromatin, either permitting or restricting transcription factor access to the promoter [4,38]. ...
Forkhead box O3 (Foxo3) is a member of the FOXO subfamily within the forkhead box (FOX) family, which has been shown to be essential for ovarian follicular development and maturation. Previous studies have shown the abundant expression of miR-195-5p in the nuclei of porcine granulosa cells (GCs), suggesting its potential role during ovarian follicle growth. In this study, a conditional immortalized porcine granulosa cell (CIPGC) line was used to determine whether the expression of Foxo3 could be regulated by the nuclear-enriched miR-195-5p. Through silico target prediction, we identified a potential binding site of miR-195-5p within the Foxo3 promoter. The over-expression of miR-195-5p increased Foxo3 expression at both mRNA and protein levels, while the knockdown of miR-195-5p decreased the expression of Foxo3. Furthermore, driven by the Foxo3 promoter, luciferase reporter activity was increased in response to miR-195-5p, while the mutation of the miR-195-5p binding site in the promoter region abolished this effect. In addition, the siRNA knockdown of Argonaute (AGO) 2, but not AGO1, significantly decreased Foxo3 transcript level. However, miR-195-5p failed to upregulate Foxo3 expression when AGO2 was knocked down. Moreover, chromatin immunoprecipitation (CHIP) assay showed that anti-AGO2 antibody pulled down both AGO2 and the Foxo3 promoter sequence, suggesting that AGO2 may be required for miR-195-5p to regulate Foxo3 expression in the nucleus. Additionally, Foxo3 expression was significantly increased by valproic acid (VPA), the inhibitor of deacetylase, as well as by methyltransferase inhibitor BIX-01294, indicating the involvement of histone modification. These effects were further enhanced in the presence of miR-195-5p and were decreased when miR-195-5p was knocked down. Overall, our results suggest that nuclear-enriched miR-195-5p regulates Foxo3 expression, which may be associated with AGO2 recruitment, as well as histone demethylation and acetylation in ovarian granulosa cells.
... No evidence yet RNA-RNA hybrid RNA-DNA-DNA hybrid [45] RNA-RNA hybrid [69] RNA-RNA hybrid RNA-DNA hybrid RNA-DNA-DNA hybrid [69] Cis and trans activities No evidence yet Yes [67] No evidence yet Yes [70] Transcriptional activity ...
... No evidence yet RNA-RNA hybrid RNA-DNA-DNA hybrid [45] RNA-RNA hybrid [69] RNA-RNA hybrid RNA-DNA hybrid RNA-DNA-DNA hybrid [69] Cis and trans activities No evidence yet Yes [67] No evidence yet Yes [70] Transcriptional activity ...
miRNAs are well known to be gene repressors. A newly identified class of miRNAs termed nuclear activating miRNAs (NamiRNAs), transcribed from miRNA loci that exhibit enhancer features, promote gene expression via binding to the promoter and enhancer marker regions of the target genes. Meanwhile, activated enhancers produce endogenous non-coding RNAs (named enhancer RNAs, eRNAs) to activate gene expression. During chromatin looping, transcribed eRNAs interact with NamiRNAs through enhancer-promoter interaction to perform similar functions. Here, we review the functional differences and similarities between eRNAs and NamiRNAs in myogenesis and disease. We also propose models demonstrating their mutual mechanism and function. We conclude that eRNAs are active molecules, transcriptional regulators, and partners of NamiRNAs, rather than mere RNAs produced during enhancer activation.
... One type of interaction (Figure 2(A1)) leads to the formation of a direct miRNA-DNA double helix complex. In this case, miRNA binds the promoter in association with AGO proteins and recruits histone modifiers that increase the levels of activating markers, such as H3K4me3, while decreasing the inhibitory histone marks [47]. In a second model the miRNA-AGO complex interacts directly with the TATA box motif region or sites on the promoter also bound by transcription factors (Figure 2(A2)). ...
MicroRNAs are small non-coding RNAs, which contribute to the regulation of many physiolog-ical and pathological processes. Conventionally, miRNAs perform their activity in the cyto-plasm, where they regulate gene expression by interacting in a sequence-specific manner with mature messenger RNAs. Recent studies point to the presence of mature miRNAs in the nucleus. This review summarizes current findings regarding the molecular activities of nuclear miRNAs. These molecules can regulate gene expression at the transcriptional level by directly binding DNA on the promoter or the enhancer of regulated genes. MiRNAs recruit to these regions dif-ferent protein complexes, resulting both in activation or repression of transcription, through a number of molecular mechanisms. Haematopoiesis is presented as a paradigmatic biological process whereby nuclear miRNAs possess a relevant regulatory role. Nuclear miRNAs may af-fect gene expression also acting on nuclear mRNA processing and on the biogenesis of miRNA themselves by regulating pri-miRNA maturation. Overall, nuclear miRNAs are biologically ac-tive molecules that can be critical for the fine tuning of gene expression and deserve further studies in a number of physiological and pathological conditions
... [8] Furthermore, microRNAs (miR-NAs) have been shown to regulate transcription through miRNA · DNA : DNA triplexes, which are further stabilized by Argonaute proteins. [9] In RNA · DNA : DNA triplex formation, the single-stranded RNA binds to the major groove of the double-stranded DNA. This RNA · DNA : DNA interaction is stabilized by Hoogsteen or reverse Hoogsteen hydrogen bonds. ...
Long non‐coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single‐molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex‐forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex‐forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration‐dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature‐dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease‐relevant lncRNAs and their DNA target genes.
... These regulatory mechanisms were frequently observed in plants and fungi [13]. In contrast, in mammals, when the 3 ′ UTR of the target gene only partially complements with the seed sequence of miRNA, it results in the translational inhibition of mRNA [14,15]. In most studies, it has been demonstrated that miRNAs bind to target mRNAs and function as regulators by suppressing the expression of the latter. ...
Simple Summary
The substantial impact of heat stress on the milk yield of dairy cows necessitates improving the physiological condition of their mammary glands to enhance dairy production efficiency. The present study observed that heat stress exerted a suppressive effect on the proliferation of bovine mammary epithelial cells (BMECs) and significantly downregulated the expression of endogenous miR-425-5p. The experimental findings confirmed that overexpression of miR-425-5p promotes the proliferation of BMECs. It was identified that Transducer of ERBB2, 2 (TOB2) is a target gene of miR-425-5p. Overexpression of TOB2 inhibited the proliferative effect of miR-425-5p on BMECs. These results demonstrate that miR-425-5p promotes the proliferation of BMECs by targeting TOB2. These findings offer a dependable reference for addressing the issue of reduced milk production in dairy cows experiencing heat stress at the molecular level.
Abstract
In recent years, rising temperatures have caused heat stress (HS), which has had a significant impact on livestock production and growth, presenting considerable challenges to the agricultural industry. Research has shown that miR-425-5p regulates cellular proliferation in organisms. However, the specific role of miR-425-5p in bovine mammary epithelial cells (BMECs) remains to be determined. The aim of this study was to investigate the potential of miR-425-5p in alleviating the HS-induced proliferation stagnation in BMECs. The results showed that the expression of miR-425-5p significantly decreased when BMEC were exposed to HS. However, the overexpression of miR-425-5p effectively alleviated the inhibitory effect of HS on BMEC proliferation. Furthermore, RNA sequencing analysis revealed 753 differentially expressed genes (DEGs), comprising 361 upregulated and 392 downregulated genes. Some of these genes were associated with proliferation and thermogenesis through enrichment analyses. Further experimentation revealed that TOB2, which acts as a target gene of miR-425-5p, is involved in the regulatory mechanism of BMEC proliferation. In summary, this study suggests that miR-425-5p can promote the proliferation of BMECs by regulating TOB2. The miR-425-5p/TOB2 axis may represent a potential pathway through which miR-425-5p ameliorates the proliferation stagnation of BMECs induced by HS.
... For RNA-DNA triplex interactions, miRNA-Ago complexes directly alter the topography of chromatin, thereby changing its accessibility to transcription factors and resulting in either activation or suppression of transcription. The triplex can also suppress transcription by overlapping the target site of transcription factors on the promoter [66]. The accuracy of the triplex binding silent status is generated by miRNA by recruiting PcG members (YY1 and Suz12), and H3K27me3 is increased while H3K4me3 is hindered. ...
MicroRNAs (miRNAs) are small noncoding RNAs (ncRNAs) that play their roles in the regulation of physiological and pathological processes. Originally, it was assumed that miRNAs only modulate gene expression posttranscriptionally in the cytoplasm by inducing target mRNA degradation. However, with further research, evidence shows that mature miRNAs also exist in the cell nucleus, where they can impact gene transcription and ncRNA maturation in several ways. This review provides an overview of novel models of nuclear miRNA functions. Some of the models remain to be verified by experimental evidence, and more details of the miRNA regulation network remain to be discovered in the future.
... The miR-223 localises inside the nucleus and targets the NFI-A promoter region at miR-223 complementary DNA sequences, recruiting the polycomb complex (PcG) to induce NFI-A transcriptional silencing (Zardo et al. 2012). Due to their length and sequence composition, a subset of miRNAs has been proposed to form triplexes and to bind to regulatory elements in different species (Paugh et al. 2016;Toscano-Garibay and Aquino-Jarquin 2014). ...
Mammalian genomes are extensively transcribed, producing a large number of coding and non-coding transcripts. A large fraction of the nuclear RNAs is physically associated with chromatin, functioning in gene activation and silencing, shaping higher-order genome organisation, such as involvement in long-range enhancer-promoter interactions, transcription hubs, heterochromatin, nuclear bodies and phase transitions. Different mechanisms allow the tethering of these chromatin-associated RNAs (caRNA) to chromosomes, including RNA binding proteins, the RNA polymerases and R-loops. In this review, we focus on the sequence-specific targeting of RNA to DNA by forming triple helical structures and describe its interplay with chromatin. It turns out that nucleosome positioning at triple helix target sites and the nucleosome itself are essential factors in determining the formation and stability of triple helices. The histone H3-tail plays a critical role in triple helix stabilisation, and the role of its epigenetic modifications in this process is discussed.
... The RNA-TFO (sometimes used as miRNA) can efficiently silence gene in-vitro and regulate a large number of target set of genes in treatment of heterogenic disease [4]. This can also be used in practice to enter in the cell for clinical trials in gene therapy [5]. ...
Locked nucleic acid (LNA) has suggested as a potential substitute of Ribose nucleotide in the application of RNA-TFO in blood clearance in cellular processes due to early degradation of Ribose nucleotide. In this work we designed different Recombinant triplexes by substituting LNA in two different sequences of DNA and verify the stability of such type of modified triplexes and determined the binding affinity of TFO with natural DNA duplex. The molecular dynamics principles were used in the simulation. Binding free energies were determined by Molecular Mechanics Generalized Born Surface Area (MMGBSA) approach. The results of simulation suggest that substitution of LNA in DNA sequence can be used as a better substitute for RNA in application modified TFO
... While their role in the nucleus is not fully understood yet, two main mechanisms have been established, i.e., PTGS mediated by nuclear RISC for miRNAs and other endonuclear non-coding RNAs and direct interactions with the promoters, where the resulting down-or upregulation depends on location and methylation status of target sides [113,114]. For the latter, three mechanisms have been proposed: (i) RNA-RNA model, where direct interaction of minimal RISC with the transcript resulting from recruitment of histone modifiers [115][116][117][118], (ii) RNA-DNA hybrid model, in which Ago is guided by miRNA to the TATA box or binding sites of transcription factors (TFs) triggering recruitment of histone modifiers or TFs [119][120][121][122][123] and (iii) RNA-DNA triplex model, where topology of DNA is changed due to binding of miRNA to major groove using Hoogsteen or reverse Hoogsteen bonds, which recruit TFs [124,125]. ...
Recent studies imply that there is a tight association between epigenetics and a molecular mechanism of major depressive disorder (MDD). Epigenetic modifications, i.e., DNA methylation, post-translational histone modification and interference of microRNA (miRNA) or long non-coding RNA (lncRNA), are able to influence the severity of the disease and the outcome of the therapy. This article summarizes the most recent literature data on this topic, i.e., usage of histone deacetylases as therapeutic agents with an antidepressant effect and miRNAs or lncRNAs as markers of depression. Due to the noteworthy potential of the role of epigenetics in MDD diagnostics and therapy, we have gathered the most relevant data in this area.
... function as antisense regulators of other RNAs (32). The mechanisms of miRNA action have been elucidated, although not completely (33). Their capacity to regulate mRNAs and other miRNAs is being reported with increasing frequency, and they have become powerful tools for gene regulation. ...
Angiogenesis is a tightly regulated biological process by which new blood vessels are formed from pre-existing blood vessels. This process is also critical in diseases such as cancer. Therefore, angiogenesis has been explored as a drug target for cancer therapy. The future of effective anti-angiogenic therapy lies in the intelligent combination of multiple targeting agents with novel modes of delivery to maximize therapeutic effects. Therefore, a novel approach is proposed that utilizes dumbbell RNA (dbRNA) to target pathological angiogenesis by simultaneously targeting multiple molecules and processes that contribute to angiogenesis. In the present study, a plasmid expressing miR-34a-3p and -5p dbRNA (db34a) was constructed using the permuted intron-exon method. A simple protocol to purify dbRNA from bacterial culture with high purity was also developed by modification of the RNASwift method. To test the efficacy of db34a, pancreatic cancer cell lines PANC-1 and MIA PaCa-2 were used. Functional validation of the effect of db34a on angiogenesis was performed on human umbilical vein endothelial cells using a tube formation assay, in which cells transfected with db34a exhibited a significant reduction in tube formation compared with cells transfected with scrambled dbRNA. These results were further validated in vivo using a zebrafish angiogenesis model. In conclusion, the present study demonstrates an approach for blocking angiogenesis using db34a. The data also show that this approach may be used to targeting multiple molecules and pathways.
