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Vatolin S, Navaratne K, Weil RJ. A novel method to detect functional microRNA targets
Abstract
MicroRNA (miRNA) molecules are non-coding RNAs, 19 to 24 nt in length that have been identified recently as important regulators of gene expression. Several computational methods have been developed to describe the target recognition mechanism by miRNA. We propose here a novel method to detect miRNA-mRNA complexes in eukaryotic cells. As a first step, we synthesize cDNA on an mRNA template using miRNAs as the endogenous cytoplasmic primer. This step extends miRNA and overcomes the problem of low complementary binding of miRNAs to their targets. Purified hybrid 3'-cDNA-miRNA-5' molecules are used in a second round of reverse transcription to anneal to target mRNA in a highly gene-specific manner. The 5'-end analysis of these cDNA molecules demonstrated that primers for cDNAs were "signatures" of miRNA molecules, and over-expression of their full-length mature miRNAs resulted in functional inhibition of target protein expression.
... The first approach exploiting this conceptual framework aimed to identify miRNAs that were associated with unique candidate targets. 241 In this instance, a short RT reaction on the hTERT-RPE1 cytoplasmic fraction was used to extend bound miR-NAs and generate a DNA-miRNA hybrid. The resulting chimeras were then purified and used for a second round of RT at a higher temperature to further extend the hybrids. ...
... Based on hybrid analysis, a number of possible miRNA-target interactions were proposed including, miR-32/miR-129:β-actin, miR-33/miR-17/let-7a:K-Ras, and let-7a:N-Ras. 241 The second study used a nearly identical concept, but aimed to discover new targets of a specific miRNA in C. elegans extracts. 242 In this case, RBPs and components of the miRISC were initially removed by a short treatment with a strong detergent at low temperature. ...
The discovery over two decades ago of short regulatory microRNAs (miRNAs) has led to the inception of a vast biomedical research field dedicated to understanding these powerful orchestrators of gene expression. Here we aim to provide a comprehensive overview of the methods and techniques underpinning the experimental pipeline employed for exploratory miRNA studies in animals. Some of the greatest challenges in this field have been uncovering the identity of miRNA–target interactions and deciphering their significance with regard to particular physiological or pathological processes. These endeavors relied almost exclusively on the development of powerful research tools encompassing novel bioinformatics pipelines, high‐throughput target identification platforms, and functional target validation methodologies. Thus, in an unparalleled manner, the biomedical technology revolution unceasingly enhanced and refined our ability to dissect miRNA regulatory networks and understand their roles in vivo in the context of cells and organisms. Recurring motifs of target recognition have led to the creation of a large number of multifactorial bioinformatics analysis platforms, which have proved instrumental in guiding experimental miRNA studies. Subsequently, the need for discovery of miRNA–target binding events in vivo drove the emergence of a slew of high‐throughput multiplex strategies, which now provide a viable prospect for elucidating genome‐wide miRNA–target binding maps in a variety of cell types and tissues. Finally, deciphering the functional relevance of miRNA post‐transcriptional gene silencing under physiological conditions, prompted the evolution of a host of technologies enabling systemic manipulation of miRNA homeostasis as well as high‐precision interference with their direct, endogenous targets. WIREs Dev Biol 2016, 5:311–362. doi: 10.1002/wdev.223
For further resources related to this article, please visit the WIREs website.
... Application of computational methods resulted in a large set of predicted miRNA-mRNA duplexes in which the 3 0 end of the miRNA was complementary to the target (Bentwich, 2005;Enright et al., 2003;John et al., 2004;Lewis et al., 2003). To verify biocomputational methods and to derive a rapid, reproducible, and inexpensive method to detect miRNA targets, we postulated that a miRNA may prime cDNA synthesis from a target mRNA in the presence of reverse transcriptase and dNTPs (Vatolin et al., 2006). Recently, a technique similar to this has been described by Andachi. ...
... This chapter concentrates on a detailed description of these two steps of reverse transcription, as well as on amplification of the final cDNA molecules, which can be cloned readily. The final steps of cDNA sequencing, computational analysis of the resulting cDNA library, and functional analyses use conventional methods that are not addressed here (Vatolin et al., 2006). ...
Mammalian cells express a large number of small, noncoding RNAs, including micro-RNAs (miRNAs), that can regulate both the level of a target mRNA and the protein produced by the target mRNA. Recognition of miRNA targets is a complicated process, as a single target mRNA may be regulated by several miRNAs. The potential for combinatorial miRNA-mediated regulation of miRNA targets complicates diagnostic and therapeutic applications of miRNAs. Despite significant progress in understanding the biology of miRNAs and advances in computational predictions of miRNA targets, methods that permit direct physical identification of miRNA-mRNA complexes in eukaryotic cells are still required. Several groups have utilized coimmunoprecipitation of RNA associated with a protein(s) that is part of the RNA silencing macromolecular complex. This chapter describes a detailed but straightforward strategy that identifies miRNA targets based on the assumption that small RNAs base paired with a complementary target mRNA can be used as a primer to synthesize cDNA that may be used for cloning, identification, and functional analysis.
... The pre-miRNA is exported from the nucleus to the cytoplasm by Exportin5/RanGTP. Once in the cytoplasm, a second RNase III, termed ''Dicer'' in conjunction with a double-stranded RNA binding domain (dsRBD) cleaves the pre-miRNA, releasing a 19-to 25-nt RNA duplex (mature miRNA and its complement miRNA*) (17). A single strand of the miRNA/miRNA* duplex is released and incorporated into the miRNA-induced silencing complex (miRISC) while the other strand is degraded. ...
... The miRISC represents a regulatory complex consisting of several protein factors, including the Argonaut protein, and is the focus of extensive study (18,19). The miRISC guides miRNAs to the target mRNA to affect either mRNA degradation or translational inhibition (17). This is partially accomplished through core base-pair complementarity between the miRNA 59 region (termed the ''seed sequence'') and target mRNA 39 UTR. ...
Over the last 15 years, investigators have identified small noncoding RNAs as regulators of gene expression. One type of noncoding RNAs are termed microRNAs (miRNAs). miRNAs are evolutionary conserved, approximately 22-nucleotide single-stranded RNAs that target genes by inducing mRNA degradation or by inhibiting translation. miRNAs are implicated in many critical cellular processes, including apoptosis, proliferation, and differentiation. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Despite the identification of greater than 500 mature miRNAs, very little is known about their biological functions and functional targets. In the last 5 years, researchers have increasingly focused on the functional relevance and role that miRNAs play in the pathogenesis of human disease. miRNAs are known to be important in solid organ and hematological malignancies, heart disease, as potential modulators of the immune response, and organ development. It is anticipated that miRNA analysis will emerge as an important complement to proteomic and genomic studies to further our understanding of disease pathogenesis. Despite the application of genomics and proteomics to the study of human lung disease, few studies have examined miRNA expression. This perspective is not meant to be an exhaustive review of miRNA biology but will provide an overview of both miRNA biogenesis and our current understanding of the role of miRNAs in lung disease as well as a perspective on the importance of integrating this analysis as a tool for identifying and understanding the biological pathways in lung-disease pathogenesis.
... This strategy employs miRNA extension with a reverse transcriptase on endogenous target mRNAs. Purified hybrid 3'-cDNA-miRNA-5' molecules are used in a second round of reverse transcription and sequenced (Vatolin et al., 2006). However, this method is prone to artifacts stemming from the variability of miRNA:target mRNA base pairing, which would result in highly variable efficiency of reverse transcription priming. ...
This review systematically covers sequence-specific gene regulation by miRNAs and dsRNA-derived siRNAs in animals and plants from the perspective of target RNA recognition, potential for non-target (off-target) effects, and reliable determination of biological effects of small RNAs in animals and plants. I will review sequence complementarity between target RNA and small RNA (siRNA or miRNA), including tolerance to mismatches, parameters influencing sequence complementarity (and target recognition and repression) and discuss specificity of targeting by miRNAs and off-targeting by siRNAs. In addition, I will discuss reliable identification of target RNAs (and, eventually, biological effects). Accordingly, the text is divided into the following four sections: (I) Small RNA:target RNA base pairing, (II) Other key factors influencing target recognition and repression, (III) Off-targeting – causes and remedies, (III) Small RNA target identification.
NOTE: This is a chapter from an open source book. The full text is available directly through the DOI link above.
... An interesting alternative method on target detection was proposed by Vatolin et al. [111]. Instead of trying to find the mRNA expression changes of the mRNA seeds complementary to the miRNA of interest, Vatolin et al. used the sequence of the miRNA to generate primers via reverse transcription matching of the mRNA targets. ...
RNAs are small noncoding RNAs that regulate gene expression. Their biological role was recently discovered and immediately raised scientific interest. In a relatively small period of time the research on microRNAs revealed their key role and involvement in many pathologies by either deregulating proteins responsible for causing diseases or deregulation of microRNAs themselves and affecting the physiology. The small size of these molecules is a challenge for their research but paved the road to development of new technologies and methods which led to the understanding of microRNAs function and biological role and setting them as potential biomarkers and therapeutic targets. In this chapter discusses the biological role and function of the microRNAs, as well as the latest developments and methods of study, and gives examples of some microRNAs with proven key role in pathologies and are on the way to be biomarkers and therapeutic targets.
... For example, miR124a target is enriched with immunoprecipitation of Ago2 gene (Karginov et al. 2007). Also, synthesis of cDNA clones of known mRNA templates with miR primers allows identification of miRs as well as binding sites for mRNA (Vatolin et al. 2006) ...
Ovarian cancer (OC) is the sixth most common cancer in women globally. However, even with the advances in detection andtherapeutics it still represents the most dangerous gynecologic malignancy in women of the industrialized countries. The discovery of micro- RNAs (miRNA), a small noncoding RNA molecule targeting multiple mRNAs and regulation of gene expression by triggering translation repression and/or RNA degradation, has revealed the existence of a new array for regulation of genes involved in cancer. This review summarizes the current knowledge regarding the role of miRNAs expression in OC. It also provides information about potential clinical relevance of circulating miRNAs for OC diagnosis, prognosis, and therapeutics. The identification of functional targets for miRNAs represents a major obstacle in our understanding of microRNA function in OC, but significant progress is being made. The better understanding of the role of microRNA expression in ovarian cancer may provide new array for the detection, diagnosis, and therapy of the OC. This article is protected by copyright. All rights reserved.
... Je¿eli miRNA zwi¹¿e siê z docelowym transkryptem w sposób czêciowo komplementarny, to dochodzi do inhibicji procesu translacji. Z kolei je¿eli cz¹steczki te po³¹cz¹ siê ze sob¹ w sposób ca³kowicie komplementarny, wówczas mRNA jest kierowane na drogê degradacji [80]. ...
The objective of this study was to evaluate the morphology of BOECs cocultured with cattle embryos at the elevated temperature of 41°C. Bovine oviducts and ovaries were collected post mortem from slaughtered cattle. Oviduct epithelial cells were isolated from oviducts, which were obtained from cattle between days 0 and 4 of estrous cycle. Cattle embryos were obtained based on in vitro fertilization of oocytes matured in vitro. BOECs were cocultured with cattle embryos at control (38.5°C) and elevated (41°C) temperatures for 168 h. Statistical analyses were performed by Statgraphics 5.0 Centurion using T-test. After 168 h, the percentage of blastocysts was significantly higher at 38.5°C than at 41°C (30.03 ± 2.07 vs. 0), (p < 0.001). The percentage of viable cells was similar in the BOECs from control (84 ± 3.02) and elevated (82 ± 2.16) temperatures. Analysis of the cilia movement of ciliated cells indicated no difference between control and elevated temperatures. The SEM micrographs’ analysis indicated that the length of cilia on BOECs surface at 38.5°C was similar to 41°C (5.5 μm ± 0.3 vs. 5.3 μm ± 0.2). The number of microvilli on secretory cells was also similar at control (44.5 ± 1.3) and elevated (43.7 ± 1.2) temperatures. The TEM micrographs’ analysis showed that the number of secretory cells of BOECs cultured at control temperature (12 ± 3.6) was similar to elevated temperature (11.9 ± 4.1). In conclusion, the elevated temperature has no effect on the morphology of BOECs but negatively influences cattle embryo development. (GRANT 453/N-COST/2009/0)
... Je¿eli miRNA zwi¹¿e siê z docelowym transkryptem w sposób czêciowo komplementarny, to dochodzi do inhibicji procesu translacji. Z kolei je¿eli cz¹steczki te po³¹cz¹ siê ze sob¹ w sposób ca³kowicie komplementarny, wówczas mRNA jest kierowane na drogê degradacji [80]. ...
RNA degradation in yeast and mammalian cells take place in cytoplasm, nucleus and mitochondria. Degradation of transcripts with a premature termination codon or without stop codon results in repression of aberrant protein synthesis. The mRNA decay pathway, which degrades mRNAs containing a stem-loop structure has been discovered and characterized in the yeast Saccharomyces cerevisiae. Eukaryotic transcripts with AU-rich elements in their 3'-untranslated region have a very short half-life. These elements regulate mRNA level in cells. The multi-enzymatic nuclear exosome complex, Rat I p and cap binding complex degrade mRNAs in the yeast nucleus. The mitochondria! degradosome (mtEXO) is an enzymatic complex which has a major function in the mitochondrial genome post-transcription gene expression regulation. RNA interference cleaves mRNA after a previous induction by dsRNA. The 2-5A/RNase L system inhibits replication of viral RNA.
... It is known that while each mRNA can be targeted by more than one miRNA (Vatolin et al. 2006 ), a single miRNA can regulate many different transcripts. In this regard, miR -15a and miR -16 have been shown to regulate expression of ≈14 % of the human genome in a leukemic cell line . ...
In eukaryotes, DNA packaging into nucleosomes and higher-order chromatin structures is able to prevent the operation of the nuclear factors in charge of genetic functions. For this reason, during all DNA-templated cellular processes, chromatin structures must undergo dynamic remodeling (opening and closing of higher-order structures) in order to regulate access to their corresponding DNA segments. Epigenetics comprises a highly connected, dynamic set of mechanisms through which cells can execute this key remodeling: DNA methylation, covalent histone modifications, histone variants, and ATP-dependent chromatin-remodeling complexes. Additionally, microRNAs are usually incorporated into the same category, as their regulation can directly affect, and be affected by, the former. Disruption of any of these processes, which are essential for cell renewal, differentiation, and stemness, is intimately linked with cancer. © 2014 Springer-Verlag Berlin Heidelberg. All rights are reserved.
... [19] In addition, the same mRNA can be targeted by several miRNAs. [20] miRNAs as tumor suppressors and oncogenes miRNA expression patterns are highly specific for cell type and cellular differentiation status. It is therefore likely that much of the aberrant miRNA expression observed in tumors is a secondary consequence of the loss of normal cellular identity that accompanies malignant transformation. ...
In spite of advances in diagnostic techniques, surgery, chemotherapy and radiotherapy, colorectal cancers remain undefeated. In the absence of screening, colorectal cancers are diagnosed in an advanced stage when regional and distant metastasis is present. Hence, the hope for control is primary prevention or early diagnosis. Western lifestyle and diet have been implicated in the causation of colon cancers. However, it is still a controversy whether this is due to excess calories, high fat content, genotoxic agents, or lack of protective agents present in vegetables and fruits. Therefore, recommending a specific cancer prevention diet can have fallacies. In this context reduction in cancer mortality can be achieved by screening population at high risk. The colorectal cancers require investigative modalities like colonoscopy, sigmoidoscopy or fecal occult blood testing (FOBT) for screening. Colonoscopy is the most sensitive and specific of all the available colorectal screening tests, whereas the sensitivity and specificity for FOBT and sigmoidoscopy are much lower. Although performance of FOBT is relatively inexpensive, sigmoidoscopy and colonoscopy must be performed by trained endoscopists and are more expensive. Moreover, lack of awareness that colorectal cancer is a prevalent and serious disease, concerns about the potential discomforts of colorectal cancer procedures or of the preparations for screening appear to be potential barriers for colorectal cancer screening. MicroRNAs (miRNAs) have roles in colon carcinogenesis; therefore, may be useful biomarkers for colorectal carcinoma (CRC). They are short ribonucleic acid (RNA) molecules having very few nucleotides compared with other RNAs. miRNAs have been studied intensively in the field of oncological research, and emerging evidence suggests that altered miRNA regulation is involved in the pathogenesis of cancers. This review summarizes the use of miRNA in the early diagnosis of colorectal cancers.
... Dweep et al. (2013) reviewed a number of databases and software for their sensitivity and accuracy for predicting miRNAs and their target sites. The two primary issues that arise when studying miRNAs in Drosophila is the relative lack of bioinformatic tools, in addition to the fact that that miRNA/mRNA interaction is still so poorly understood it makes prediction complicated and requires experimental confirmation (Betel et al., 2008, Dweep et al., 2013, Ebert et al., 2007, Jin et al., 2010, Kertesz et al., 2007, Krek et al., 2005, Vatolin et al., 2006. As well as testing for miRNAs that target an intron, in some cases, an intron can form a hairpin that would code for a miRNA and so the possibility that dmpi8 generates a miRNA was also tested ...
When an organism migrates from one area to another it comes into contact with many boundaries to its survival and fitness. The fruitfly Drosophila melanogaster has migrated from Africa, into Europe and colonised much of the rest of the world. The subject of this thesis is to better understand how Drosophila has adapted to survive the temperate climates in Europe. The variability of temperature and light from one season to the next makes adaptation of the circadian clock and life history strategy all the more important. Drosophila appear to have adjusted to the new conditions by exhibiting diapause in low temperatures when the nights are long and by altering several other characteristics of its circadian clock that may be related to diapause. One of these is a novel European single nucleotide polymorphism in the timeless gene that allows flies to maintain a more robust diapause than flies carrying the ancestral allele. This variant exists in all European populations and winter simulation experiments reveal that it maintains its diapause for longer than the ancestral variant. These experiments also supported the possibility that Drosophila diapause can be maintained for much longer periods than previous studies have indicated. Experiments with ancestral African D. melanogaster lines alongside several closely related species indicates that diapause may not be a recent adaptation, but an ancient response to stressful conditions that has adapted in Europe to be more sensitive to low temperatures and short photoperiods. I also discover that a splice variant of the period gene has a dramatic, further cementing the controversial relationship between clock genes and diapause. In addition I have performed a study of putative functional polymorphisms in this untranslated region around this splice site from European populations. Finally, a study of putative clock genes reported in Chapter 3 provides a cautionary tale as to the dangers of using RNAi.
... Each mRNA can be targeted by more than one miRNA [13], and each miRNA can target hundreds of different transcripts. For instance, it was shown that a cluster of two miRNAs (miR-15a and miR-16) can affect the expression of about 14% of the human genome in a leukemic cell line [14]. ...
Epigenetic alterations have been reported to deregulate the expression of many transcripts, including noncoding RNAs that have no apparent protein-coding capacity. Recently, as the result of numerous studies focused on miRNAs, novel sequencing technologies have made available the transcription profile of the entire human genome. miRNAs as drivers of tumor-suppressive and oncogenic functions have been found to be dysregulated in numerous cancer types. However, the functions of epigenetically regulated genetic elements other than protein-coding genes are still a matter of debate. In this review, the authors focus mainly on describing the epigenetic regulation of miRNAs in cancer. They also discuss the role of miRNAs as potential diagnostic and/or prognostic biomarkers.