... The sequence-specific binding of an RNA to the major groove of a Triplex Targeting Sequence (TTS) according to the Hoogsteen and reverse Hoogsteen base-pairing rules results in a triple helix, one kind of site specific RNA targeting mechanism [1][2][3]. Functional triple helix formation has been described for a number of long non-coding RNAs, including MEG3, HOTAIR, SPHK1, promoter-associated ncRNAs [4][5][6][7][8], and has been suggested for a subset of micro-RNAs [9,10]. ...
A significant fraction of non-coding RNAs (ncRNAs) is associated with chromatin, shown to regulate gene expression and to organize nuclear architecture. Mechanisms of direct and indirect RNA-chromatin interactions have been described, including the sequence-specific formation of triple helix structures. Triplexes are formed by the sequence-specific binding of RNA to the bases located in the major groove of DNA. We recently showed that triplexes do exist in the context of cellular chromatin and that these structures are stabilized by the histone H3 tail of adjacent nucleosomes. The in vitro characterization of the specificity and binding affinity of triplex sequences next to nucleosomes are essential parameters to identify potential sites of RNA-chromatin interaction in vivo. Here we provide a detailed protocol to determine the influence of nucleosome positioning on triple helix formation. This assay allows the comparative quantification of triplex formation and specificity for triplex targeting sequences relative to the spatial nucleosome position.
... Fully complementary duplex RNAs that target the COX-2 promoter transcript, in addition to miR-589, activate COX-2 transcription [84]. In addition, miRs can bind to a gene promoter by forming triple-helical structures with purine-rich duplex DNA via Hoogsteen or reverse Hoogsteen interactions in the major groove of the duplex DNA (reviewed elsewhere [104]). This interaction may alter the DNA topography by steric effects and may allow binding of transcription factors that in turn affect transcriptional activation or suppression [105]. ...
A myriad of signaling molecules in a heuristic network of the tumor microenvironment (TME) pose a challenge and an opportunity for novel therapeutic target identification in human cancers. MicroRNAs (miRs), due to their ability to affect signaling pathways at various levels, take a prominent space in the quest of novel cancer therapeutics. The role of miRs in cancer initiation, progression, as well as in chemoresistance, is being increasingly investigated. The canonical function of miRs is to target mRNAs for post-transcriptional gene silencing, which has a great implication in first-order regulation of signaling pathways. However, several reports suggest that miRs also perform non-canonical functions, partly due to their characteristic non-coding small RNA nature. Examples emerge when they act as ligands for toll-like receptors or perform second-order functions, e.g., to regulate protein translation and interactions. This review is a compendium of recent advancements in understanding the role of miRs in cancer signaling and focuses on the role of miRs as novel regulators of the signaling pathway in the TME.
... The Fendrr•gDNA triple helix silences Foxf1 (encodes forkhead box F1) and Pitx2 (encodes paired-like homeodomain 2) genes by recruiting either polycomb repressive complex 2 or trithorax group/mixed lineage leukemia complex to the promoter region (13,14). lncRNAs are not the only RNA class proposed to form triple helices with gDNA, as microRNAs may also form triple helices (miRNA•gDNA) to regulate gene expression (15,16). More recently, it was shown that intronic -globin RNA interacts with an upstream regulator element through R•D-D triple helix formation, displacing transcription factors and RNA polymerase II to downregulate -globin gene expression (17). ...
Recent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA-DNA triple helix that regulates gene expression. However, base triplet composition of pyrimidine motif RNA•DNA-DNA triple helices is not well understood beyond the canonical U•A-T and C•G-C base triplets. Using native gel-shift assays, the relative stability of 16 different base triplets at a single position, Z•X-Y (where Z = C, U, A, G and X-Y = A-T, G-C, T-A, C-G), in an RNA•DNA-DNA triple helix was determined. The canonical U•A-T and C•G-C base triplets were the most stable, while three non-canonical base triplets completely disrupted triple-helix formation. We further show that our RNA•DNA-DNA triple helix can tolerate up to two consecutive non-canonical A•G-C base triplets. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA-DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triplets in DNA•DNA-DNA and RNA•RNA-RNA triple helices was distinctly different from those in RNA•DNA-DNA triple helices, showing that base triplet stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.
... Indeed, the solved T. thermophilus RISC structure contains a hybrid duplex [5], and T. thermophilus Argonaute is now understood to load guide and target DNA in host defense against foreign DNAs in vivo [55,56]. A role for Argonaute-mediated RNA-DNA interactions in transcriptional regulation has also been reported [57]. We currently lack computational methods to predict targets involving RNA-DNA interactions due partly to a paucity of structural data on hybrid duplexes. ...
Translational repression and degradation of transcripts by microRNAs (miRNAs) is mediated by a ribonucleoprotein complex called the miRNA-induced silencing complex (miRISC, or RISC). Advances in experimental determination of RISC structures have enabled detailed analysis and modeling of known miRNA targets, yet a full appreciation of the structural factors influencing target recognition remains a challenge, primarily because target recognition involves a combination of RNA–RNA and RNA–protein interactions that can vary greatly among different miRNA–target pairs. In this chapter, we review progress toward understanding the role of tertiary structure in miRNA target recognition using computational approaches to assemble RISC complexes at known targets and physics-based methods for computing target interactions. Using this framework to examine RISC structures and dynamics, we describe how the conformational flexibility of Argonautes plays an important role in accommodating the diversity of miRNA–target duplexes formed at canonical and noncanonical target sites. We then discuss applications of tertiary structure-based approaches to emerging topics, including the structural effects of SNPs in miRNA targets and cooperative interactions involving Argonaute–Argonaute complexes. We conclude by assessing the prospects for genome-scale modeling of RISC structures and modeling of higher-order Argonaute complexes associated with miRNA biogenesis, mRNA regulation, and other functions.
... The transmigration into the nucleus was visualised by transfection with a FITC-labelled miR-15a and ET-1 treatment, leading to the detection of FITC signals in the nucleus, while without treatment they remained in the cytoplasm. Since Ago2 is needed for the presence of miRNA15a in the nucleus [Toscano-Garibay and Aquino-Jarquin, 2014], by immunoprecipitation it was shown that the Ago2 protein is present in the cytoplasm as well as in the nuclear fraction and is increased after ET-1 treatment. Furthermore, it binds miRNA15 showing that Ago2 is part of the newly identified miRNA: DNA complex. ...
... Regulation through the formation of triplex structure mediated by AGO proteins have also been reported in other cases such as the promoter region of dihydrofolate reductase (DHFR) (Martianov et al., 2007). Thus, a triplex formation mediated by miRNA:AGO complex could account for a noncanonical gene regulation strategy targeting various nuclear events, such as transcription, RNA splicing, or chromatinic modification (Toscano-Garibay et al., 2014). AGO mediated RNA-induced transcriptional silencing has also been reported in the nucleus of S. pombe. ...
Small RNAs govern almost every biological process in eukaryotes associating with the Argonaute proteins to form the RNA-induced silencing complex (mRISC). Argonaute proteins constitute the core of RISCs with different members having variety of protein binding partners and biochemical properties. This review focuses on the AGO subfamily of the Argonautes that are ubiquitously expressed and are associated with small RNAs. The structure, function and the role of the AGO proteins in the cell is discussed in detail.
... (B) Indicating a practical value of negative control as a standard, each sample value was presented as above by percentage. [30,33,34]. To date, this direct transcriptional regulation is due to the decreased association of RNA polymerase-II, the increased association of heterochromatising proteins [30]. ...
... In support, a nuclear localization of miR-138 has been reported by Zhang et al. (Zhang et al. 2014). Furthermore, miRNAs can form triplex structures with doublestranded DNA (miRNA-DNA·DNA) as well as a duplex if the promoter binding site is in a single-stranded open conformation (miRNA-DNA) (Paugh et al. 2016;Toscano-Garibay and Aquino-Jarquin 2014). Transcriptional gene activation mediated by miRNAs is accompanied by the localization of Argonaute proteins to the promoter target site, an enhanced RNA polymerase II promoter occupancy and the recruitment of chromatin-remodeling components leading to increased markers of active chromatin (Majid et al. 2010;Matsui et al. 2013;Place et al. 2008;Qu et al. 2015;Zhang et al. 2014). ...
Purpose:
Alpha-methylacyl-CoA racemase (AMACR) is highly overexpressed in prostate cancer (PCa) and its transcriptional regulators include various transcription factors and CTNNB1/β-catenin. Our previous findings suggested a post-transcriptional regulation by the tumor-suppressive microRNA miR-138 in PCa. Thus, the aim of this study was to demonstrate the direct interaction of miR-138 with the 3'-UTR of AMACR. Furthermore, the influence of miR-138 on the expression of AMACR and selected AMACR regulators was investigated in PCa cells.
Methods:
Using DU-145, PC-3, and LNCaP PCa cells, the effect of exogenous miR-138 on AMACR and selected AMACR regulators was determined by quantitative PCR and Western blot. Luciferase reporter assays were used to verify target and promoter interaction.
Results:
Using a luciferase reporter assay a direct interaction of miR-138 with the AMACR-3'-UTR was confirmed. Surprisingly, AMACR expression was up-regulated by up to 125% by exogenous miR-138 in PCa cells. The lack of any miR-138 binding sites within the AMACR promoter suggested an indirect mechanism of up-regulation. Therefore, the effect of miR-138 on selected AMACR regulators including CTNNB1/β-catenin, RELA, SMAD4, SP1, and TCF4 was evaluated. MiR-138 solely evoked an up-regulation of CTNNB1 mRNA expression and β-catenin protein levels by up to 75%. Further in silico analysis revealed a binding site for miR-138 within the CTNNB1 promoter. MiR-138 could enhance the activity of the CTNNB1 promoter, which in turn could contribute to the observed AMACR up-regulation.
Conclusions:
The present findings suggest that miR-138 can indirectly up-regulate AMACR via transcriptional induction of CTNNB1, at least in vitro in PCa cells.
... Experimental procedures using synthetic gapmers or siRNA approaches to inactivate pRNA function support results from the genomic analysis [89,[95][96][97]. However, it is well known that small RNAs are capable of hybridizing not only with other RNAs, but also with double-stranded DNA to form relatively stable RNA*DNA:DNA triplexes via Hoogsteen or reversed Hoogsteen base-pairing [107][108][109][110]. Therefore, alternative mechanisms of miRNA directed TGS should be investigated and taken into consideration. ...
The finding that small non-coding RNAs (ncRNAs) are able to control gene expression in a sequence specific manner has had a massive impact on biology. Recent improvements in high throughput sequencing and computational prediction methods have allowed the discovery and classification of several types of ncRNAs. Based on their precursor structures, biogenesis pathways and modes of action, ncRNAs are classified as small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs or esiRNAs), promoter associate RNAs (pRNAs), small nucleolar RNAs (snoRNAs) and sno-derived RNAs. Among these, miRNAs appear as important cytoplasmic regulators of gene expression. miRNAs act as post-transcriptional regulators of their messenger RNA (mRNA) targets via mRNA degradation and/or translational repression. However, it is becoming evident that miRNAs also have specific nuclear functions. Among these, the most studied and debated activity is the miRNA-guided transcriptional control of gene expression. Although available data detail quite precisely the effectors of this activity, the mechanisms by which miRNAs identify their gene targets to control transcription are still a matter of debate. Here, we focus on nuclear functions of miRNAs and on alternative mechanisms of target recognition, at the promoter lavel, by miRNAs in carrying out transcriptional gene silencing.
... This high abundance of triplex-forming sequences has captured the attention of researchers, since it suggests the existence of a potential gene regulation mechanism based on a triplex formation [52]. The discovery of small non-coding RNA with regulatory properties such as miRNA has triggered the hypothesis of gene regulation mechanisms by miRNA [53][54][55][56]. The present work confirms the potential of small RNA molecules to act as triplex-forming compounds in molecular crowding conditions. ...
Triplex stability is studied in crowding conditions using small cosolutes (ethanol,
acetonitrile and dimethylsulfoxide) by ultraviolet (UV), circular dichroism (CD) and nuclear magnetic
resonance (NMR) spectroscopies. The results indicate that the triplex is formed preferentially when
the triplex forming oligonucleotide (TFO) is RNA. In addition, DNA triplexes (D:D.D) are clearly less
stable in cosolute solutions while the stability of the RNA triplexes (R:D.D) is only slightly decreased.
The kinetic of triplex formation with RNA-TFO is slower than with DNA-TFO and the thermal
stability of the triplex is increased with the salt concentration in EtOH-water solutions. Accordingly,
RNA could be considered a potential molecule to form a stable triplex for regulatory purposes in
molecular crowding conditions.
... The association between noncoding RNA transcripts and genomic DNA is particularly relevant in the context of this review due to the formation of biologically active RNA-dsDNA triplex structures [34]. One such structure, described at the mouse Foxf1 locus, is thought to serve a scaffolding role for the delivery of site-specific epigenetic modifications leading to gene silencing. ...
Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA-RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA-RNA and RNA-RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA-RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review.
... One possibility would be that the duplex RNAs bind directly to chromosomal DNA. This binding could be through triple helix formation (135) or Watson-Crick base pairing (136). Zhang and colleagues have reported that cellular microRNAs can associate with RNA polymerase II and TATA-binding protein (TBP) and bind to TATA box motifs at gene promoters (137). ...
RNA interference (RNAi) is well known as a mechanism for controlling mammalian mRNA translation in the cytoplasm, but what
would be the consequences if it also functions in cell nuclei? Although RNAi has also been found in nuclei of plants, yeast,
and other organisms, there has been relatively little progress towards understanding the potential involvement of mammalian
RNAi factors in nuclear processes including transcription and splicing. This review summarizes evidence for mammalian RNAi
factors in cell nuclei and mechanisms that might contribute to the control of gene expression. When RNAi factors bind small
RNAs, they form ribonucleoprotein complexes that can be selective for target sequences within different classes of nuclear
RNA substrates. The versatility of nuclear RNAi may supply a previously underappreciated layer of regulation to transcription,
splicing, and other nuclear processes.
... Stable triplex have been described between double helix DNA and miRNA. There is evidence that transcriptional gene silencing over promoter regions mediated by triplex structures occurs in the cell [30,31] and the complex miRNA-DNA.DNA triplex has been found that is stabilized by proteins [32]. In particular, Triplex-forming complexes between human miRNAs (hsa-miR) that are complementary to human immunodeficiency virus (HIV-1) have been explored as antiviral therapeutic agents [33]. ...
The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.
... This same laboratory also established that miR-208a-5p and endoglin expression was up-regulated in an in vivo volume overloadinduced heart failure rat model (Table 8). Importantly, several recent studies have shown that, in addition to targeting mRNAs for translational repression and/or destabilization by the miRISC, miR-NAs may also function to induce gene expression by direct interactions with MRE sequences harbored within active promoters or by triplex structure formation (doublestranded DNA/RNA) stabilized by AGO2 (Dharap et al., 2013;Ma et al., 2010;Majid et al., 2010;Place et al., 2008;Toscano-Garibay et al., 2014;Zhang et al., 2014). Shyu et al. (2013) speculate that miR-208a-5p may interact with a MRE located in the promoter region of the rat Eng gene, which in turn induces rat endoglin gene expression. ...
Functionally matured microRNAs (miRNAs) are small single-stranded non-coding RNA molecules which are emerging as important post-transcriptional regulators of gene expression and consequently are central players in many physiological and pathological processes. Since the biological roles of individual miRNAs will be dictated by the mRNAs that they regulate, the identification and validation of miRNA/mRNA target interactions is critical for our understanding of the regulatory networks governing biological processes. We promulgate the combined use of prediction algorithms, the examination of curated databases of experimentally supported miRNA/ mRNA interactions, manual sequence inspection of cataloged miRNA binding sites in specific target mRNAs, and review of the published literature as a reliable practice for identifying and prioritizing biologically important miRNA/mRNA target pairs. Once a preferred miRNA/mRNA target pair has been selected, we propose that the authenticity of a functional miRNA/mRNA target pair be validated by fulfilling four well-defined experimental criteria. This review summarizes our current knowledge of miRNA biology, miRNA/mRNA target prediction algorithms, validated miRNA/mRNA target data bases, and outlines several experimental methods by which miRNA/mRNA targets can be authenticated. In addition, a case study of human endoglin is presented as an example of the utilization of these methodologies. © 2015, Leibniz Research Centre for Working Environment and Human Factors. All rights reserved.
... (B) sncRNAs in complex with AGO/PIWI also associate directly with DNA. This results in the recruitment of CMEs and epigenetic regulation [95]. AGO/PIWI indicates a member of either the argonaute or PIWI family of proteins. ...
The diverse functions of noncoding RNAs (ncRNAs) can influence virtually every aspect of the transcriptional process including epigenetic regulation of genes. In the CNS, regulatory RNA networks and epigenetic mechanisms have broad relevance to gene transcription changes involved in long-term memory formation and cognition. Thus, it is becoming increasingly clear that multiple classes of ncRNAs impact neuronal development, neuroplasticity, and cognition. Currently, a large gap exists in our knowledge of how ncRNAs facilitate epigenetic processes, and how this phenomenon affects cognitive function. In this review, we discuss recent findings highlighting a provocative role for ncRNAs including lncRNAs and piRNAs in the control of epigenetic mechanisms involved in cognitive function. Furthermore, we discuss the putative roles for these ncRNAs in cognitive disorders such as schizophrenia and Alzheimer's disease.
... Researches on ncRNAs have now gained the No.1 ranking in the top ten scientific breakthroughs in the early decades of the twentyfirst century (News, 2010;Pennisi, 2010). Over the past fifteen years, small regulatory ncRNA (<200 nucleotides in length), such as small interfering RNA (siRNAs) and mi-croRNAs (miRNAs), have been extensively investigated and the underlying molecular mechanisms have been well documented, suggesting that these ncRNAs play major roles in many cellular processes (Chitwood and Timmermans, 2010;Stuwe et al., 2014;Toscano-Garibay and Aquino-Jarquin, 2014). The surprises didn't stop at small ncRNAs, and an expanding body of evidence reveals that long noncoding RNAs (lncRNAs, >200 nucleotides in length), once were described as 'dark matter', act as essential regulators in diverse cellular progresses. ...
Cells can adapt to environment and development by reconstructing their transcriptional networks to regulate diverse cellular processes without altering the underlying DNA sequences. These alterations, namely epigenetic changes, occur during cell division, differentiation and cell death. Numerous evidences demonstrate that epigenetic changes are governed by various types of determinants, including DNA methylation patterns, histone posttranslational modification signatures, histone variants, chromatin remodeling, and recently discovered chromosome conformation characteristics and non-coding RNAs (ncRNAs). Here, we highlight recent efforts on how the two latter epigenetic factors participate in the sophisticated transcriptional process and describe emerging techniques which permit us to uncover and gain insights into the fascinating genomic regulation.
... This might regulate the rate or progression of transcription at these sites, even if the DNA is not cleaved. It has also been proposed that Ago-miRNA complexes might bind to specific double-stranded sites on promoters to form triple-helix structures [24]. b) DNA in the cytoplasm. ...
When a field shares the consensus that a particular phenomenon does NOT occur, this may reflect extensive experimental investigations with negative outcomes, or may represent the “common sense” position based on current knowledge and established ways of thinking. The current consensus of the RNA field is that eukaryotic Argonaute (Ago) proteins employ RNA guides and target other RNAs. The alternative -- that eukaryotic Ago has biologically important interactions with DNA in vivo – has not been seriously considered, in part because the only role contemplated for DNA was as a guide strand, and in part because it did not seem plausible that any natural source of suitable DNAs exists in eukaryotic cells. However, eukaryotic Argonaute domains bind DNA in the test tube, and several articles report that small inhibitory double-stranded DNAs do have the ability to silence target RNAs in a sequence-dependent (though poorly characterized) manner. A search of the literature identified potential DNA binding partners for Ago, including (among others) single-stranded DNAs residing in extracellular vesicles, and cytoplasmic satellite-repeat DNA fragments that are associated with the plasma membrane and transcribed by Pol II. It is interesting to note that both cytoplasmic and extracellular vesicle DNA are expressed at greatly elevated levels in cancer cells relative to normal cells. In such a pathological scenario, if not under normal conditions, there may be appreciable binding of Ago to DNA despite its lower affinity compared to RNA. If so, DNA might displace Ago from binding to its normal partners (miRNAs, siRNAs and other short ncRNAs), disrupting tightly controlled post-transcriptional gene silencing processes that are vital to correct functioning of a normal cell. The possible contribution to cancer pathogenesis is a strong motivator for further investigation of Ago-DNA binding. More generally, this case underscores the need for better informatics tools to allow investigators to analyze the state of a given scientific question at a high-level and to identify possible new research directions.
Reviewers: This article was reviewed by Eugene Koonin, Kira S. Makarova, Alexander Maxwell Burroughs (nominated by L Aravind), and Isidore Rigoutsos.
Open peer review: Reviewed by Eugene Koonin, Kira S. Makarova, Alexander Maxwell Burroughs (nominated by L Aravind), and Isidore Rigoutsos. For the full reviews, please go to the Reviewers’ comments section.
Satellite cells are bona fide muscle stem cells that are indispensable for successful post-natal muscle growth and regeneration after severe injury. These cells also participate in adult muscle adaptation in several capacities. microRNA (miRNA) are post-transcriptional regulators of mRNA that are implicated in several aspects of stem cell function. There is evidence to suggest that miRNAs affect satellite cell behavior in vivo and myogenic progenitor behavior in vitro, but the role of miRNAs in adult skeletal muscle satellite cells is less studied. In this review, we provide evidence for how miRNAs control satellite cell behavior with emphasis on satellite cells of adult muscle in vivo. We first outline how miRNAs are indispensable for satellite cell viability and control the phases of myogenesis. Next, we discuss the interplay between miRNAs and myogenic cell redox status, senescence, and communication to other muscle-resident cells during muscle adaptation. Results from recent satellite cell miRNA profiling studies are also summarized. In vitro experiments in primary myogenic cells and cell lines have been invaluable for exploring the influence of miRNAs, but we identify a need for novel genetic tools to further interrogate how miRNAs control satellite cell behavior in adult skeletal muscle in vivo.
As shown in Chap. 2, the short interfering RNA (siRNA) system was thought to be the same as the microRNA (miRNA) system. The miRNA genes are information because they are protein-noncoding RNAs. RNA informational genes (Rigs) are usually located in approximately 98% of the noncoding regions on the human genome. In contrast, miRNAs are deeply connected to the protein system. Rig is the master regulator of Central Dogma. Fine-tuning of protein expression by Rigs has been implicated in infection and cancer disease control. Traditional biogenesis of miRNAs is now a common pathway, but after biosynthesis, linear single-stranded miRNAs target linear single-stranded messenger RNA (mRNA), and miRNA seed regions target the 3′ untranslated region (3′ UTR) of mRNAs with a helix-like shape. The mature miRNA duplexes with mRNA in the Argonaute (Ago) protein. However, many human loop RNAs (loRNAs) have recently been reported, and the miRNA loop (lomiRNA) can be associated with Ago. Therefore, it is unknown whether helix recognition structures are common between miRNAs and mRNAs. Its helix geometry is unlikely to serve circular mRNA–miRNA interactions, and miRNA biogenesis or function has many exceptions, as does RNA interference. Thus, the miRNA machinery in circularity differs from RNA interference by Fire and Mellow. Due to the complexity of miRNA biogenesis and function, the biological relevance of circular mRNAs may be superimposed on circular torus miRNAs in RNA Wave 2000, suggesting that the coherence of torus miRNAs and torus mRNAs has implications for human health life. Beyond helices, the biosynthetic potential of tori could be a keystone for demonstrating this idea according to the quantum RNA language. In reality, circular RNA (circRNA) has been detected. Additionally, the miRNA quantum code was created from circular miRNA/miRNA relationships. Therefore, miRNA differs from RNA interference.
Non-canonical secondary structures (NCSs) are alternative nucleic acid structures that differ from the canonical B-DNA conformation. NCSs often occur in repetitive DNA sequences and can adopt different conformations depending on the sequence. The majority of these structures form in the context of physiological processes, such as transcription-associated R-loops, G4s, as well as hairpins and slipped-strand DNA, whose formation can be dependent on DNA replication. It is therefore not surprising that NCSs play important roles in the regulation of key biological processes. In the last years, increasing published data have supported their biological role thanks to genome-wide studies and the development of bioinformatic prediction tools. Data have also highlighted the pathological role of these secondary structures. Indeed, the alteration or stabilization of NCSs can cause the impairment of transcription and DNA replication, modification in chromatin structure and DNA damage. These events lead to a wide range of recombination events, deletions, mutations and chromosomal aberrations, well-known hallmarks of genome instability which are strongly associated with human diseases. In this review, we summarize molecular processes through which NCSs trigger genome instability, with a focus on G-quadruplex, i-motif, R-loop, Z-DNA, hairpin, cruciform and multi-stranded structures known as triplexes.
Non-coding DNA accounts for approximately 98.5% of the human genome. Once labeled as “junk DNA”, this portion of the genome has undergone a progressive re-evaluation and it is now clear that some of its transcriptional products, belonging to the non-coding RNAs (ncRNAs), are key players in cell regulatory networks. A growing body of evidence demonstrates the crucial impact of regulatory ncRNAs on mammalian gene expression. Here, we focus on the defined relationship between chromatin-interacting RNAs, particularly long non-coding RNA (lncRNA), enhancer RNA (eRNA), non-coding natural antisense transcript (ncNAT), and circular RNA (circRNA) and epigenome, a common ground where both protein and RNA species converge to regulate cellular functions. Through several examples, this review provides an overview of the variety of targets, interactors, and mechanisms involved in the RNA-mediated modulation of loci-specific epigenetic states, a fundamental evolutive strategy to orchestrate mammalian gene expression in a timely and reversible manner. We will discuss how RNA-mediated epigenetic regulation impacts development and tissue homeostasis and how its alteration contributes to the onset and progression of many different human diseases, particularly cancer.