... The pri-miRNA is cleaved by the Drosha ribonuclease III enzyme to generate an approximately 70nt stem-loop precursor miRNA (pre-miRNA), which is exported into the cytoplasm by the transporter exportin 5 [4]. Subsequently, it is further cleaved by the Dicer ribonuclease to produce the mature miRNA and antisense miRNA star (miRNA*) products [5]. The mature miRNA is incorporated into a RNA-induced silencing complex (RISC), which most commonly results in translational inhibition or destabilization of the target mRNAs [6,7]. ...
The miR-451 was found to be frequently down-regulated in tumors, indicating that miR-451 could play an important role in carcinogenesis. This study uncovered the mechanism by which the miR-451 functions as a tumor suppressor. The target genes of miR-451 were determined using target gene prediction softwares. Then the miR-451 mimics were introduced into RKO and Hela cells respectively. The proliferation and invasion of cells were monitored by MTT, cell cycle and in vitro extracellular matrix invasion assays. Also the angiogenesis of HUVEC cells transfected with miR-451 mimics was examined. Subsequently, IL6R, a predicted target gene of miR-451, was studied by real time PCR, Western blotting, and siRNA technologies. The mRNA and protein levels of IL6R gene were found to be down-regulated in the RKO and Hela cells transfected with miR-451 mimics. Consequently, the cell proliferation was inhibited. Also, the invasion of RKO cells was suppressed. Furthermore, the angiogenesis of HUVEC cells transfected with miR-451 mimics was assayed and the decreased angiogenic ability was detected compared to the controls. All these results were validated by IL6R siRNA experiments. The IL6R gene is a target gene of miR-451. The miR-451 behaves as a tumor suppressor, probably by targeting the IL6R pathway.
... Finally, although the paper uses the three most popular algorithms for demonstrating the methods proposed for comparisons and rank aggregation, the methods are applicable to a different set of, or including additional, algorithms, such as the recent additions from Vatolin et al. (2006) and Wang and Wang (2006). ...
Motivation: MicroRNAs (or miRNAs) are short noncoding RNAs whose primary role is to repress translations by negatively regulating gene expressions post-transcriptionally through binding to their mRNA targets. Currently, there exist a few computational algorithms for human miRNA target predictions, but their results may vary widely. Therefore, it would be useful to consolidate and lter through these discrepant results so that researchers may have a greater degree of certainty in the validity of these targets before engaging in costly experiments. Results: We studied three of the most popular target prediction algorithms, miRanda, Tar- getScan, and PicTar, systematically through the use of three measures of similarity/distance and a statistical test on the gene ontology categories that are enriched in the target lists. Furthermore, two composite statistics were devised to combine and rank the composite tar- get list. We applied the methods developed to all human miRNAs that had been identied. Our results indicate that TargetScan and PicTar tend to have a greater degree of similarity, based on all measures considered. We also demonstrate that our composite statistics and the program implementing them can be useful tools for ltering through large target sets to short list genes for downstream experiments. Contact: shili@stat.ohio-state.edu. Supplementary Information: The program implementing the composite statistics (R codes) and supplementary materials are at
... Recognition of mRNAs by the loaded miRNA/RISC occurs at target sites that are complementary to the seed sequence, a $8-nucleotide long sequence in the 5' end of the microRNA [134,135]. Target sites have historically most often been searched for in the 3' untranslated region (3'UTR) of the messenger RNA; however this may neglect a novel mechanism of how miRNAs function as recent results indicate that functional targets may be found across the length of the mRNA [136,137]. The short length of the seed sequence has the consequence that target sites to each microRNA are present in hundreds of different mRNAs, thereby complicating the elucidation of miRNA regulation of cellular functions. ...
... Each mRNA can be targeted by more than one miRNA (Vatolin et al., 2006), and each miRNA can target hundreds different transcripts. For instance, it has been demonstrated that a cluster of two miRNAs (namely miR-15a and miR-16) can affect the expression of about 14% of the human genome in a leukemic cell line . ...
MicroRNAs (miRNAs) are short noncoding RNAs with gene regulatory functions. It has been demonstrated that the genes encoding for miRNAs undergo the same regulatory epigenetic processes of protein coding genes. In turn, a specific subgroup of miRNAs, called epi-miRNAs, is able to directly target key enzymatic effectors of the epigenetic machinery (such as DNA methyltransferases, histone deacetylases, and polycomb genes), therefore indirectly affecting the expression of epigenetically regulated oncogenes and tumor suppressor genes. Also, several of the epigenetic drugs currently approved as anticancer agents affect the expression of miRNAs and this might explain part of their mechanism of action. This chapter focuses on the tight relationship between epigenetics and miRNAs and provides some insights on the translational implications of these findings, leading to the upcoming introduction of epigenetically related miRNAs in the treatment of cancer.
... A single miRNA can regulate expression of multiple genes, and expression of a single gene may be regulated by several distinct miRNAs, creating complicated regulatory networks. It is estimated that roughly 60% of human protein-coding genes are regulated by miRNAs25262728. In this study, we evaluated whether miRNAs are modulated by XMRV in cultured cells and if so, can they be identified to see whether a single or a set of miRNAs specific to the infection can be detected early that could serve as biomarker(s) of XMRV infection. Our results demonstrate that a) two miRNAs, miR- 193a-3p and miRPlus-E1245 (a proprietary sequence of Exiqon Inc, Denmark and named as such to differentiate from miR-1245) were commonly regulated among all 4 cell types infected with XMRV used in the study, and b) while miR-193a-3p is down regulated, miRPlus-E1245 exhibited varied expression profile in the four cell types infected with XMRV. ...
XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture.
Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray.
MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types.
The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.
... In a method for detecting miRNA–mRNA complexes developed by Vatolin and colleagues (74), endogenous miRNAs are used as primers for cDNA synthesis of target mRNAs. Because miRNAs bind their mRNA target within the RISC, a strong detergent is used to disassociate these proteins to allow reverse transcriptase to bind and synthesize cDNA. ...
MicroRNAs (miRNAs) are important regulators of eukaryotic gene expression in most biological processes. They act by guiding
the RNAi-induced silencing complex (RISC) to partially complementary sequences in target mRNAs to suppress gene expression
by a combination of translation inhibition and mRNA decay. The commonly accepted mechanism of miRNA targeting in animals involves
an interaction between the 5′-end of the miRNA called the ‘seed region’ and the 3′ untranslated region (3′-UTR) of the mRNA.
Many target prediction algorithms are based around such a model, though increasing evidence demonstrates that targeting can
also be mediated through sites other than the 3′-UTR and that seed region base pairing is not always required. The power and
validity of such in silico data can be therefore hindered by the simplified rules used to represent targeting interactions. Experimentation is essential
to identify genuine miRNA targets, however many experimental modalities exist and their limitations need to be understood.
This review summarizes and critiques the existing experimental techniques for miRNA target identification.
... Each mRNA can be targeted by more than one miRNA (Vatolin et al., 2006), and each miRNA can target hundreds different transcripts. For instance, it has been demonstrated that a cluster of two miRNAs (namely miR-15a and miR-16) can affect the expression of about 14% of the human genome in a leukemic cell line . ...
Epigenetic factors and microRNAs (miRNAs) are regulators of gene expression. Their regulatory function is frequently aberrant in cancer. In this chapter, we show that a tight connection occurs between miRNAs and epigenetics. Epigenetic factors can be responsible for the aberrancies of the miRNome (defined as the full spectrum of miRNAs for a specific genome) observed in cancer. Indeed, miRNAs undergo the same epigenetic regulatory laws like any other protein-coding gene. Moreover, a specific group of miRNAs (defined as epi-miRNAs) can directly target effectors of the epigenetic machinery (such as DNA methyltransferases, histone deacetylases, and polycomb repressive complex genes) and indirectly affect the expression of tumor suppressor genes, whose expression is controlled by epigenetic factors. The result of this epigenetic-miRNA interaction is a new layer of complexity in gene regulation, whose comprehension opens new avenues to understand human cancerogenesis and to achieve new cancer treatments.
... Interestingly, the mRNAs that were significantly enriched by immunoprecipitation included targets that were also down-regulated in total mRNA, and these targets were very likely to contain the seed site. Another approach consists of synthesizing cDNA clones from known mRNA templates using endogenous miRNAs as primers (Vatolin, et al. 2006). Sequencing of the synthesized cDNAs then allows the identification of the miRNAs as well as the binding sites on the mRNA. ...
The molecular mechanisms involved in epithelial ovarian cancer initiation and progression are just beginning to be elucidated. In particular, it has become evident that microRNAs (miRNAs or miRs), a class of molecules that post-transcriptionally regulate gene expression, play a major role in ovarian tumorigenesis. Several microRNA profiling studies have identified changes in microRNA patterns that take place during ovarian cancer development. While most deregulated microRNAs are down-regulated in cancer, and may therefore act as tumor suppressors, others are elevated and may represent novel oncogenes in this disease. A number of microRNAs identified as aberrantly expressed in ovarian carcinoma have been shown to have important functional roles in cancer development and may therefore represent targets for therapy. In addition, some of the microRNA patterns may have prognostic significance. The identification of functional targets represents a major hurdle in our understanding of microRNA function in ovarian carcinoma, but significant progress is being made. It is hoped that a better understanding of the microRNA expression and roles in ovarian cancer may provide new avenues for the detection, diagnosis, and therapy of this deadly disease.
... Usually miRNAs exert their regulatory function by binding to a ''seed sequence'' in the 3 0 untranslated region (3 0 -UTR) of their target mRNAs. However, binding to the 5 0 -UTR and to the coding regions of the messengers has also been described (Vasudevan et al. 2007; Vatolin et al. 2006). The disruption of the miRNA processing machinery contributes to impaired miRNA production and carcinogenesis. ...
Cancer is a genetic and epigenetic disease. MicroRNAs (miRNAs), a class of small noncoding RNAs, have been shown to be deregulated in many diseases including cancer. An intertwined connection between epigenetics and miRNAs has been supported by the recent identification of a specific subgroup of miRNAs called "epi-miRNAs" that can directly and indirectly modulate the activity of the epigenetic machinery. The complexity of this connection is enhanced by the epigenetic regulation of miRNA expression that generates a fine regulatory feedback loop. This review focuses on how epigenetics affects the miRNome and how the recently identified epi-miRNAs regulate the epigenome in human cancers, ultimately contributing to human carcinogenesis.
... The method essentially gives no information on miRNA-target combinations. Another method for detecting miRNA–mRNA complexes was proposed by Vatolin et al. (2006), in which cytoplasmic extract was reacted with reverse transcriptases to indicate that an endogenous miRNA can be a primer for cDNA synthesis on a target mRNA template. The cDNA fragment was ligated to an adapter oligonucleotide at the 59 terminus and amplified by polymerase chain reaction (PCR) with a primer from the adapter and a genespecific primer corresponding to the target mRNA. ...
MicroRNAs (miRNAs) are roughly 22-nucleotide regulatory RNAs that play important roles in many developmental and physiological processes. Animal miRNAs down-regulate target genes by forming imperfect base pairs with 3' untranslated regions (3' UTRs) of their mRNAs. Thousands of miRNAs have been discovered in several organisms. However, the target genes of almost all of these miRNAs remain to be identified. Here, we describe a method for isolating cDNA clones of target mRNAs that form base pairs in vivo with an endogenous miRNA of interest, in which the cDNAs are synthesized from the mRNAs using the miRNA as a reverse-transcription primer. The application of this method to Caenorhabditis elegans miRNA lin-4 under test conditions yielded many clones of the known target gene lin-14 that correspond to partial sequences 5' to lin-4 binding sites in the 3' UTR. The method was also applied to C. elegans miRNA let-7 and a new target gene responsible for the lethal phenotype in let-7 mutants was identified. These results demonstrate that the method is a useful way to identify targets on the basis of base pairing with individual miRNAs.
MicroRNAs (miRNAs) are small noncoding RNA which regulate gene expression. In addition to their “classical” mechanism of action (targeting of messenger RNAs by binding to partially complementary sequences in their 3′-untranslated region), it has been shown that miRNAs can also regulate gene expression by affecting the epigenetics status of genes. These miRNAs, called epi-miRNAs, act by directly silencing key effectors of the epigenetic machinery, such as DNA methyltransferases (DNMTs), histone deacetylases (HDACs), and Polycomb Repressive Complex (PRC) genes. Moreover, miRNAs, like any other protein coding gene, are also regulated by epigenetic mechanisms. Overall, this two-way interaction between the miRNome and the epigenome is critically involved in the dysregulation of miRNAs observed in human cancer, and at least in part responsible for human carcinogenesis. A better understanding of the dynamic between miRNAs and epigenetics in cancer is leading to the identification of new molecular anticancer targets.
Objectives
MicroRNAs have critical roles in cancer development by regulating the expression of oncogenes or tumor suppressor genes. We identified and characterized a novel miRNA, smR-164, in human lung cancer cells. The aim of this study was to investigate its novel function in human lung cancer by targeting XIAP
Material and methods
Novel miRNA cloning, Real-time qRT-PCR, western blotting, dual luciferase assay, miRNA transfection, proliferation and apoptosis assay were carried on human lung cancer cell line A549. Fifteen paired NSCLC tissues and noncancerous lung tissues were collected. In vivo xenograft assay was performed.
Results
Expression of smR-164 was downregulated in human lung cancer cell lines and tissues compared with normal cells and tissues. We identified a putative target gene, XIAP, whose expression was regulated by smR-164 overexpression. XIAP is an inhibitor of apoptosis that represses the activation of caspase 3 and 9. XIAP mRNA and protein levels were directly suppressed by smR-164. XIAP has an important role in carcinogenesis, and previous studies suggest that it may regulate cell survival and proliferation by its anti-apoptotic ability.
Conclusion
Taken together, smR-164 inhibited cell proliferation and induced apoptosis in vitro and in vivo by targeting XIAP. These data can be applied to identify novel therapeutic targets for lung cancer therapy.
This report is the outcome of an EFSA procurement aiming at investigating and summarising the state of knowledge on (I) the mode-of-action of dsRNA and miRNA pathways, (II) the potential for nontarget gene regulation by dsRNA-derived siRNAs or miRNAs, (III) the determination of siRNA pools in plant tissues and the importance of individual siRNAs for silencing. The report is based on a comprehensive and systematic literature search, starting with the identification and retrieval of~190,000 publications related to the research area and further filtered down with keywords to produce focused collections used for subsequent screening of titles and abstracts. The report is comprised of an (I) Introduction to the field of small RNAs, (II) a Data and Methodologies section containing strategies used for literature search and study selection, and (III) the Results of the literature review organized according to the three main procurement tasks. The outcome of the first task reviews dsRNA and miRNA pathways in mammals (including humans), birds, fish, arthropods, annelids, molluscs, nematodes, and plants. Eight taxon-dedicated chapters are based on ~1,400 cumulative references chosen from ~10,000 inspected titles and abstracts. We review conserved and divergent aspects of small RNA pathways and dsRNA responses in animals and plants including structure and function of key proteins as well as four basic mechanisms: genome-encoded posttranscriptional regulations (miRNA), degradation of RNAs by short interfering RNA pools generated from long dsRNA (RNAi), transcriptional silencing, and sequence-independent responses to dsRNA. The outcome of the second task focuses on base pairing between small RNAs and their target RNAs and predictability of biological effects of small RNAs in animals and plants. The outcome of the last task reviews methodology, siRNA pools, and movement of small RNAs in plants. Potential transfer of small RNAs between species and circulating miRNAs in mammals is described in the final chapter.
full text: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1246/pdf
Needed: large scale experimental validation of predicted microRNA targets.
Naturally occurring microRNAs (miRNAs) are small noncoding RNAs 19–24 nucleotides (nt) long, typically cleaved from 60- to 110-nt hairpin precursors (pre-miRNAs) that are produced from large precursors (pri-miRNAs) and are encoded in the genomes of invertebrates, vertebrates, and plants. miRNAs act as regulators of gene expression during development and differentiation at the transcriptional, posttranscriptional, and/or translational level, although most target genes have yet to be identified. Many miRNAs are conserved in sequence between distantly related organisms, suggesting that these molecules participate in essential processes. In this chapter, we present principles related to the basic and translational research that has emerged in the last decade, a period that can be truly considered the “miRNA revolution” in molecular oncology. Furthermore, we present a compendium of information about the main miRNAs that have been identified in the last several years as playing important roles in cancer. Also, we alert the reader to several additional reviews that may provide a deeper understanding of this new and exciting field of research.
MicroRNAs (miRNAs) are small noncoding RNA which regulate gene expression. In addition to their “classical” mechanism of action (targeting of messenger RNAs by binding to partially complementary sequences in their 3′-untranslated region), it has been shown that miRNAs can also regulate gene expression by affecting the epigenetics status of genes. These miRNAs, called epi-miRNAs, act by directly silencing key effectors of the epigenetic machinery, such as DNA methyltransferases (DNMTs), histone deacetylases (HDACs), and Polycomb Repressive Complex (PRC) genes. Moreover, miRNAs, like any other proteic coding gene, are also regulated by epigenetic mechanisms. Overall, this two-way interaction between the miRNome and the epigenome is critically involved in the dysregulation of miRNAs observed in human cancer, and at least in part responsible for human carcinogenesis. A better understanding of the dynamic between miRNAs and epigenetics in cancer is leading to the identification of new molecular anticancer targets.
MicroRNAs (miRNAs) are a group of regulatory RNAs that regulate gene expression post-transcriptionally by the degradation or translational inhibition of their target messenger RNAs (mRNAs). Regulation is accomplished when the 22-25 nucleotide miRNAs bind to complementary sequences in the 3'-untranslated regions (UTR). One barrier to miRNA research is to find target genes. Although computational target predictions have shed light on important aspects of microRNA target recognition, questions remain concerning the rates of false positives. In addition, we do not completely understand how microRNAs can recognize and regulate their targets. As such, experimental approaches are required, which can reflect in vivo processes, eliminating false positive predictions and allow for an unbiased study of microRNA target recognition. In this review, we summarized experimental approaches that have been described for the identification and validation of mRNA targets associated with specific miRNAs.
Little is known about how a neuron undergoes site-specific changes in intrinsic excitability during neuronal activity. We provide evidence for a novel mechanism for mTORC1 kinase-dependent translational regulation of the voltage-gated potassium channel Kv1.1 messenger RNA (mRNA). We identified a microRNA, miR-129, that repressed Kv1.1 mRNA translation when mTORC1 was active. When mTORC1 was inactive, we found that the RNA-binding protein, HuD, bound to Kv1.1 mRNA and promoted its translation. Unexpectedly, inhibition of mTORC1 activity did not alter levels of miR-129 and HuD to favor binding to Kv1.1 mRNA. However, reduced mTORC1 signaling caused the degradation of high affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA. Hence, mTORC1 activity regulation of mRNA stability and high affinity HuD-target mRNA degradation mediates the bidirectional expression of dendritic Kv1.1 ion channels.