Once considered to solely function to modify gene expression by modulating mRNA stability, the repertoire of miRNA function has expanded to include not only cytoplasmic but also nuclear effects. While most miRNA biogenesis is canonical in which the initial cleavage of a primitive miRNA by the nuclear microprocessor complex yields a precursor miRNA (pre-miRNA), which undergoes Exportin-5/RanGTP-mediated nucleocytoplasmic shuttling followed by further processing into a mature miRNA, several noncanonical pathways of both synthesis and nucleocytoplasmic shuttling are now evident. This includes functionally mature miRNAs and components of the miRNA-induced silencing complex undergoing shuttling back into the nucleus to further modulate gene expression at the levels of both transcription and mRNA processing. In this chapter, we will review the process of miRNA biogenesis in light of key regulatory nuclear steps, the evidence supporting a nuclear role for mature miRNAs, and the mechanisms by which miRNA are degraded, thus terminating their activity.
Die vorliegende Arbeit untersucht molekulare Mechanismen, die in Verbindung mit der Expression des Proteins Argonaut 2 (AGO2) zelluläre Eigenschaften des malignen Melanoms beeinflussen. AGO2 ist Teil des sogenannten „RNA induced silencing complex“ (RISC). Mit Hilfe von kleinen, nicht-codierenden RNAs (miRNAs oder siRNAs) reguliert der RISC spezifisch die Translation und Stabilität von mRNAs. Vordaten zu dieser Arbeit konnten zeigen, dass die Expression von AGO2 im malignen Melanom im Vergleich zu normalen humanen epidermalen Melanozyten oder Zelllinien anderer Tumorarten post-transkriptionell herunterreguliert ist. Die verminderte AGO2-Proteinexpression führt in Melanomzellen zu einer allgemein schlechteren Effizienz von siRNAs. Außerdem konnte in vorhergehenden Studien beobachtet werden, dass die AGO2-Reduktion die tumorigenen Eigenschaften von Melanomzellen begünstigt, wie zum Beispiel die Migration. Eine Analyse des molekularen Mechanismus, der zur AGO2-Reduktion im Melanom führt, stellt den ersten Teil dieser Arbeit dar. Dabei konnte zunächst mit Hilfe eines AGO2-Expressionsplasmids gezeigt werden, dass sich die Regulation von AGO2 auf Bereiche in der codierenden Sequenz (engl.: „coding sequence“; CDS) bezieht und transkriptions-, sowie transfektions-unabhängig abläuft. Ein erhöhter proteasomaler Proteinabbau oder eine verstärkte Aktivität des PI3K-vermittelten lysosomalen Abbauwegs konnten als Ursache für die AGO2-Reduktion in Melanomzellen ausgeschlossen werden. Weiterhin wurde keine melanomspezifisch verringerte AGO2-mRNA-Halbwertszeit gefunden. Um den für die Regulation verantwortlichen Sequenzbereich von AGO2 weiter einzugrenzen, wurden einzelne Teile der AGO2-CDS, die den funktionellen AGO2-Domänen entsprechen, separat in Expressionsvektoren kloniert. Dabei konnte eine gesteigerte Translation der AGO2-N+PAZ-mRNA im Vergleich zur AGO2-MID+PIWI-mRNA beobachtet werden. Eine weitere Unterteilung von AGO2-MID+PIWI ergab eine verbesserte Expression von sowohl AGO2-MID, als auch AGO2-PIWI im Vergleich zu AGO2-MID+PIWI in Melanomzellen. Dies deutet darauf hin, dass sich die regulatorische Sequenz genau am Übergang zwischen AGO2-MID und AGO2-PIWI befindet oder mehrere Sequenzen in AGO2-MID und AGO2-PIWI für die AGO2-Regulation im Melanom verantwortlich sind. Die Mutation der Bindestelle der miR-1265, die genau an diesem Übergang bindet, konnte die AGO2-Expression in Melanomzellen nicht verbessern. Ebenso wenig die Mutation von 24 weiteren miRNA-Bindestellen, die laut Computervorhersage an die AGO2-CDS binden und im Melanom im Vergleich zu normalen humanen epidermalen Melanozyten hochreguliert sind. Im zweiten Teil dieser Arbeit konnte die Existenz einer neuen AGO2-Spleißvariante identifiziert werden. Diese Variante, AGO2-Exon1/3, der Exon 2 fehlt, wird in verschiedenen Melanomzelllinien bis zu 15 % der normalen AGO2-mRNA exprimiert. Eine Expression von AGO2-Exon1/3 konnte weiterhin auch in Gewebeproben von Melanompatienten nach-gewiesen werden. Im weiteren Verlauf dieser Arbeit wurde die zelluläre Funktion von AGO2-Exon1/3 charakterisiert. Dazu wurden zunächst die Auswirkungen einer spezifischen, siRNA- vermittelten Reduktion der Expression von AGO2-Exon1/3 auf verschiedene Melanomzelllinien untersucht. Die gewonnenen Daten zeigen, dass sich aufgrund der reduzierten Expression von AGO2-Exon1/3 die Zellzahl verringert und die im „xCELLigence Real Time Cell Analyzer“ gemessene Proliferationsrate verlangsamt. Des Weiteren besitzen die Zellen eine verminderte Fähigkeit Kolonien aus Einzelzellen zu bilden. Der beobachtete Wachstumsdefekt der Zellen mit einer reduzierten Expression von AGO2-Exon1/3 lässt sich nicht auf zelluläre Seneszenz, einen Zellzyklusarrest oder eine langsamere Zellteilungsrate zurückführen. Jedoch führt die siRNA-vermittelte Reduktion von AGO2-Exon1/3 zu einer Induktion von Apoptose, aus der sich die geringere Zellzahl und Proliferationsrate erklärt. Eine Überexpression von AGO2-Exon1/3 zeigt einen gegenteiligen Effekt und steigert das Zellwachstum und die Klonogenität von Melanomzellen. Daraus lässt sich schließen, dass der Spleißmechanismus, der zur Bildung von AGO2-Exon1/3 führt, den Tumorzellen einen Überlebensvorteil bietet. In einer mittels der CRISPR/Cas-Technik generierten AGO2-Knockout Melanomzelllinie führt der Verlust von AGO2 zu einer gesteigerten Proliferation und einer vermehrten Fähigkeit Einzelzellkolonien zu bilden. In dieser Zelllinie wird AGO2-Exon1/3, das als Spleißprodukt nicht durch den CRSIPR/Cas-Knockout betroffen ist, verstärkt exprimiert. Ein siRNA-„knockdown“ von AGO2-Exon1/3 in diesen Zellen führt ebenfalls zu einem verringerten Zellwachstum. Dies deutet darauf hin, dass der Wachstumsvorteil der Zellen nur von der Expression von AGO2-Exon1/3, nicht aber von AGO2 abhängig ist. Die in dieser Arbeit neu identifizierte und charakterisierte AGO2-Spleißvariante AGO2-Exon1/3 stellt daher, unabhängig von AGO2, ein interessantes neues Zielgen zur Inhibition des Wachstums von Melanomzellen dar. Zusammen mit der Kenntnis des Mechanismus, der die AGO2-Expression in Melanomzellen reguliert, könnte eine neue, siRNA-basierte Therapiemöglichkeit für das maligne Melanom entwickelt werden. Somit stellen die in dieser Arbeit gewonnenen Daten einen wichtigen Schritt zum besseren Verständnis der Entstehung des malignen Melanoms dar und können helfen, der Tumorigenese entgegenzuwirken und mögliche siRNA-basierte Therapieoptionen zu verbessern.
L'épissage alternatif est le mécanisme permettant la production de plusieurs isoformes d'ARNs messagers à partir du même gène. La majorité des gènes humains sont concernés par ce processus. Epissage et transcription étant simultanés, les deux processus sont co-régulés. Plusieurs études récentes ont proposé que l'organisation tridimensionnelle du génome, qui régule la transcription, pourrait également moduler l'épissage. DDX5 et DDX17 sont deux hélicases à ARN impliquées dans plusieurs étapes de la biogenèse et de la maturation des ARNs, y compris la transcription et l'épissage. Des travaux de notre équipe ont montré que leur expression est réprimée lors de la différenciation cellulaire, ce qui contribue à établir des programmes d'épissage spécifiques. DDX5 et DDX17 interagissent avec CTCF et le complexe Cohésine, deux régulateurs de l’organisation 3D de la chromatine, suggérant un rôle des hélicases dans la topologie du génome, et potentiellement dans la connexion éventuelle entre organisation 3D et épissage. Nous avons dans un premier temps évalué l’impact à large échelle de DDX5/17 sur l’épissage par RNA-Seq, et montré que la co-déplétion de CTCF et de Cohésine avec les hélicases augmente leur effet sur l'inclusion de certains exons. De plus, nos résultats indiquent que la déplétion de DDX5/17 impacte la terminaison de la transcription de centaines de gènes. Enfin, nous avons sélectionné deux exons régulés par DDX5/17 et CTCF pour étudier l'organisation tridimensionnelle de leurs gènes par des expériences de 3C (Chromosome Conformation Capture). Le premier candidat est un exon interne du gène NCS1 et le second un exon situé just en aval du promoteurdu gène PRMT2. Nos résultats de 3C indiquent la présence d'une boucle entre le promoteur du gène NCS1 et l'exon alternatif interne. De plus, nous montrons pour les 2 gènes testés la proximité physique entre leur promoteur et leur région terminatrice, et une déstabilisation de cette boucle en absence de DDX5/17, ce qui pourrait expliquer les défauts observés de terminaison. . Enfin, une stabilisation contrainte de la boucle promoteur-terminateur du gène PRMT2 altère l'inclusion de l'exon promoteur proximal de ce gène. Ainsi, nos résultats appuient l'hypothèse d'un lien mécanistique entre l'organisation 3D des gènes et la régulation de l'épissage alternatif et plus généralement de la fidélité de leur transcription
Irritable bowel syndrome (IBS) is one of the commonest gastrointestinal disorders. Although long-time considered a pure functional disorder, intense research in past years has rendered a very complex and varied array of observations indicating the presence of structural and molecular abnormalities underlying characteristic motor and sensitive changes and clinical manifestations. Analysis of gene and protein expression in the intestinal mucosa has shed light on the molecular mechanisms implicated in IBS physiopathology. This analysis uncovers constitutive and inductive genetic and epigenetic marks in the small and large intestine that highlight the role of epithelial barrier, immune activation, and mucosal processing of foods and toxins and several new molecular pathways in the origin of IBS. The incorporation of innovative high-throughput techniques into IBS research is beginning to provide new insights into highly structured and interconnected molecular mechanisms modulating gene and protein expression at tissue level. Integration and correlation of these molecular mechanisms with clinical and environmental data applying systems biology/medicine and data mining tools emerge as crucial steps that will allow us to get meaningful and more definitive comprehension of IBS-detailed development and show the real mechanisms and causality of the disease and the way to identify more specific diagnostic biomarkers and effective treatments.
MicroRNAs (miRNAs) are key regulatory elements encoded by the genome. A single miRNA can downregulate the expression of multiple genes involved in diverse functions. Because cancer is a disease with multiple gene aberrations, developing novel approaches to identify and modulate miRNA pathways may result in a breakthrough for cancer treatment. With a special focus on glioblastoma (GBM), this review provides an up-to-date summary of miRNA biogenesis, the role of miRNA in cancer resistance, and essential tools for modulating miRNA expression, as well as of clinically promising RNAi delivery systems and how they can be adapted for therapy.
MicroRNAs are small non-coding RNAs with regulatory biological activity, by modulating target genes on epigenetic, transcriptional, post-transciptional and translational levels. Hundreds of reports indicated that miRNAs play important roles in non-small cell lung cancer (NSCLC). Actually, microRNAs are both regulation targets and regulators targeting effector genes. This article reviewed multifaceted role of microRNAs associated to NSCLC, not only targeting to but also targeted by tumor related genes, to help us understand microRNAs related complex regulation networks. Aberrant expressed micoRNAs and their targets were summarized; the statistical results showed that several microRNAs may play key roles by targeting multiple tumor associated targets. On the other hand, Oncogenes and tumor repressors represented by PTEN were also shown to be the most popular targets of microRNAs. Additionally, ZEB1/2 may be a featured pathway in NSCLC, with significant frequency modulated by microRNAs.
The phenomenon of RNA activation (RNAa) was initially discovered by Li and colleagues about a decade ago. Subsequently, gene activation by exogenously expressed small activating RNA has been demonstrated in different cellular contexts by a number of laboratories. Conceivably, endogenously expressed microRNAs may also utilize RNA activation as a cellular mechanism for gene regulation, which may be dysregulated in disease states such as cancer. RNA activation can be applied to gain-of-function studies and holds great promise for disease intervention. This chapter will discuss examples of promoter-targeting microRNAs discovered in recent years and their pathophysiological relevance. I will also briefly touch upon other novel classes of microRNAs with positive gene regulatory roles, including TATA-box-activating microRNAs and enhancer-associated microRNAs.
The various DNA conformational changes are correlation with biological event. In particular, DNA B-Z equilibrium was a high correlation with translation and transcription. In this study, we developed DNA probe containing 5-trifluoromethylcytidine or 5-trifluoromethylthymidine to detect DNA B-Z equilibrium using 19F-NMR. Its probe enable to detect the B-, Z-, and ss- DNA quantitatively based on 19F-NMR chemical shift change.