MicroRNAs (miRNAs) are de-regulated in cancer versus the normal tissue counterpart and actively participate in human carcinogenesis. Among the genes whose expression is under their control there are both oncogenes and tumor suppressor genes, revealing that it is not only limiting but simply wrong to assign them a function just as oncogenes or as tumor suppressor genes. In addition to primary tumors, miRNAs can be detected in almost all human body fluids and effectively help to diagnose cancer and to prognosticate clinical outcome and response to treatment of tumors. The advent of miRNA mimic and miRNA silencing molecules has allowed to modulate miRNA expression in tumors, showing that miRNAs can be effectively used as therapeutic agents. This review will focus on those findings that have provided the rationale for the use of miRNAs as patient "tailored" anti-cancer agents.
The accurate prediction and validation of microRNA targets is essential to understanding the function of microRNAs. Computational predictions indicate that all human genes may be regulated by microRNAs, with each microRNA possibly targeting thousands of genes. Here we discuss computational and experimental methods for identifying mammalian microRNA targets. We describe microRNA target prediction resources and procedures that are suitable for experiments where more accurate prediction of microRNA targets is more important than detecting all putative targets. We then discuss experimental methods for identifying and validating microRNA target genes, with an emphasis on the target reporter assay as the method of choice for specifically testing functional microRNA target sites.
Non-coding RNAs are important actors in human biology. A massive amount of data has been created and manipulated, and important findings have been extracted thanks in part to bioinformatics approaches and consequent experimental validation; many of these results are for a specific class of non-coding RNAs, the microRNAs (miRNAs), that are important regulators of gene expression although their transcriptional regulation is not yet well understood. Their involvement in cancer development and progression makes the related research field an integrated one, composed of bioinformaticians, clinicians, statisticians and biologists, as well as informaticians and data miners that cure data manipulation and storage especially due to the output of the latest technologies, like the Next Generation Sequencers.
In this chapter we report the main miRNA findings of the last 10 years in terms of identification and prediction techniques, data generation and manipulation methods, as well as possible use in clinical practice.
In this paper, a novel, label-free amperometric biosensor for the detection of microRNA-155 based on the conductive self-assembled multilayer comprised of Nafion, thionine (Thi) and Pd nanoparticles was successfully prepared. Nafion was firstly dropped on to a bare glass carbon electrode. Then Thi was absorbed by the cation exchanger Nafion. Furthermore, a Pd nanoparticles layer which was used to immobilize target biomolecules was constructed by the amino group of Thi as linker. Moreover, the proposed biosensor showed excellent electrocatalytic activity towards H(2)O(2) and enhanced the current response of the biosensor. Cyclic voltammetry was used for the characterization and electrochemical properties of the stepwise self-assembly process of the biosensor, and scanning electron microscopy was used for observing the microstructure of the modified film. Using microRNA-155 as a model, the resulting biosensor presented high sensitivity, good stability and a broad linear response from 5.6 to 5.6 × 10(5) pM with the detection limit of 1.87 pM under optimization of the assay conditions.
miRNAs, a recently discovered family of small noncoding RNAs, are emerging as major controllers of gene expression and key determinants of pancreatic β-cell function. These 19-22-nucleotide molecules govern gene expression by partially pairing to 3´-untranslated regions of target mRNAs and by inhibiting their translation. The elucidation of the role of miRNAs promises to unravel new aspects of β-cell biology and to clarify the mechanisms leading to defective insulin secretion in diabetes mellitus. This information is expected to favor the design of new approaches for preserving functional β-cells in prediabetic stages and the development of strategies for engineering insulin-secreting cells capable of replacing endogenous β-cells in diabetic patients.
Hepatocellular carcinoma (HCC) is one of the few cancers with a worldwide increasing trend of incidence, representing the third largest cause of cancer-related death. The initiation and progression of HCC depend on progressive accumulation of genetic and epigenetic defects that alter an array of signaling cascades via deregulation of signal activators and inhibitors. MicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally repress gene expression including those signal molecules and thus critical for many cellular pathways. However, the balance of this fine-tuning function is broken by the abnormal expression of miRNAs in various cancers through genomic alterations or epigenetic mechanisms. This review summarizes the current knowledge of the role of epigenetic aberrations, including histone methylation and deacetylation as well as DNA hypermethylation and hypomethylation in the aberrant regulation of miRNAs leading to activation of signaling pathways such as Ras, STAT3 and AKT/mTOR in HCC. Conceivably, the therapeutic efficacy of current chromatin-modifying drugs might be related to their capacity to reactivate previously silenced tumor-suppressive miRNAs and cause down-regulation of target oncogenes. Better understanding of the epigenetics-miRNA regulatory cascades in the control of the functionally significant pathways will provide new opportunities for the development of more effective therapeutic modality for HCC.
Micro-RNAs (miRNAs) are small noncoding RNAs with gene regulatory functions. Abnormal expression of miRNAs distinguishes normal
from tumoral tissues in both solid and hematological malignancies. These aberrations parallel abnormal expression of miRNA
target genes, which ultimately are responsible for the development of the malignant phenotype. Intriguingly, miRNAs expression
levels are variable during normal hematopoiesis, revealing that their up/downregulation is required during the physiologic
differentiation of the hematopoietic multipotent precursor. Therefore, variations in miRNA levels mirror abnormal expression
of their target genes and are involved in the development of lymphomas and leukemias. This chapter will describe how miRNAs
affect normal hematopoiesis and contribute to the genesis of hematological malignancies, and how they affect the prognosis,
and harbor therapeutic implications for patients affected by lymphomas and leukemias.
KeywordsMicro-RNAs-Leukemias-Lymphomas-Noncoding RNAs-Polyadenylated precursor pri-miRNA-Precursor pre-miRNA-RISC complex-Cancer-associated genomic regions (CAGRs), Tumor suppressor genes-Fragile sites-Loss of heterozygosity (LOH)-Insulin-like growth factor receptor (IGFR)-Chemokine receptor 4 (CXCR4)-Promyelocytic leukemia zinc finger (PLZF)-Burkitt lymphoma
REV3Lp, the catalytic subunit of DNA polymerase zeta, is the major participant in translesion DNA synthesis. Recent evidence suggests that REV3L has an important role in the maintenance of genome stability despite its mutagenic characteristics. Such a function makes it a cancer susceptibility candidate gene. To investigate association between REV3L polymorphisms and lung cancer risk in a Chinese population, we first genotyped 15 common polymorphisms of the REV3L gene and found that three single nucleotide polymorphisms (rs465646, rs459809 and rs1002481) were significantly associated with lung cancer risk. One of the strongest associations observed was for the 3'-terminal untranslated region (3'UTR) 460 T>C polymorphism (rs465646) (adjusted odds ratio (OR)=0.69 for TC/CC; P=0.007, compared with TT). Similar results were obtained in a subsequent replication study (adjusted OR=0.72; P=0.016). Combined data from the two studies of 1072 lung cancer patients and 1064 cancer-free controls generated an even stronger association (adjusted OR=0.71; P=3.04 × 10(-4)). This 3'UTR 460 T>C variant was predicted to modulate the binding of several micro RNAs. Surface plasmon resonance analysis and luciferase assays showed that the T allele demonstrated a stronger binding affinity for miR-25 and miR-32, resulting in significantly weaker reporter expression levels. Additional experiments revealed that miR-25/32 could downregulate endogenous REV3L. Furthermore, the tumor-suppressing role of REV3L was confirmed by the foci formation assay. These results support our hypothesis that the REV3L rs465646 variant modifies lung cancer susceptibility in Chinese Han population by affecting miRNA-mediated gene regulation.Oncogene advance online publication, 20 February 2012; doi:10.1038/onc.2012.32.
Epigenetic silencing of tumor suppressor genes is a salient feature of tumor cells. Re-expression of epigenetically silenced genes is a feasible and achievable strategy for cancer treatment. DNA methylation is the most characterized epigenetic silencing mechanism and the reversal of DNA methylation, genetically or pharmacologically, induces gene re-expression and proliferation arrest in tumor cells. Other epigenetic targets, such as histone acetylation and methylation, are also rational drug targets, and several small-molecule modulators of histone acetylation and methylation are currently under development or already in clinical trials. Epigenetic deregulation of miRNAs induces aberrant expression of miRNAs, which have been associated with the development and progression of cancer. The reversal of DNA methylation can induce the re-expression of miRNAs, and oligonucleotides can silence aberrantly expressed miRNAs. Evaluating the combination of different epigenetic modifiers and ensuring their optimization are the next challenges towards the establishment of epigenetic therapy.
MicroRNAs (miRNAs) are small, single-stranded RNA molecules encoded by genes that are transcribed from DNA but not translated into protein (noncoding RNA). The ability of miRNA to regulate the expression of, as yet, an unknown quantity of targets has recently become an area of huge interest to researchers studying many different areas in many species. Identifying miRNA targets provides functional insights and strategies for therapy. Furthermore, the recent advent of high-throughput methods for profiling miRNA expression and for the identification of miRNA targets has ushered in a new era in the research of gene regulation. miRNA profiling further adds a new dimension of information for the molecular profiling of disease. Summarized herein are the methods used to query the expression of miRNAs at both an individual and global level. We have also described modern computational approaches to identifying miRNA target transcripts.
The discovery in mammalian cells of hundreds of small RNA molecules, called microRNAs, with the potential to modulate the expression of the majority of the protein-coding genes has revolutionized many areas of biomedical research, including the diabetes field. MicroRNAs function as translational repressors and are emerging as key regulators of most, if not all, physiological processes. Moreover, alterations in the level or function of microRNAs are associated with an increasing number of diseases. Here, we describe the mechanisms governing the biogenesis and activities of microRNAs. We present evidence for the involvement of microRNAs in diabetes mellitus, by outlining the contribution of these small RNA molecules in the control of pancreatic beta-cell functions and by reviewing recent studies reporting changes in microRNA expression in tissues isolated from diabetes animal models. MicroRNAs hold great potential as therapeutic targets. We describe the strategies developed for the delivery of molecules mimicking or blocking the function of these tiny regulators of gene expression in living animals. In addition, because changes in serum microRNA profiles have been shown to occur in association with different human diseases, we also discuss the potential use of microRNAs as blood biomarkers for prevention and management of diabetes.