Cancer associated fibroblasts (CAFs) are a key component of the tumor microenvironment (TME). They play critical roles in the occurrence and development of gastric cancer (GC) through controlling various cytokines secretion and direct cell-to-cell interaction. However, the underlying mechanism of CAFs in tumor progression has not been entirely elucidated. MicroRNAs (miRNAs) as important factors have a central role in the interplay between tumor cell and TME. Recent studies also highlight that the aberrant expression of miRNAs in CAFs is involved in multiple functions in tumorigenesis and malignant process of GC. In this article, we shortly introduce the miRNAs biogenesis and provide an overview of the mechanisms and emerging roles of CAFs-related miRNAs. Focusing on these miRNAs as potential therapeutic targets may bring better treatment effect on GC and other diseases.
Long noncoding RNAs (lncRNAs) play a pivotal role in the regulation of biological processes through various mechanisms that are not fully understood. Proposed mechanisms include regulation based on RNA-protein interactions, as well as RNA-RNA interactions and RNA-DNA interactions. Here, we focus on one possible mechanism that lncRNA might be using to impact biological function, the RNA-DNA triplex formation. We summarize currently available examples of lncRNA triplex formation and discuss the details surrounding orientation of triplex formation as one of the key properties guiding this process. We propose that symmetrical triplex-forming motifs, especially those in cis-acting lncRNAs, favor triplex formation. We also consider the effects of lncRNA structures, protein or ligand binding, and chromatin structures on the lncRNAs triplex formation.
The emergence of psychiatric disorders relies on the interaction between genetic vulnerability and environmental adversities. Several studies have demonstrated a crucial role for epigenetics (e.g. DNA methylation, post-translational histone modifications and microRNA-mediated post-transcriptional regulation) in the translation of environmental cues into adult behavioural outcome, which can prove to be harmful thus increasing the risk to develop psychopathology. Within this frame, non-coding RNAs, especially microRNAs, came to light as pivotal regulators of many biological processes occurring in the Central Nervous System, both during the neuronal development as well as in the regulation of adult function, including learning, memory and neuronal plasticity. On these basis, in recent years it has been hypothesised a central role for microRNA modulation and expression regulation in many brain disorders, including neurodegenerative disorders and mental illnesses. Indeed, the aim of the present review is to present the most recent state of the art regarding microRNA involvement in psychiatric disorders. We will first describe the mechanisms that regulate microRNA biogenesis and we will report evidences of microRNA dysregulation in peripheral body fluids, in postmortem brain tissues from patients suffering from psychopathology as well as in animal models. Last, we will discuss the potential to consider microRNAs as putative target for pharmacological intervention, using common psychotropic drugs or more specific tools, with the aim to normalize functions that are disrupted in different psychiatric conditions.
Caenorhabditis elegans development is very tightly regulated, leading to the same number of cells in each individual. Part of this regulation network relies on small single strand RNAs (miRNAs), which can target homologous sequences in the 3’ untranslated regions (3’UTR) of messenger RNAs. We want to investigate the contribution of miRNAs during neurons differentiation. In order to study the miRNA contribution to gene regulation we use double fluorescent reporters that allow us to visualize the posttranscriptional contribution to regulation throughout development. The GFP and the mCherry are expressed under the control of the gene promoter, but followed by either the 3’UTR of interest, or a control 3’UTR. We first chose as a control 3’UTR the unc-54 3’UTR (myosin class II). The gene unc-54 is expressed through all larval stages and in the adult worms. The two colors reporter system showed that unc-54 3’UTR undergoes a regulation in the ADF pair of neurons and partially in the body wall muscle. The characterization of this regulation pointed out a potential role for mir-1820. The GFP was cloned between mir1820 5’ and 3’ sequences and the construction displayed an expression profile overlapping with the regulation pattern observed on unc-54 3’UTR.
Heparanase (HPSE) is the endogenous endoglycosidase that degrades heparan sulfate proteoglycans and promotes the tumor growth, invasion, metastasis, and angiogenesis. Our previous studies have shown that HPSE is highly expressed in neuroblastoma (NB), the most common extracranial solid tumor in childhood. However, the underlying regulatory mechanisms remain largely unknown. In this study, we identified one binding site of microRNA-558 (miR-558) within the HPSE promoter. In NB tissues and cell lines, miR-558 was up-regulated and positively correlated with HPSE expression. Gain- and loss-of-function studies demonstrated that miR-558 facilitated the transcript and protein levels of HPSE and its downstream gene, vascular endothelial growth factor, in NB cell lines. In addition, miR-558 enhanced the promoter activities of HPSE, and these effects were abolished by the mutation of miR-558 binding site. Mechanistically, miR-558 induced the enrichment of active epigenetic marker and RNA polymerase II on HPSE promoter in NB cells in an Argonaute 1-dependent manner, which was abolished by repressing the miR-558-promoter interaction. Knockdown of endogenous miR-558 decreased the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. In contrast, over-expression of miR-558 promoted the growth, invasion, metastasis, and angiogenesis of SH-SY5Y and SK-N-SH cells. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by knockdown or over-expression of miR-558. These data indicate that miR-558 induces the transcriptional activation of HPSE via the binding site within promoter, thus facilitating the tumorigenesis and aggressiveness of NB.
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MicroRNAs (miRNAs) are short RNA molecules that modulate post-transcriptional gene expression by partial or incomplete base-pairing to the complementary sequences on their target genes. Sequence-based miRNA target gene recognition enables the utilization of computational methods, which are highly informative in identifying a subset of putative miRNA targets from the genome. Subsequently, single miRNA-target gene binding is evaluated experimentally by in vitro assays to validate and quantify the transcriptional or post-transcriptional effects of miRNA-target gene interaction. Although ex vivo approaches are instructive in providing a basis for further analyses, in vivo genetic studies are critical to determine the occurrence and biological relevance of miRNA targets under physiological conditions. In the present review, we summarize the important features of each of the experimental approaches, their technical and biological limitations, and future challenges in light of the complexity of miRNA target gene recognition.
Although many long non-coding RNAs (lncRNAs) have been discovered, their function and their association with RNAi factors
in the nucleus have remained obscure. Here, we identify RNA transcripts that overlap the cyclooxygenase-2 (COX-2) promoter and contain two adjacent binding sites for an endogenous miRNA, miR-589. We find that miR-589 binds the promoter
RNA and activates COX-2 transcription. In addition to miR-589, fully complementary duplex RNAs that target the COX-2 promoter transcript activate COX-2 transcription. Activation by small RNA requires RNAi factors argonaute-2 (AGO2) and GW182, but does not require AGO2-mediated
cleavage of the promoter RNA. Instead, the promoter RNA functions as a scaffold. Binding of AGO2 protein/small RNA complexes
to the promoter RNA triggers gene activation. Gene looping allows interactions between the promoters of COX-2 and phospholipase A2 (PLA2G4A), an adjacent pro-inflammatory pathway gene that produces arachidonic acid, the substrate for COX-2 protein. miR-589 and
fully complementary small RNAs regulate both COX-2 and PLA2G4A gene expression, revealing an unexpected connection between key steps of the eicosanoid signaling pathway. The work demonstrates
the potential for RNA to coordinate locus-dependent assembly of related genes to form functional operons through cis-looping.
GW182 family proteins play important roles in microRNA (miRNA)-mediated gene silencing. They interact with Argonaute (Ago) proteins and localize in processing bodies, which are cytoplasmic foci involved in mRNA degradation and storage. Here, we demonstrated that human GW182 paralog, TNRC6A, is a nuclear-cytoplasmic shuttling protein, and its subcellular localization is conducted by a nuclear export signal (NES) and a nuclear localization signal (NLS) identified in this study. TNRC6A with mutations in its NES region was predominantly localized in the nucleus in an Ago-independent manner. However, it was found that TNRC6A could bring Ago protein into the nucleus via its Ago-interacting motif(s). Furthermore, miRNAs were also colocalized with nuclear TNRC6A-Ago and exhibited gene silencing activity. These results proposed the possibility that TNRC6A plays an important role in navigating Ago protein into the nucleus to lead miRNA-mediated gene silencing.
Bloom syndrome and Werner syndrome are genome instability disorders, which result from mutations in two different genes encoding
helicases. Both enzymes are members of the RecQ family of helicases, have a 3′ → 5′ polarity, and require a 3′ single strand
tail. In addition to their activity in unwinding duplex substrates, recent studies show that the two enzymes are able to unwind
G2 and G4 tetraplexes, prompting speculation that failure to resolve these structures in Bloom syndrome and Werner syndrome
cells may contribute to genome instability. The triple helix is another alternate DNA structure that can be formed by sequences
that are widely distributed throughout the human genome. Here we show that purified Bloom and Werner helicases can unwind
a DNA triple helix. The reactions are dependent on nucleoside triphosphate hydrolysis and require a free 3′ tail attached
to the third strand. The two enzymes unwound triplexes without requirement for a duplex extension that would form a fork at
the junction of the tail and the triplex. In contrast, a duplex formed by the third strand and a complement to the triplex
region was a poor substrate for both enzymes. However, the same duplex was readily unwound when a noncomplementary 5′ tail
was added to form a forked structure. It seems likely that structural features of the triplex mimic those of a fork and thus
support efficient unwinding by the two helicases.
Argonaute proteins play a major part in transcriptional gene silencing in many organisms, but their role in the nucleus of somatic mammalian cells remains elusive. Here, we have immunopurified human Argonaute-1 and Argonaute-2 (AGO1 and AGO2) chromatin-embedded proteins and found them associated with chromatin modifiers and, notably, with splicing factors. Using the CD44 gene as a model, we show that AGO1 and AGO2 facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Proper AGO1 and AGO2 recruitment to CD44 transcribed regions required the endonuclease Dicer and the chromobox protein HP1γ, and resulted in increased histone H3 lysine 9 methylation on variant exons. Our data thus uncover a new model for the regulation of alternative splicing, in which Argonaute proteins couple RNA polymerase II elongation to chromatin modification.
Advances in the understanding of the epigenetic events underlying the regulation of developmental genes expression and cell lineage commitment are revealing novel regulatory networks. These also involve distinct components of the epigenetic pathways, including chromatin histone modification, DNA methylation, repression by polycomb complexes and microRNAs. Changes in chromatin structure, DNA methylation status and microRNA expression levels represent flexible, reversible and heritable mechanisms for the maintenance of stem cell states and cell fate decisions. We recently provided novel evidence showing that microRNAs, besides determining the post-transcriptional gene silencing of their targets, also bind to evolutionarily conserved complementary genomic seed-matches present on target gene promoters. At these sites, microRNAs can function as a critical interface between chromatin remodeling complexes and the genome for transcriptional gene silencing. Here, we discuss our novel findings supporting a role of the transcriptional chromatin targeting by polycomb-microRNA complexes in lineage fate determination of human hematopoietic cells.
One of the common forms of multidrug resistance (MDR) is caused by activation of the mdr1 (ABCB1) gene, resulting in overexpression of P-glycoprotein (P-gp) and conferring cancer cell resistance to a broad range of chemotherapeutics. Recently, P-gp-mediated MDR has been associated with aberrant expression of microRNAs (miRNAs) in several types of cancer. miRNAs are small noncoding RNAs that regulate gene expression in a posttranscriptional manner through partial or total hybridization with specific sequences in the 3'-UTR of target mRNAs. Interestingly, there are at least two reports that suggest an additional regulation by miRNAs at the mdr1 promoter level. Here, we critically analyzed some of the miRNAs that regulate P-gp expression at two different levels: posttranscriptional and transcriptional. We proposed that the latter may occur through two possible scenarios: (1) direct miRNA hybridization with an active promoter and (2) triplex structure formation (double-stranded DNA/RNA) stabilized by Argonaute 2. Also, we classified transcriptional gene silencing (1) by homology, represented by small interfering RNAs directed to viral promoters, and (2) by complementarity (Watson-Crick/Hoogsteen base pairing), mediated by miRNAs. Transcriptional regulation could represent a new avenue of knowledge applicable to the modulation of other genes mediated by these noncoding RNAs.
Background:
microRNAs are generally understood to regulate gene expression through binding to target sequences within 3'-UTRs of mRNAs. Therefore, computational prediction of target sites is usually restricted to these gene regions. Recent experimental studies though have suggested that microRNAs may alternatively modulate gene expression by interacting with promoters. A database of potential microRNA target sites in promoters would stimulate research in this field leading to more understanding of complex microRNA regulatory mechanism.
Methodology:
We developed a database hosting predicted microRNA target sites located within human promoter sequences and their associated genomic features, called microPIR (microRNA-Promoter Interaction Resource). microRNA seed sequences were used to identify perfect complementary matching sequences in the human promoters and the potential target sites were predicted using the RNAhybrid program. >15 million target sites were identified which are located within 5000 bp upstream of all human genes, on both sense and antisense strands. The experimentally confirmed argonaute (AGO) binding sites and EST expression data including the sequence conservation across vertebrate species of each predicted target are presented for researchers to appraise the quality of predicted target sites. The microPIR database integrates various annotated genomic sequence databases, e.g. repetitive elements, transcription factor binding sites, CpG islands, and SNPs, offering users the facility to extensively explore relationships among target sites and other genomic features. Furthermore, functional information of target genes including gene ontologies, KEGG pathways, and OMIM associations are provided. The built-in genome browser of microPIR provides a comprehensive view of multidimensional genomic data. Finally, microPIR incorporates a PCR primer design module to facilitate experimental validation.