The advent of blood component storage revolutionized health care by allowing for a managed supply of transfusion quality blood products. During storage, blood components undergo a series of physiological changes that affect the product quality, which ultimately can interfere with the safety and efficacy of such products after transfusion. Despite continuous improvements in blood component quality and safety, it is still desirable to have in vitro standard markers of measurable characteristics that predict blood component safety and efficacy in vivo following their transfusion. Over the last decade, research on the feasibility of using microRNAs as biomarkers for various clinical manifestations and cellular pathologies has exploded. Here, we review the literature on blood cell microRNAs and discuss the potential of these molecules to act as measurable characteristics (product biomarkers) for stored blood component quality and safety.
Neuroblastoma is the most common extra-cranial tumour disease in children, and accounts for 15% of all childhood cancer deaths. MYCN is a transcription factor and a proto-oncogene that most often initiates transcription of target genes involved in increased proliferation and inhibition of differentiation. MYCN amplified neuroblastoma represents the most aggressive form of this disease. Silencing of MYCN is an attractive approach for understanding the role of this gene in development and disease. MYCN is involved in development, proliferation and differentiation. In order to explore its functions in detail, it is a great advantage to be able to silence its expressions in all cells present for an indefinite period of time, and at any suitable time-point. In paper I we describe a novel design of an inducible H1 promoter capable of delivering shRNA in a conditional manner. This versatile system allows inducible expression of any desired shRNA. In paper II this promoter is used to control the conditional expression of anti-MYCN shRNAs from cassettes stably integrated in two MYCN-amplified cell lines. Here MYCN is silenced when the inducer doxycycline is added to the media, resulting in increased differentiation and reduced proliferation of the MYCN amplified cell lines. MiRNAs are small non-coding RNA shown to regulate the expression of a vast amount of proteins in humans. In paper III we investigate the effect of MYCN suppression on the global miRNA expression in MYCN-amplified neuroblastoma. We find that reduced MYCN expression leads to both increased and reduced expression of several miRNAs. The function of mir-21 is investigated further, and no effect on neither proliferation nor differentiation is found. Other miRNAs demonstrated to be regulated by MYCN, is mir-92a and mir-92b. In paper IV we demonstrate how these miRNAs suppresses the expression of DKK3, a tumour suppressor gene often found to be down-regulated in MYCN-amplified neuroblastoma. Paper 4 of the thesis is not available in Munin: 4. Bjørn Helge Haug, Jørn Remi Henriksen, Jochen Buechner, Per Kogner,Tommy Martinsson, Trond Flægstad, Baldur Sveinbjørnsson and Christer Einvik: 'MYCN-regulated miRNAs inhibit secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma' (unpublished manuscript)
Epigenetics research is one of the emerging research fields in biomedical research. During the last few decades, a collection of useful tools (both to design the experiments and to analyze the results) and databases are developed. This review chapter discusses basic tools which are used to detect CpG islands and the Transcription Start Site (TSS) and discusses experimental design and analysis, mainly of DNA-methylation experiments. During the last years, an enormous amount of experimental data had been generated and published. Therefore, we describe some epigenetic databases, with a special focus on DNA methylation and cancer. Some general cancer databases are discussed as well, as they might reveal the link between the results from epigenetic experiments and their biological influence on the development or progression of cancer. Next, some novel computational approaches in epigenetics are discussed, for instance used to predict the methylation state of a promoter in certain circumstances. To show a possible data analysis strategy of an epigenetic dataset in cancer research, there is a showcase where a DNA-methylation dataset, generated on colorectal cancer samples, is analyzed. This demonstrates how a DNA-methylation dataset might look like and the different steps in a possible analysis strategy and how to interpret the results.
MicroRNAs (miRNAs) are small regulatory RNA molecules functioning to modulate gene expression at the post-transcriptional level, and playing an important role in many developmental and physiological processes. Ten thousand miRNAs have been discovered in various organisms. Although considerable progress has been made in computational methodology to identify miRNA targets, most predicted miRNA targets may be false positive. Due to the lack of effective tools to identify miRNA targets, the study of miRNAs is seriously retarded. In recent years, some molecular cloning strategies of miRNA targets have been developed, including RT-PCR using miRNAs as endogenous primers, labeled miRNA pull-down assay (LAMP) and RNA ligase-mediated amplification of cDNA end (RLM-RACE). The identified miRNA targets should be further validated via effects of miRNA alteration on the target protein levels and bioactivity. This review summarizes advances in strategies to identify miRNA targets and methods by which miRNA targets are validated.
miRNAs are noncoding RNAs that regulate gene expression. The advent of high-throughput techniques has revealed that miRNA expression is deregulated in almost all human tumors (both solid and hematologic) with respect to the normal tissue counterpart. These differences frequently recur in tumor-specific miRNA signatures, which are very helpful to diagnose the tissue of origin of the neoplasia, and sometimes also specific tumor subtypes. Increasing evidence also supports a role for miRNAs as prognostic biomarkers of human cancers. Finally, miRNAs are differentially expressed in the blood of cancer patients versus healthy donors, providing a rationale for the detection of miRNAs and diagnostic and prognostic circulating biomarkers.
The traditional understanding that proteins are the only effectors of gene function has been challenged by the discovery of a group of genes that do not encode proteins (non-coding genes [ncGs]). The role of ncGs in the pathogenesis and potentially the treatment of several human diseases is increasingly being confirmed. A robust collection of literature exists to support the theory of the involvement of ncGs and their non-coding RNA (ncRNA) transcripts in the pathogenesis of cancer. This review focuses on the role of ncRNAs in human carcinogenesis and describes why deciphering the function of these RNAs might lead to the development of new anticancer drugs.
We review the pertinent literature on methods used in high-throughput experimental identification of microRNA (miRNA) "targets" with emphasis on neurochemical studies. miRNAs are short regulatory noncoding RNAs that play important roles in the mammalian brain. The functions of miRNAs are related to their binding of RNAs including mRNAs. Since mammalian miRNAs tend to bind to target mRNAs via imperfect complementarity, understanding exactly which target mRNAs are recognized by which specific miRNAs is a challenge. Based on early experimental evidence, a set of "binding rules" for miRNAs has been described. These have focused on the 5' "seed" region of miRNAs binding to the 3' untranslated region of targeted mRNAs. Bioinformaticians have applied these algorithms for theoretical miRNA target prediction. To date, the different computational methods are not in agreement with each other and do not explain all miRNA targets as defined using high-throughput experimental methods. We consider these latter techniques which identify putative miRNA targets directly. Each experimental approach involves specific assumptions and potential technical pitfalls. Some of these direct experimental methods for miRNA target identification have used co-immunoprecipitation (RIP-Chip and others) and transfection-based experimental design. Topics related to experimentally identified miRNA targets are discussed, with special emphasis on studies pertinent to the mammalian brain.
Men have a higher incidence of hepatocellular carcinoma (HCC) than women, which is believed to partly be because of protective effects of estrogen. We sought to determine whether there were differences in levels of microRNA (miRNA) molecules between male and female HCC samples.
The expression profiles of a panel of candidate miRNAs were compared between male and female HCC tissues using the TaqMan miRNA assay. A luciferase reporter assay was used to identify mRNA targets recognized by specific miRNAs. The levels of pri- and pre-miRNA for each specific miRNA were assayed by quantitative reverse-transcription polymerase chain reaction to delineate the step deregulated in the biogenesis process. Finally, a colorimetric assay was used to determine the effect of specific miRNAs on hepatoma cell proliferation.
The miR-18a miRNA increased specifically in samples from female HCC patients (female/male ratio, 4.58; P = .0023). The gene ESR1, which encodes the estrogen receptor-alpha (ERalpha), was identified as a target of miR-18a. miR-18a can repress ERalpha translation by binding to its mRNA at the 3' untranslated region. Increased levels of miR-18a in female HCC tissues correlated with reduced ERalpha expression; the level of pre-miR-18a changed in concordance with that of mature miR-18a in these tissues. Overexpression of miR-18a decreased ERalpha levels but stimulated the proliferation of hepatoma cells.
This study provides a novel miRNA-mediated regulatory mechanism for controlling ERalpha expression in hepatocytes. miR-18a prevents translation of ERalpha, potentially blocking the protective effects of estrogen and promoting the development of HCC in women.
The nucleotide sequence of an RNA primer molecule for initiation of Rous sarcoma virus DNA synthesis in vitro has been determined. The sequence can be drawn in a cloverleaf structure typical of tRNAs with an anticodon for tryptophan. Aminoacylation of the molecule confirms that it is tRNA-Trp. The same sequence and aminoacylation results are obtained regardless of whether the RNA is isolated from virions or from cells of chickens, the natural host for this virus. It is the only species of tRNA-Trp that is dectected in chicked cell tRNA.
MicroRNAs (miRNAs) are 21-23 nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs. miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation and apoptosis. Although strict tissue- and developmental-stage-specific expression is critical for appropriate miRNA function, mammalian transcription factors that regulate miRNAs have not yet been identified. The proto-oncogene c-MYC encodes a transcription factor that regulates cell proliferation, growth and apoptosis. Dysregulated expression or function of c-Myc is one of the most common abnormalities in human malignancy. Here we show that c-Myc activates expression of a cluster of six miRNAs on human chromosome 13. Chromatin immunoprecipation experiments show that c-Myc binds directly to this locus. The transcription factor E2F1 is an additional target of c-Myc that promotes cell cycle progression. We find that expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. These findings expand the known classes of transcripts within the c-Myc target gene network, and reveal a mechanism through which c-Myc simultaneously activates E2F1 transcription and limits its translation, allowing a tightly controlled proliferative signal.