Conclusions:
The proposed microPIR database is a useful integrated resource of microRNA-promoter target interactions for experimental microRNA researchers and computational biologists to study the microRNA regulation through gene promoter. The database can be freely accessed from: http://www4a.biotec.or.th/micropir.
Cellular senescence is a tumour-suppressor mechanism that is triggered by cancer-initiating or promoting events in mammalian cells. The molecular underpinnings for this stable arrest involve transcriptional repression of proliferation-promoting genes regulated by the retinoblastoma (RB1)/E2F repressor complex. Here, we demonstrate that AGO2, RB1 and microRNAs (miRNAs), as exemplified here by let-7, physically and functionally interact to repress RB1/E2F-target genes in senescence, a process that we call senescence-associated transcriptional gene silencing (SA-TGS). Herein, AGO2 acts as the effector protein for let-7-directed implementation of silent-state chromatin modifications at target promoters, and inhibition of the let-7/AGO2 effector complex perturbs the timely execution of senescence. Thus, we identify cellular senescence as the an endogenous signal of miRNA/AGO2-mediated TGS in human cells. Our results suggest that miRNA/AGO2-mediated SA-TGS may contribute to tumour suppression by stably repressing proliferation-promoting genes in premalignant cancer cells.
microRNAs (miRNAs), defined as 21-24 nucleotide non-coding RNAs, are important regulators of gene expression. Initially, the functions of miRNAs were recognized as post-transcriptional regulators on mRNAs that result in mRNA degradation and/or translational repression. It is becoming evident that miRNAs are not only restricted to function in the cytoplasm, they can also regulate gene expression in other cellular compartments by a spectrum of targeting mechanisms via coding regions, 5' and 3'untransalated regions (UTRs), promoters, and gene termini. In this point-of-view, we will specifically focus on the nuclear functions of miRNAs and discuss examples of miRNA-directed transcriptional gene regulation identified in recent years.
Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoiesis or granulopoiesis, respectively. Here we show that NFI-A promoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin "bivalent domains," hypermethylation, recruitment of polycomb (PcG)-RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-A promoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-A transcriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes heterochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors.
RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21-30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.
DNA triplexes can naturally occur, co-localize and interact with many other regulatory DNA elements (e.g. G-quadruplex (G4) DNA motifs), specific DNA-binding proteins (e.g. transcription factors (TFs)), and micro-RNA (miRNA) precursors. Specific genome localizations of triplex target DNA sites (TTSs) may cause abnormalities in a double-helix DNA structure and can be directly involved in some human diseases. However, genome localization of specific TTSs, their interconnection with regulatory DNA elements and physiological roles in a cell are poor defined. Therefore, it is important to identify comprehensive and reliable catalogue of specific potential TTSs (pTTSs) and their co-localization patterns with other regulatory DNA elements in the human genome.
"TTS mapping" database is a web-based search engine developed here, which is aimed to find and annotate pTTSs within a region of interest of the human genome. The engine provides descriptive statistics of pTTSs in a given region and its sequence context. Different annotation tracks of TTS-overlapping gene region(s), G4 motifs, CpG Island, miRNA precursors, miRNA targets, transcription factor binding sites (TFBSs), Single Nucleotide Polymorphisms (SNPs), small nucleolar RNAs (snoRNA), and repeat elements are also mapped based onto a sequence location provided by UCSC genome browser, G4 database http://www.quadruplex.org and several other datasets. The results pages provide links to UCSC genome browser annotation tracks and relative DBs. BLASTN program was included to check the uniqueness of a given pTTS in the human genome. Recombination- and mutation-prone genes (e.g. EVI-1, MYC) were found to be significantly enriched by TTSs and multiple co-occurring with our regulatory DNA elements. TTS mapping reveals that a high-complementary and evolutionarily conserved polypurine and polypyrimidine DNA sequence pair linked by a non-conserved short DNA sequence can form miR-483 transcribed from intron 2 of IGF2 gene and bound double-strand nucleic acid TTSs forming natural triplex structures.
TTS mapping provides comprehensive visual and analytical tools to help users to find pTTSs, G-quadruplets and other regulatory DNA elements in various genome regions. TTS Mapping not only provides sequence visualization and statistical information, but also integrates knowledge about co-localization TTS with various DNA elements and facilitates that data analysis. In particular, TTS Mapping reveals complex structural-functional regulatory module of gene IGF2 including TF MZF1 binding site and ncRNA precursor mir-483 formed by the high-complementary and evolutionarily conserved polypurine- and polypyrimidine-rich DNA pair. Such ncRNAs capable of forming helical triplex structures with a polypurine strand of a nucleic acid duplexes (DNA or RNA) via Hoogsteen or reverse Hoogsteen hydrogen bonds. Our web tool could be used to discover biologically meaningful genome modules and to optimize experimental design of anti-gene treatment.
The ability of double-stranded DNA to form a triple-helical structure by hydrogen bonding with a third strand is well established, but the biological functions of these structures remain largely unknown. There is considerable albeit circumstantial evidence for the existence of nucleic triplexes in vivo and their potential participation in a variety of biological processes including chromatin organization, DNA repair, transcriptional regulation, and RNA processing has been investigated in a number of studies to date. There is also a range of possible mechanisms to regulate triplex formation through differential expression of triplex-forming RNAs, alteration of chromatin accessibility, sequence unwinding and nucleotide modifications. With the advent of next generation sequencing technology combined with targeted approaches to isolate triplexes, it is now possible to survey triplex formation with respect to their genomic context, abundance and dynamical changes during differentiation and development, which may open up new vistas in understanding genome biology and gene regulation.
Synthetic small duplex RNAs that are fully complementary to gene promoters can silence transcription in mammalian cells. microRNAs (miRNAs) are endogenous small regulatory RNAs that sequence specifically regulate gene expression. We have developed a computational method to identify potential miRNA target sites within gene promoters. Ten candidate miRNAs predicted to target the human progesterone receptor (PR) gene promoter were tested for their ability to modulate gene expression. Several miRNA mimics inhibited PR gene expression and miR-423-5p, which targets a highly conserved region of the PR promoter, was chosen for detailed analysis. Chromatin immunoprecipitation revealed that the miR-423-5p mimic decreased RNA polymerase II occupancy and increased histone H3 lysine 9 dimethylation (H3K9me2) at the PR promoter, indicative of chromatin-level silencing. Transcriptional silencing was transient, independent of DNA methylation, and associated with recruitment of Argonaute 2 (AGO2) to a non-coding RNA (ncRNA) transcript that overlaps the PR gene promoter. The miR-423-5p mimic also silenced expression of immunoglobulin superfamily member 1 (IGSF1), an additional gene with a predicted target site within its promoter. While additional investigations of endogenous miRNA function will be necessary, these observations suggest that recognition of gene promoters by miRNAs may be a natural and general mechanism for regulating gene transcription.
Noncoding RNAs are important components of regulatory networks controlling the epigenetic state of chromatin. We analyzed the role of pRNA (promoter-associated RNA), a noncoding RNA that is complementary to the rDNA promoter, in mediating de novo CpG methylation of rRNA genes (rDNA). We show that pRNA interacts with the target site of the transcription factor TTF-I, forming a DNA:RNA triplex that is specifically recognized by the DNA methyltransferase DNMT3b. The results reveal a compelling new mechanism of RNA-dependent DNA methylation, suggesting that recruitment of DNMT3b by DNA:RNA triplexes may be a common and generally used pathway in epigenetic regulation.
MicroRNAs (miRNAs) are approximately 22-nt small non-coding regulatory RNAs that have generally been considered to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in the nucleus.
To determine the number of miRNAs localized to the nucleus, we systematically investigated the subcellular distribution of small RNAs (sRNAs) by independent deep sequencing sequenced of the nuclear and cytoplasmic pools of 18- to 30-nucleotide sRNAs from human cells. We identified 339 nuclear and 324 cytoplasmic known miRNAs, 300 of which overlap, suggesting that the majority of miRNAs are imported into the nucleus. With the exception of a few miRNAs evidently enriched in the nuclear pool, such as the mir-29b, the ratio of miRNA abundances in the nuclear fraction versus in the cytoplasmic fraction vary to some extent. Moreover, our results revealed that a large number of tRNA 3' trailers are exported from the nucleus and accumulate in the cytoplasm. These tRNA 3' trailers accumulate in a variety of cell types, implying that the biogenesis of tRNA 3' trailers is conserved and that they have a potential functional role in vertebrate cells.
Our results provide the first comprehensive view of the subcellular distribution of diverse sRNAs and new insights into the roles of miRNAs and tRNA 3' trailers in the cell.
An efficient -1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote -1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several -1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of -1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the -1 PRF efficiency of this stimulator can be modulated by sequential removal of base-triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem-loop interactions and their relative positions to the helical junction play crucial roles for the -1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate -1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate -1 PRF. Furthermore, it suggests that -1 PRF, induced by an in-trans RNA via specific base-triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs.
Small RNAs targeted to gene promoters in human cells can mediate transcriptional gene silencing (TGS) by directing silent
state epigenetic modifications to targeted loci. Many mechanistic details of this process remain poorly defined, and the ability
to stably modulate gene expression in this manner has not been explored. Here we describe the mechanisms of establishment
and maintenance of long-term transcriptional silencing of the human ubiquitin C gene (UbC). Sustained targeting of the UbC promoter with a small RNA for a minimum of 3 days resulted in long-term silencing which correlated with an early increase
in histone methylation and a later increase in DNA methylation at the targeted locus. Transcriptional silencing of UbC required the presence of a promoter-associated RNA. The establishment and maintenance of the TGS were shown to require distinct
protein factors. Argonaute 1 (Ago1), DNA methyltransferase 3a (DNMT3a) and histone deacetylase 1 (HDAC1) were required for
the initiation of silencing, and DNA methyltransferase 1 (DNMT1) was necessary for maintenance. Taken together the data presented
here highlight the cellular pathway with which noncoding RNAs interact to epigenetically regulate gene expression in human
cells.
MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level in the cytoplasm, but recent findings suggest additional roles for miRNAs in the nucleus. To address whether miRNAs might transcriptionally silence gene expression, we searched for miRNA target sites proximal to known gene transcription start sites in the human genome. One conserved miRNA, miR-320, is encoded within the promoter region of the cell cycle gene POLR3D in the antisense orientation. We provide evidence of a cis-regulatory role for miR-320 in transcriptional silencing of POLR3D expression. miR-320 directs the association of RNA interference (RNAi) protein Argonaute-1 (AGO1), Polycomb group (PcG) component EZH2, and tri-methyl histone H3 lysine 27 (H3K27me3) with the POLR3D promoter. Our results suggest the existence of an epigenetic mechanism of miRNA-directed transcriptional gene silencing (TGS) in mammalian cells.
• miRNA
• POLR3D
• RNAi
• Argonaute
• epigenetic
MicroRNAs (miRNAs) are short RNAs that direct messenger RNA degradation or disrupt mRNA translation in a sequence-dependent manner. For more than a decade, attempts to study the interaction of miRNAs with their targets were confined to the 3' untranslated regions of mRNAs, fuelling an underlying assumption that these regions are the principal recipients of miRNA activity. Here we focus on the mouse Nanog, Oct4 (also known as Pou5f1) and Sox2 genes and demonstrate the existence of many naturally occurring miRNA targets in their amino acid coding sequence (CDS). Some of the mouse targets analysed do not contain the miRNA seed, whereas others span exon-exon junctions or are not conserved in the human and rhesus genomes. miR-134, miR-296 and miR-470, upregulated on retinoic-acid-induced differentiation of mouse embryonic stem cells, target the CDS of each transcription factor in various combinations, leading to transcriptional and morphological changes characteristic of differentiating mouse embryonic stem cells, and resulting in a new phenotype. Silent mutations at the predicted targets abolish miRNA activity, prevent the downregulation of the corresponding genes and delay the induced phenotype. Our findings demonstrate the abundance of CDS-located miRNA targets, some of which can be species-specific, and support an augmented model whereby animal miRNAs exercise their control on mRNAs through targets that can reside beyond the 3' untranslated region.
UV-absorption spectrophotometry and molecular modeling have been used to study the influence of the chemical nature of sugars (ribose or deoxyribose) on triple helix stability. For the Pyrimidine.purine* Pyrimidine motif, all eight combinations were tested with each of the three strands composed of either DNA or RNA. The chemical nature of sugars has a dramatic influence on triple helix stability. For each double helix composition, a more stable triple helix was formed when the third strand was RNA rather than DNA. No stable triple helix was detected when the polypurine sequence was made of RNA with a third strand made of DNA. Energy minimization studies using the JUMNA program suggested that interactions between the 2'-hydroxyl group of the third strand and the phosphates of the polypurine strand play an important role in determining the relative stabilities of triple-helical structures in which the polypyrimidine third strand is oriented parallel to the polypurine sequence. These interactions are not allowed when the third strand adopts an antiparallel orientation with respect to the target polypurine sequence, as observed when the third strand contains G and A or G and T/U. We show by footprinting and gel retardation experiments that an oligoribonucleotide containing G and A or G and U fails to bind double helical DNA, while the corresponding DNA oligomers form stable triple-helical complexes.
A monoclonal antibody, Jel 466, was prepared from mice immunized with poly[d(Tm5C)].poly[d(GA)]. The binding of Jel 466 to nucleic acids was characterized by solid phase radioimmunoassays and competition experiments. There was no binding to single-stranded DNAs or to duplexes which could not form triplexes. In addition, the antibody preferred the triplex form of poly[d(TC)].poly[d(GA)]; it bound weakly to the triplex derived from poly[d(G)].poly[d(C)], but there was no interaction with poly[d(T)].poly[d(A)].poly[d(T)]. This pattern of specificity is very different from that of Jel 318, a triplex-specific antibody that will bind to poly[d(T)].poly[d(A)].poly[d(T)]. The amino acid sequence of Jel 466 also showed very little homology with Jel 318, although both contain many positively charged amino acids. The immunofluorescent staining of mouse and human chromosomes with Jel 466 was studied. In all cases, there was a marked reciprocal relationship between the pattern of Jel 466 on the one hand and that of Hoechst 33258 and Jel 318 on the other. Jel 466 was negative for C-band and G-band but positive for R-band, whereas the opposite was found for Hoechst and Jel 318. Since C and G-bands are AT-rich and R-bands are GC-rich, these staining patterns match the sequence preferences of the two antibodies. Thus the base composition of triplex-forming DNA differs from domain to domain.
A DNA triplex is formed when pyrimidine or purine bases occupy the major groove of the DNA double Helix forming Hoogsteen pairs with purines of the Watson-Crick basepairs. Intermolecular triplexes are formed between triplex forming oligonucleotides (TFO) and target sequences on duplex DNA. Intramolecular triplexes are the major elements of H-DNAs, unusual DNA structures, which are formed in homopurine-homopyrimidine regions of supercoiled DNAs. TFOs are promising gene-drugs, which can be used in an anti-gene strategy, that attempt to modulate gene activity in vivo. Numerous chemical modifications of TFO are known. In peptide nucleic acid (PNA), the sugar-phosphate backbone is replaced with a protein-like backbone. PNAs form P-loops while interacting with duplex DNA forming triplex with one of DNA strands leaving the other strand displaced. Very unusual recombination or parallel triplexes, or R-DNA, have been assumed to form under RecA protein in the course of homologous recombination.
Identification of proteins binding specifically to peculiar nucleic acid structures can lead to comprehension of their role in vivo and contribute to the discovery of structure-related gene regulation. This work was devoted to establishing a reliable procedure to select proteins on the basis of their interaction with a nucleic acid probe chosen to fold into a given structure. 2D-electrophoresis and mass spectrometry were combined for protein identification. We applied this procedure to select and identify triplex-binding activities in HeLa nuclear extracts. To achieve this, we used a panel of deoxyribonucleic probes adopting intramolecular triple-helices, varying in their primary sequence, structure or triple-helix motif. A limited number of spots was reproducibly revealed by South-western blotting. Spots of interest were localised among a complex population of (35)S-labelled proteins according to their (32)P-specific emission. Position of the same spots was extrapolated on a preparative gel coloured with Coomassie blue, allowing excision and purification of the corresponding proteins. The material was subjected to mass spectrometry upon trypsin digestion and MALDI-TOF peptide fingerprinting was used for research in databases: five of them were identified and found to belong to the hnRNP family (K, L, A2/B1, E1 and I). The identities of several of them were confirmed by comparing western and South-western blots on the same membrane using specific antibodies. The recognition specificity of most of these proteins is large, according to previous reports and our own experiments. It includes pyrimidine-rich DNA sequences in different contexts: single strand to a small extent, triplex and possibly other higher-order structures.
Multifunctional proteins challenge the conventional ‘one protein–one function’ paradigm. Here we note apparent multifunctional
proteins with nucleic acid partners, tabulating eight examples. We then focus on eight additional cases of transcription factors
that bind double‐stranded DNA with sequence specificity, but that also appear to lead alternative lives as RNA‐binding proteins.
Exemplified by the prototypic Xenopus TFIIIA protein, and more recently by mammalian p53, this list of transcription factors includes WT‐1, TRA‐1, bicoid, the
bacterial σ70 subunit, STAT1 and TLS/FUS. The existence of transcription factors that bind both DNA and RNA provides an interesting puzzle.
Little is known concerning the biological roles of these alternative protein–nucleic acid interactions, and even less is known
concerning the structural basis for dual nucleic acid specificity. We discuss how these natural examples have motivated us
to identify artificial RNA sequences that competitively inhibit a DNA‐binding transcription factor not known to have a natural
RNA partner. The identification of such RNAs raises the possibility that RNA binding by DNA‐binding proteins is more common
than currently appreciated.
MicroRNAs (miRNAs) are small RNAs that regulate the expression of complementary messenger RNAs. Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved. With miRNA roles identified in developmental timing, cell death, cell proliferation, haematopoiesis and patterning of the nervous system, evidence is mounting that animal miRNAs are more numerous, and their regulatory impact more pervasive, than was previously suspected.
RNA interference (RNAi) is an evolutionarily conserved mechanism that uses short antisense RNAs that are generated by 'dicing' dsRNA precursors to target corresponding mRNAs for cleavage. However, recent developments have revealed that there is also extensive involvement of RNAi-related processes in regulation at the genome level. dsRNA and proteins of the RNAi machinery can direct epigenetic alterations to homologous DNA sequences to induce transcriptional gene silencing or, in extreme cases, DNA elimination. Furthermore, in some organisms RNAi silences unpaired DNA regions during meiosis. These mechanisms facilitate the directed silencing of specific genomic regions.
Alternative promoters within the same gene are a general phenomenon in gene expression. Mechanisms of their selective regulation vary from one gene to another and are still poorly understood. Here we show that in quiescent cells the mechanism of transcriptional repression of the major promoter of the gene encoding dihydrofolate reductase depends on a non-coding transcript initiated from the upstream minor promoter and involves both the direct interaction of the RNA and promoter-specific interference. The specificity and efficiency of repression is ensured by the formation of a stable complex between non-coding RNA and the major promoter, direct interaction of the non-coding RNA with the general transcription factor IIB and dissociation of the preinitiation complex from the major promoter. By using in vivo and in vitro assays such as inducible and reconstituted transcription, RNA bandshifts, RNA interference, chromatin immunoprecipitation and RNA immunoprecipitation, we show that the regulatory transcript produced from the minor promoter has a critical function in an epigenetic mechanism of promoter-specific transcriptional repression.
The function of microRNAs are well characterised in the cytoplasm, where they direct an Argonaute-containing complex to target and repress mRNAs. More recently, regulatory roles for microRNAs and Argonaute have also been reported in the nucleus where microRNAs guide Argonaute to target gene promoters and directly regulate transcription in either a positive or a negative manner. Deep sequencing has revealed a high abundance of endogenous microRNAs within the nucleus, and in silico target prediction suggest thousands of potential microRNA:promoter interaction sites. The predicted high frequency of miRNA:promoter interactions is supported by chromatin immunoprecipitation, indicating the microRNA-dependent recruitment of Argonaute to thousands of transcriptional start sites and the subsequent regulation of RNA Polymerase-II occupancy and chromatin modifiers. In this review we discuss the evidence for, and mechanisms associated with, direct transcriptional regulation by microRNAs which may represent a significant and largely unexplored aspect of microRNA function.
Despite billions of dollars allocated to cancer research, cancer remains the number 2 cause of death in the United States with less than 50% of advanced cancer patients living one year following standard treatment. Cancer is a complex disease both intrinsically and in relation to its host environment. From a molecular standpoint no two cancers are the same despite histotypic similarity. As evidenced by the recent advances in molecular biology, treatment for advanced cancer is headed towards specific targeting of vulnerable signaling nodes within the reconfigured pathways created by "omic" rewiring. With advancements in proteo-genomics and the capacity of bioinformatics, complex tumor biology can now be more effectively and rapidly analyzed to discover the vulnerable high information transfer nodes within individual tumors. RNA interference (RNAi) technology, with its capability to knock down the expression of targeted genes (the vulnerable nodes), is moving into the clinic to target these nodes, which are integral to tumor maintenance, with a low risk of side-effects and to block intrinsic immunosuppressors thereby priming the tumor for immune attack. An RNAi based sequential approach, a so called "one-two punch," is being advocated comprising tumor volume reduction (ideally to minimal residual disease status) effected by integrated multi-target knockdown followed by immune activation. Examples and recent developments are provided to illustrate this highly powerful approach heralding the future of personalized cancer therapy.
A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability.
MicroRNAs regulate self-renewal, differentiation, and division of cells via post-transcriptional gene silencing. Aberrant microRNA levels, specifically an overall downregulation, are present in many cancers, as compared to their normal tissue counterparts. Therefore, a potential therapeutic use of microRNAs is to correct these aberrant transcript levels involved in the signaling pathways of cancer. This review focuses on the current knowledge of microRNAs and their involvement with cancer cells and cancer stem cells. The methods currently being used to develop miRNA-based cancer therapeutics are examined, and the limitations halting further progress are also discussed.
One of the most fascinating discoveries in biology in recent years is unquestionably the identification of the family of small, noncoding RNAs known as microRNAs (miRNAs). Each miRNA targets multiple mRNA species through recognition of complementary sequences, typically located at multiple sites within the 3 untranslated region. In animals, single-stranded miRNA binds specific messenger RNA (mRNA) by a mechanism that is yet to be fully characterized. The bound mRNA remains untranslated resulting in reduced levels of the corresponding protein; however, if the sequence match between the miRNA and its target is precise, the bound mRNA can be degraded resulting in reduced levels of the corresponding transcript. Eukaryotic genes are also regulated by triplex formation between double helix and a third small RNA or DNA molecule. Thousands of triplex-forming (TF) islands in human genomes are mapped. However, the role of TF miRNAs within the hairpin structures of miRNA and the target mRNA has not been reported. We have explored TF complexes between human miRNAs (hsa-miR) that are complementary to human immunodeficiency virus (HIV)-1 and their antiviral potential as therapeutic agents.
We downloaded mature miRNA sequences from the human miRBase Sequence Database (http://microrna.sanger.ac.uk/sequences/), and computationally analyzed miRNAs that have significant homologies to HIV-1 genome (pNL 4-3 Accession #AF324493). We developed an algorithm to look for triplex-binding motifs (C+CG and T AT) and selected 4 miRNAs with 3 negative controls. TF stability was tested by using fluorophore-labeled duplexes connected by a single hexaethylene glycol moiety, representing HIV-1 proviral motifs, and black-hole quencher-1 labeled oligonucleotides, representing miRNA.
Fifty miRNAs were discovered that showed greater than 80% homology to HIV-1, of which 4 hsa-miR that exhibited an ability to form stable triplex with double stranded-HIV-1 sequences were selected. Three negative controls were used. The TF stability of the 4 hsa-miRs and the negative controls were confirmed and measured. Stably transfected Hela-CD4+ cell lines expressing each of the hsa-miR were developed. All 4 miRNAs exhibited a significant inhibition of HIV-1 as measured by HIV-1 p24 enzyme-linked immunosorbent assay (>90%; P>0.001) when compared with the 3 negative controls. By using immunohistochemical staining with triplex binding monoclonal antibodies, significant expression of TF miRNAs was detected in the cell lines, but not in the negative controls (P<0.001).
In this study, we demonstrated for the first time that besides the well-established post-transcriptional silencing based on mRNA degradation, miRNAs may be responsible for long-term latency of HIV-1 by TF, a different mechanism. We provide a possible molecular mechanism by which HIV-1 homologous miRNAs may impart resistance to HIV-1 and suggest a new miRNA-based therapeutic strategy for HIV-1.
Although a large portion of the human genome is actively transcribed into RNA, less than 2% encodes proteins. Most transcripts are non-coding RNAs (ncRNAs), which have various cellular functions. Small ncRNAs (or small RNAs) – ncRNAs that are characteristically ~20-35 nucleotides long – are
Many viruses use programmed -1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stem-loop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate frameshifting. Better understanding of the molecular determinants of frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting frameshifting.
Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.
Studies of a series of short oligonucleotide double and triple helices containing either all RNA, all DNA, or a mixture of the two show strand-dependent variation in their stability and structure. The variation in stability for both groups falls over a range of greater than 10 kilocalories per mole. In forming the triple helix, RNA is favored on both pyrimidine strands, whereas DNA is favored on the purine strand. In general, relatively unstable duplexes form particularly stable triplexes and vice versa. Structural data indicate that the strands in hybrid helices adopt a conformation that is intermediate between molecules containing all DNA and all RNA. Thus, RNA-DNA hybrids were not forced into the conformation of the RNA (A-form). The provocative stability of the triplex with an RNA third strand+DNA duplex points to novel antisense strategies and opens the possibility of an in vivo role of these structures. Overall, the data emphasize the fundamental role of sugars in determining the properties of nucleic acid complexes.