Of the over 200 identified mammalian microRNAs (miRNAs), only a few have known biological activity. To gain a better understanding of the role that miRNAs play in specific cellular pathways, we utilized antisense molecules to inhibit miRNA activity. We used miRNA inhibitors targeting miR-23, 21, 15a, 16 and 19a to test efficacy of antisense molecules in reducing miRNA activity on reporter genes bearing miRNA-binding sites. The miRNA inhibitors de-repressed reporter gene activity when a miRNA-binding site was cloned into its 3'-untranslated region. We employed a library of miRNA inhibitors to screen for miRNA involved in cell growth and apoptosis. In HeLa cells, we found that inhibition of miR-95, 124, 125, 133, 134, 144, 150, 152, 187, 190, 191, 192, 193, 204, 211, 218, 220, 296 and 299 caused a decrease in cell growth and that inhibition of miR-21 and miR-24 had a profound increase in cell growth. On the other hand, inhibition of miR-7, 19a, 23, 24, 134, 140, 150, 192 and 193 down-regulated cell growth, and miR-107, 132, 155, 181, 191, 194, 203, 215 and 301 increased cell growth in lung carcinoma cells, A549. We also identified miRNA that when inhibited increased the level of apoptosis (miR-1d, 7, 148, 204, 210, 216 and 296) and one miRNA that decreased apoptosis (miR-214) in HeLa cells. From these screens, we conclude that miRNA-mediated regulation has a complexity of cellular outcomes and that miRNAs can be mediators of regulation of cell growth and apoptosis pathways.
In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs:
microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction
via the RNA interference (RNAi) pathway. Here we show that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target
determines its function. Humanlet-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which we show is an RNAi enzyme complex.
Each let-7–containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway
in human cells.
Among the 3 billion base pairs of the human genome, there are approximately 30,000-40,000 protein-coding genes, but the function of at least half of them remains unknown. A new tool - short interfering RNAs (siRNAs) - has now been developed for systematically deciphering the functions and interactions of these thousands of genes. siRNAs are an intermediate of RNA interference, the process by which double-stranded RNA silences homologous genes. Although the use of siRNAs to silence genes in vertebrate cells was only reported a year ago, the emerging literature indicates that most vertebrate genes can be studied with this technology.
With the discovery of RNA interference (RNAi) and related phenomena, new regulatory roles attributed to RNA continue to emerge. Here we show, in mammalian tissue culture, that a short interfering RNA (siRNA) can repress expression of a target mRNA with partially complementary binding sites in its 3' UTR, much like the demonstrated function of endogenously encoded microRNAs (miRNAs). The mechanism for this repression is cooperative, distinct from the catalytic mechanism of mRNA cleavage by siRNAs. The use of siRNAs to study translational repression holds promise for dissecting the sequence and structural determinants and general mechanism of gene repression by miRNAs.
hunchback regulates the temporal identity of neuroblasts in Drosophila. Here we show that hbl-1, the C. elegans hunchback ortholog, also controls temporal patterning. Furthermore, hbl-1 is a probable target of microRNA regulation through its 3'UTR. hbl-1 loss-of-function causes the precocious expression of adult seam cell fates. This phenotype is similar to loss-of-function of lin-41, a known target of the let-7 microRNA. Like lin-41 mutations, hbl-1 loss-of-function partially suppresses a let-7 mutation. The hbl-1 3'UTR is both necessary and sufficient to downregulate a reporter gene during development, and the let-7 and lin-4 microRNAs are both required for HBL-1/GFP downregulation. Multiple elements in the hbl-1 3'UTR show complementarity to regulatory microRNAs, suggesting that microRNAs directly control hbl-1. MicroRNAs may likewise function to regulate Drosophila hunchback during temporal patterning of the nervous system.
MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in identifying their targets. The short length of miRNAs and the fact that their complementarity to target sequences is imperfect mean that target identification in animal genomes is not possible by standard sequence comparison methods. Here we screen conserved 3' UTR sequences from the Drosophila melanogaster genome for potential miRNA targets. The screening procedure combines a sequence search with an evaluation of the predicted miRNA-target heteroduplex structures and energies. We show that this approach successfully identifies the five previously validated let-7, lin-4, and bantam targets from a large database and predict new targets for Drosophila miRNAs. Our target predictions reveal striking clusters of functionally related targets among the top predictions for specific miRNAs. These include Notch target genes for miR-7, proapoptotic genes for the miR-2 family, and enzymes from a metabolic pathway for miR-277. We experimentally verified three predicted targets each for miR-7 and the miR-2 family, doubling the number of validated targets for animal miRNAs. Statistical analysis indicates that the best single predicted target sites are at the border of significance; thus, target predictions should be considered as tentative until experimentally validated. We identify features shared by all validated targets that can be used to evaluate target predictions for animal miRNAs. Our initial evaluation and experimental validation of target predictions suggest functions for two miRNAs. For others, the screen suggests plausible functions, such as a role for miR-277 as a metabolic switch controlling amino acid catabolism. Cross-genome comparison proved essential, as it allows reduction of the sequence search space. Improvements in genome annotation and increased availability of cDNA sequences from other genomes will allow more sensitive screens. An increase in the number of confirmed targets is expected to reveal general structural features that can be used to improve their detection. While the screen is likely to miss some targets, our study shows that valid targets can be identified from sequence alone.
A new paradigm of gene expression regulation has emerged recently with the discovery of microRNAs (miRNAs). Most, if not all, miRNAs are thought to control gene expression, mostly by base pairing with miRNA-recognition elements (MREs) found in their messenger RNA (mRNA) targets. Although a large number of human miRNAs have been reported, many of their mRNA targets remain unknown. Here we used a combined bioinformatics and experimental approach to identify important rules governing miRNA-MRE recognition that allow prediction of human miRNA targets. We describe a computational program, "DIANA-microT", that identifies mRNA targets for animal miRNAs and predicts mRNA targets, bearing single MREs, for human and mouse miRNAs.
MicroRNAs (miRNAs) are an extensive class of tiny RNA molecules that regulate the expression of target genes by means of complementary base pair interactions. Although the first miRNAs were discovered in Caenorhabditis elegans, >300 miRNAs were recently documented in animals and plants, both by cloning methods and computational predictions. We present a genome-wide computational approach to detect miRNA genes in the Arabidopsis thaliana genome. Our method is based on the conservation of short sequences between the genomes of Arabidopsis and rice (Oryza sativa) and on properties of the secondary structure of the miRNA precursor. The method was fine-tuned to take into account plant-specific properties, such as the variable length of the miRNA precursor sequences. In total, 91 potential miRNA genes were identified, of which 58 had at least one nearly perfect match with an Arabidopsis mRNA, constituting the potential targets of those miRNAs. In addition to already known transcription factors involved in plant development, the targets also comprised genes involved in several other cellular processes, such as sulfur assimilation and ubiquitin-dependent protein degradation. These findings considerably broaden the scope of miRNA functions in plants.
• comparative genomics
• thale cress
• rice
• noncoding RNA
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.
MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate the expression of target genes by binding to the target mRNAs. Although a large number of animal miRNAs has been defined, only a few targets are known. In contrast to plant miRNAs, which usually bind nearly perfectly to their targets, animal miRNAs bind less tightly, with a few nucleotides being unbound, thus producing more complex secondary structures of miRNA/target duplexes. Here, we present a program, RNA-hybrid, that predicts multiple potential binding sites of miRNAs in large target RNAs. In general, the program finds the energetically most favorable hybridization sites of a small RNA in a large RNA. Intramolecular hybridizations, that is, base pairings between target nucleotides or between miRNA nucleotides are not allowed. For large targets, the time complexity of the algorithm is linear in the target length, allowing many long targets to be searched in a short time. Statistical significance of predicted targets is assessed with an extreme value statistics of length normalized minimum free energies, a Poisson approximation of multiple binding sites, and the calculation of effective numbers of orthologous targets in comparative studies of multiple organisms. We applied our method to the prediction of Drosophila miRNA targets in 3'UTRs and coding sequence. RNAhybrid, with its accompanying programs RNAcalibrate and RNAeffective, is available for download and as a Web tool on the Bielefeld Bioinformatics Server (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/).
MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. The specific function of most mammalian miRNAs is unknown. We have predicted target sites on the 3' untranslated regions of human gene transcripts for all currently known 218 mammalian miRNAs to facilitate focused experiments. We report about 2,000 human genes with miRNA target sites conserved in mammals and about 250 human genes conserved as targets between mammals and fish. The prediction algorithm optimizes sequence complementarity using position-specific rules and relies on strict requirements of interspecies conservation. Experimental support for the validity of the method comes from known targets and from strong enrichment of predicted targets in mRNAs associated with the fragile X mental retardation protein in mammals. This is consistent with the hypothesis that miRNAs act as sequence-specific adaptors in the interaction of ribonuclear particles with translationally regulated messages. Overrepresented groups of targets include mRNAs coding for transcription factors, components of the miRNA machinery, and other proteins involved in translational regulation, as well as components of the ubiquitin machinery, representing novel feedback loops in gene regulation. Detailed information about target genes, target processes, and open-source software for target prediction (miRanda) is available at http://www.microrna.org. Our analysis suggests that miRNA genes, which are about 1% of all human genes, regulate protein production for 10% or more of all human genes.