A monclonal antibody (Jel 318) was produced by immunizing mice with poly[d(Tm5 C)].poly[d(GA)].poly[d(m45 CT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs
such as poly[.d(TG)] .poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing m6 A (e.g. poly[d(TC)].poly[d(Gm6A)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes
was assessed by immunofluorescent microscopy of mouse myelona cells which had been fixed in methanol/acetic acid. An antibody
specific for duplex DNA (Jel 239) served as a control. The fluorescence due to Jel 318 was much weaker than that of Jel 239
but binding to metaphase chromosomes and interphase nuclei was observed. The staining by Jel 318 was unaffected by addition
of E. coli DNA but it was obliterated in the presence of triplex. Since an acid pH favours triplex formation, nuclei were also prepared
from mouse melanoma cells by fixation in cold acetone. Again Jel 318 showed weak but consistent staining of the nuclei. Therefore
it seeas likely that triplexes are an inherent feature of the structure of eucaryotic DNA.
Three-stranded nucleic acid complexes have been shown to be formed from double-stranded DNA and single-stranded RNA polymers with restricted base sequences. Complex formation takes place only when the DNA contains all purine bases in one strand and all pyrimidine bases in the other strand and the RNA must be a polypyrimidine polymer. Hence, poly (dA)·(dT) forms a three-stranded complex with poly U, and poly d(T-C)·d(G-A)† forms a three-stranded complex with poly (U-C) in acidic solutions. No other RNA's including the random co-polymer poly (U,C) interact with poly d(T-C)·dG-A) to give a stoichiometric three-stranded structure. Under no condition is a complex detected between poly d(T-G)·d(C-A) or poly d(A-T)·d(A-T) and any polyribonucleotide. The three-stranded poly d(T-C)·d(G-A)·(U-CH+) was characterized by cesium sulfate buoyant density studies, by continuous variations study, by ultraviolet spectral properties and by optical density-temperature profiles.Addition of poly (U-C) to poly d(T-C)·d(G-A) and addition of poly U to poly (dA)·(dT), prior to the addition of Escherichia coli RNA polymerase, effectively reduced the transcription rate of both strands of these DNA's. Other combinations of RNA's with DNA's showed essentially no inhibition; all of these RNA-DNA mixtures were also found to be inert as regards triplex formation. Possible biological roles of the triplexes are discussed.
Research concerning oligonucleotide-directed triple helix formation has mainly focused on the binding of DNA oligonucleotides
to duplex DNA. The participation of RNA strands In triple helices is also of interest. For the pyrimldine motif (pyrlmldlne-
purine (pyrimldlne triplets), systematic substitution of RNA for DNA in one, two, or all three triplex strands has previously
been reported. For the purine motif (purine-purine-pyrimldlne triplets), studies have shown only that RNA cannot bind to duplex
DNA. To extend this result, we created a DNA triple helix in the purine motif and systematically replaced one, two, or all
three strands with RNA. In dramatic contrast to the general accommodation of RNA strands in the pyrimidine triple helix motif,
a stable triplex forms In the purine motif only when all three of the substltuent strands are DNA. The lack of triplex formation
among any of the other seven possible strand combinations involving RNA suggests that: (i) duplex structures containing RNA
cannot be targeted by DNA oligonucleotides In the purine motif; (ii) RNA strands cannot be employed to recognize duplex DNA
in the purine motif; and (ill) RNA tertiary structures are likely to contain only isolated base triplets in the purine motif.
A 30 nt RNA with a sequence designed to form an intramolecular triple helix was analyzed by one- and two-dimensional NMR spectroscopy
and UV absorption measurements. NMR data show that the RNA contains seven pyrimidine-purine-pyrimidine base triples stabilized
by Watson-Crick and Hoog-steen interactions. The temperature dependence of the imino proton resonances, as well as UV absorption
data, indicate that the triple helix is highly stable at acidic pH, melting in a single sharp transition centered at 62°C
at pH 4.3. The Watson-Crick and Hoogsteen pairings are disrupted simultaneously upon melting. The NMR data are consistent
with a structural model where the Watson-Crick paired strands form an A-helix. Results of model building, guided by NMR data,
suggest a possible hydrogen bond between the 2′ hydroxyl proton of the Hoogsteen strand and a phosphate oxygen of the purine
strand. The structural model is discussed in terms of its ability to account for some of the differences in stability reported
for RNA and DNA triple helices and provides insight into features that are likely to be important in the design of RNA binding
compounds.
Because of the repetitive nature of the sequence, when titrating a G,A-rich, triple helix-forming oligonucleotide (TFO) with increasing concentrations of target duplex in order to obtain the dissociation constant of the complex, a duplex is sometimes first generated at intermediate concentrations of the target. This duplex is based on an imperfect Watson-Crick pairing of the TFO to the pyrimidine-rich strand of the target. An explanation is proposed for this duplex being obtained only in a certain domain of the titration range, before the triple helix becomes predominant.
In addition to traditional drug development methods designed to modulate the activity of protein targets, knowledge of disease gene DNA sequences provides an opportunity for the highly rational design of therapeutic agents that act at the DNA level through sequence-specific interactions. Among the ligands capable of binding DNA in a precise, sequence-specific manner are oligonucleotides, peptide nucleic acids and polyamides. Various strategies employing these agents to either transiently or permanently alter gene expression have been investigated over the past decade. During the past two to three years, important steps have been taken to illustrate the therapeutic potential of these ligands. Triple-helix (triplex) forming oligonucleotides have been particularly effective DNA-targeting agents with a wide range of applications, including the positive and negative transcriptional regulation of target genes, as well as the controlled delivery of site-specific mutations. This review will focus upon recent advances involving the use of sequence-specific DNA-binding ligands to modify gene expression and/or structure, with particular emphasis on the use of triplex-forming oligonucleotides in these roles.
As previously shown, type III intermediate filaments (IFs) select from a mixture of linear mouse genomic DNA fragments mobile and repetitive, recombinogenic sequences that have also been identified in SDS-stable crosslinkage products of vimentin and DNA isolated from intact fibroblasts. Because these sequences also included homopurine.homopyrimidine (Pu.Py) tracts known to adopt triple-helical conformation under superhelical tension, and because IF proteins are single-stranded (ss) and supercoiled DNA-binding proteins, it was of interest whether they have a particular affinity for triplex DNA. To substantiate this, IF-selected DNA fragments harboring a (Pu.Py) segment and synthetic d(GA)(n) microsatellites were inserted into a vector plasmid and the constructs analyzed for their capacity to interact with IF proteins. Band shift assays revealed a substantially higher affinity of the IF proteins for the insert-containing plasmids than for the empty vector, with an activity decreasing in the order of vimentin > glial fibrillary acidic protein > desmin. In addition, footprint analyses performed with S1 nuclease, KMnO(4), and OsO(4)/bipyridine showed that the (Pu.Py) inserts had adopted triplex conformation under the superhelical strain of the plasmids, and that the IF proteins protected the triple-helical insert sequences from nucleolytic cleavage and chemical modification. All these activities were largely reduced in extent when analyzed on linearized plasmid DNAs. Because intramolecular triplexes (H-DNA) expose single-stranded loops, and the prokaryotic ssDNA-binding proteins g5p and g32p also protected at least the Pu-strand of the (Pu.Py) inserts from nucleolytic degradation, it seemed likely that the IF proteins take advantage of their ssDNA-binding activity in interacting with H-DNA. However, in contrast to g5p and E. coli SSB, they produced no clear band shifts with single-stranded d(GA)(20) and d(TC)(20), so that the interactions rather appear to occur via the duplex-triplex and triplex-loop junctions of H-DNA. On the other hand, the IF proteins, and also g32p, promoted the formation of intermolecular triplexes from the duplex d[A(GA)(20).(TC)(20)T] and d(GA)(20) and d(TC)(20) single strands, with preference of the Py (Pu.Py) triplex motif, substantiating an affinity of the proteins for the triplex structure as such. This triplex-stabilizing effect of IF proteins also applies to the H-DNA of (Pu.Py) insert-containing plasmids, as demonstrated by the preservation of intramolecular triplex-vimentin complexes upon linearization of their constituent supercoiled DNAs, in contrast to poor complex formation from free, linearized plasmid DNA and vimentin. Considering that (Pu.Py) sequences are found near MAR/replication origins, in upstream enhancer and promoter regions of genes, and in recombination hot spots, these results might point to roles of IF proteins in DNA replication, transcription, recombination, and repair.
Altered expression of c-myc is implicated in pathogenesis and progression of many human cancers. Triple helix-forming oligonucleotides (TFOs) directed
to a polypurine/polypyrimidine sequence in a critical regulatory region near the c-myc P2 promoter have been shown to inhibit c-myc transcription in vitro and in cells. However, these guanine-rich TFOs had moderate binding affinity and required high concentrations for activity.
The 23 bp myc P2 sequence is split equally into AT- and GC-rich tracts. Gel mobility analysis of a series of short TFOs directed in parallel
and anti-parallel orientation to the purine strand of each tract showed that only parallel CT and anti-parallel GT TFOs formed
stable triplex on the AT- and GC-rich tracts, respectively. A novel full-length GTC TFO was designed to bind simultaneously
in parallel and anti-parallel orientation to the polypurine strand. Gel-shift and footprinting assays showed that the new
TFO formed a triple helix in physiological conditions with significantly higher affinity than an anti-parallel TFO. Protein-binding
assays showed that 1 µM GTC TFO inhibited binding of nuclear transcription factors to the P2 promoter sequence. The novel
TFO can be developed into a potent antigene agent, and its design strategy applied to similar genomic sequences, thus expanding
the TFO repertoire.
The polypurine/polypyrimidine (PuPy) tracts present in the human genome are known to be scattered among and within chromosomes. In PuPy tract sequences, triplex formation occurs readily under physiological conditions, leaving single-stranded DNAs capable of hybridization with complementary single-stranded DNAs and RNAs. The formation of single-strands and transmolecular triplexes is thought to enable sequences spaced distantly along the genome to associate with each other and organize nuclear DNA into ordered configurations. Triplex-forming DNAs in the human interphase nucleus were analyzed by combining fluorescence in situ "nondenaturing" hybridization employing PuPy tract probes and immunodetection by antitriplex antibodies. The nondenaturing hybridization technique, which has been used to detect RNA, may detect single-stranded DNAs in nondenatured nuclei, if present. Probes such as (GA/TC)(n) and (GAA/TTC)(n) sequences gave sequence-specific signals that overlapped with or were closely associated with triplexes immunolocalized by using known antitriplex antibodies. Pretreatment of nuclei with antitriplex antibodies blocked probe signal formation. Signal formation was resistant to pretreatment of nuclei with RNases but sensitive to single strand-specific nucleases. Triplexes visualized differentially with distinct PuPy tract probes were associated spatially with centromeric sequences in the interphase nucleus in a sequence-specific manner.
MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.
In recent years, triplex-forming oligonucleotides (TFOs) have emerged as powerful tools for site-specific gene modification. Their sequence specificity, binding affinity, and ability to provoke repair and recombination make them promising reagents for altering gene expression. This chapter highlights the binding requirements for triplex formation, identifies a number of chemical modifications that have been used with some success, and discusses studies using TFOs for inhibiting transcription. It also reviews work done using TFOs and related molecules to direct site-specific DNA damage, inducing mutagenesis or sensitizing a site to recombination. TFOs were initially used as positioning devices for nonspecific mutagens but were later discovered to have mutagenic properties of their own in cells with functional nucleotide excision repair (NER) and transcription-coupled repair (TCR) pathways. In subsequent studies triplex formation was able to induce both intramolecular and intermolecular homologous recombination, revealing its potential application for gene therapy. Recent reports demonstrate the ability of these molecules to locate and modify their cognate sites in chromosomal DNA in both cell culture and live animals, laying the foundation for triplex technology in vivo.
DNA triple helix has attracted a lot of attention in the recent past because of their potential therapeutic applications to inhibit the transcription of genes associated with several human diseases. Therefore, studies on DNA triplex formation and stabilization have become very essential to understand the nature of DNA triplex both in quantitative and qualitative terms. We have studied the effect of a tetrapeptide, Pro-Lys-Arg-Trp (PKRW), containing the conserved sequence (PKR) of high mobility group (HMG) B1 DNA binding domain on the stability of purine motif DNA triplex. The triple stranded DNA complex (23R*R*Y) containing (GAA) repeats used in the present study is formed by: 5'-TCGCGAAGAAGAAGAAGAACGCT-3', 5'-AGCGTTCTTCTTCTTCTTCGCGA-3' and the third strand 5'-AAGAAGAAGAAGAAG-3'. The DNA triplex 23R*R*Y and its complex with PKRW were investigated by DNA-melting, UV-absorption, circular dichroism and fluorescence spectroscopy. The increased stability of 23R*R*Y triplex in presence of PKRW is supported by the following observations; (i) a marked increase in triplex melting temperature (ii) significant increase in the ellipticity of the characteristic negative band at 210 nm and (iii) a large change in free energy, Delta DeltaG (DeltaG of triplex-peptide complex - DeltaG of free triplex), -9.6 kcal mol(-1) on complexation with PKRW. Based on the above observations and fluorescence quenching data, we suggest a two stage model for the binding of PKRW to 23R*R*Y in which (a) PKRW binds to the minor groove of the 23R*R*Y with lysine and arginine making electrostatic interactions with phosphates of DNA, which is followed by (b) intercalation of tryptophan side chain between the A*A*T base triplets. We conclude that PKRW preferentially stabilizes Hoogsteen base pair to the Watson-Crick base pair in purine motif 23R*R*Y DNA triplex. It is tempting to speculate that HMGB1 plays an important role in recognizing the DNA triplex structures like H-DNA in the gene, which may have biological implications with respect to chromatin structure and its function in the cell.