The factors regulating the expression of microRNAs (miRNAs), a ubiquitous family of approximately 22-nt noncoding regulatory RNAs, remain undefined. However, it is known that miRNAs are first transcribed as a largely unstructured precursor, termed a primary miRNA (pri-miRNA), which is sequentially processed in the nucleus, to give the approximately 65-nt pre-miRNA hairpin intermediate, and then in the cytoplasm, to give the mature miRNA. Here we have sought to identify the RNA polymerase responsible for miRNA transcription and to define the structure of a full-length human miRNA. We show that the pri-miRNA precursors for nine human miRNAs are both capped and polyadenylated and report the sequence of the full-length, approximately 3433-nt pri-miR-21 RNA. This pri-miR-21 gene sequence is flanked 5' by a promoter element able to transcribe heterologous mRNAs and 3' by a consensus polyadenylation sequence. Nuclear processing of pri-miRNAs was found to be efficient, thus largely preventing the nuclear export of full-length pri-miRNAs. Nevertheless, an intact miRNA stem-loop precursor located in the 3' UTR of a protein coding gene only moderately inhibited expression of the linked open reading frame, probably because the 3' truncated mRNA could still be exported and expressed. Together, these data show that human pri-miRNAs are not only structurally similar to mRNAs but can, in fact, function both as pri-miRNAs and mRNAs.
Mature microRNAs (miRNAs) are generated via a two-step processing pathway to yield approximately 22-nucleotide small RNAs that regulate gene expression at the post-transcriptional level. Initial cleavage is catalysed by Drosha, a nuclease of the RNase III family, which acts on primary miRNA transcripts (pri-miRNAs) in the nucleus. Here we show that Drosha exists in a multiprotein complex, the Microprocessor, and begin the process of deconstructing that complex into its constituent components. Along with Drosha, the Microprocessor also contains Pasha (partner of Drosha), a double-stranded RNA binding protein. Suppression of Pasha expression in Drosophila cells or Caenorhabditis elegans interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs. Finally, depletion or mutation of pash-1 in C. elegans causes de-repression of a let-7 reporter and the appearance of phenotypic defects overlapping those observed upon examination of worms with lesions in Dicer (dcr-1) or Drosha (drsh-1). Considered together, these results indicate a role for Pasha in miRNA maturation and miRNA-mediated gene regulation.
MicroRNAs are small approximately 22 nucleotide regulators of numerous biological processes and bind target gene messenger RNAs to control gene expression. The C. elegans microRNA let-7 and its target lin-41 were the first microRNA::target interaction to be validated in vivo. let-7 molecules form imperfect duplexes with two required let-7 complementary sites in the lin-41 3' UTR. Here, we show that base pairing at both the 5' and 3' ends of the let-7 binding site, as well as the presence of unpaired RNA residues in the predicted duplexes, are required for lin-41 downregulation. In this study, our model for microRNA::target interactions also demonstrates that the context of a microRNA binding can be critical for function, revealing an unforeseen complexity in microRNA::target interactions.
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in animals and plants. Comparative genomic computational methods have been developed to predict new miRNAs in worms, flies, and humans. Here, we present a novel single genome approach for the detection of miRNAs in Arabidopsis thaliana. This was initiated by producing a candidate miRNA-target data set using an algorithm called findMiRNA, which predicts potential miRNAs within candidate precursor sequences that have corresponding target sites within transcripts. From this data set, we used a characteristic divergence pattern of miRNA precursor families to select 13 potential new miRNAs for experimental verification, and found that corresponding small RNAs could be detected for at least eight of the candidate miRNAs. Expression of some of these miRNAs appears to be under developmental control. Our results are consistent with the idea that targets of miRNAs encompass a wide range of transcripts, including those for F-box factors, ubiquitin conjugases, Leucine-rich repeat proteins, and metabolic enzymes, and that regulation by miRNAs might be widespread in the genome. The entire set of annotated transcripts in the Arabidopsis genome has been run through find MiRNA to yield a data set that will enable identification of potential miRNAs directed against any target gene.
MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12 h. The 3' untranslated regions of these messages had a significant propensity to pair to the 5' region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans.
Comprehensive identification of all functional elements encoded in the human genome is a fundamental need in biomedical research. Here, we present a comparative analysis of the human, mouse, rat and dog genomes to create a systematic catalogue of common regulatory motifs in promoters and 3' untranslated regions (3' UTRs). The promoter analysis yields 174 candidate motifs, including most previously known transcription-factor binding sites and 105 new motifs. The 3'-UTR analysis yields 106 motifs likely to be involved in post-transcriptional regulation. Nearly one-half are associated with microRNAs (miRNAs), leading to the discovery of many new miRNA genes and their likely target genes. Our results suggest that previous estimates of the number of human miRNA genes were low, and that miRNAs regulate at least 20% of human genes. The overall results provide a systematic view of gene regulation in the human, which will be refined as additional mammalian genomes become available.
MicroRNAs (miRNAs) are regulatory RNAs found in multicellular eukaryotes, including humans, where they are implicated in cancer. The let-7 miRNA times seam cell terminal differentiation in C. elegans. Here we show that the let-7 family negatively regulates let-60/RAS. Loss of let-60/RAS suppresses let-7, and the let-60/RAS 3'UTR contains multiple let-7 complementary sites (LCSs), restricting reporter gene expression in a let-7-dependent manner. mir-84, a let-7 family member, is largely absent in vulval precursor cell P6.p at the time that let-60/RAS specifies the 1 degrees vulval fate in that cell, and mir-84 overexpression suppresses the multivulva phenotype of activating let-60/RAS mutations. The 3'UTRs of the human RAS genes contain multiple LCSs, allowing let-7 to regulate RAS expression. let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, providing a possible mechanism for let-7 in cancer.
lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction.
The nucleotide sequence of an RNA primer molecule for initiation of Rous sarcoma virus DNA synthesis in vitro has been determined. The sequence can be drawn in a cloverleaf structure typical of tRNAs with an anticodon for tryptophan. Aminoacylation of the molecule confirms that it is tRNA-Trp. The same sequence and aminoacylation results are obtained regardless of whether the RNA is isolated from virions or from cells of chickens, the natural host for this virus. It is the only species of tRNA-Trp that is dectected in chicked cell tRNA.
Reverse transcription-polymerase chain reaction (RT-PCR) is a gene expression assay by which messenger RNA (mRNA) production can be measured. This technique involves three steps: isolation of RNA from cells or tissues, the creation of a DNA copy of the desired message (cDNA) by viral reverse transcriptase enzymes (RT), and amplification of this DNA segment by the polymerase chain reaction (PCR) for subsequent quantitation and analysis. Here we describe a one-enzyme, one-step method combining the RT and PCR steps of conventional RT-PCR by exploiting the recently documented RT properties of Taq polymerase, the thermostable enzyme used for PCR amplification of DNA. RNA was extracted from gibbon T-cells (MLA144), reverse transcribed and amplified with oligonucleotide primers (specific for the 5' portion of a spliced interleukin-2 (IL-2) messenger RNA) by Taq polymerase. A discrete fragment of correct length for IL-2 cDNA was detected. Experiments showed that this product was RNA-dependent and specific for IL-2. This fragment was sequenced by automation employing a biotin primer-streptavidin magnetic bead protocol which confirmed its origin as processed IL-2 mRNA. The modification of the RT-PCR procedure using a thermostable enzyme speeds up reaction time and increases stringency. This method should make the diagnostic screening of cells for gene expression more efficient and practical.
During C. elegans development, the temporal pattern of many cell lineages is specified by graded activity of the heterochronic gene Lin-14. Here we demonstrate that a temporal gradient in Lin-14 protein is generated posttranscriptionally by multiple elements in the lin-14 3'UTR that are regulated by the heterochronic gene Lin-4. The lin-14 3'UTR is both necessary and sufficient to confer lin-4-mediated posttranscriptional temporal regulation. The function of the lin-14 3'UTR is conserved between C. elegans and C. briggsae. Among the conserved sequences are seven elements that are each complementary to the lin-4 RNAs. A reporter gene bearing three of these elements shows partial temporal gradient activity. These data suggest a molecular mechanism for Lin-14p temporal gradient formation: the lin-4 RNAs base pair to sites in the lin-14 3'UTR to form multiple RNA duplexes that down-regulate lin-14 translation.
lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction.
We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.
Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.
Animal genomes contain an abundance of small genes that produce regulatory RNAs of about 22 nucleotides in length. These microRNAs are diverse in sequence and expression patterns, and are evolutionarily widespread, suggesting that they may participate in a wide range of genetic regulatory pathways.
Most of the RNA transcribed from your genome doesn't make protein. Carina Dennis talks to the revolutionaries who believe that it functions in gene-regulatory networks that underlie the complexity of higher organisms.
Temporal control of development is an important aspect of pattern formation that awaits complete molecular analysis. We identified lin-57 as a member of the C. elegans heterochronic gene pathway, which ensures that postembryonic developmental events are appropriately timed. Loss of lin-57 function causes the hypodermis to terminally differentiate and acquire adult character prematurely. lin-57 is hbl-1, revealing a role for the worm hunchback homolog in control of developmental time. Significantly, fly hunchback (hb) temporally specifies cell fates in the nervous system. The hbl-1/lin-57 3'UTR is required for postembryonic downregulation in the hypodermis and nervous system and contains multiple putative binding sites for temporally regulated microRNAs, including let-7. Indeed, we find that hbl-1/lin-57 is regulated by let-7, at least in the nervous system. Examination of the hb 3'UTR reveals potential binding sites for known fly miRNAs. Thus, evolutionary conservation of hunchback genes may include temporal control of cell fate specification and microRNA-mediated regulation.
MicroRNAs (miRNAs) constitute an extensive class of noncoding RNAs that are thought to regulate the expression of target genes via complementary base-pair interactions. To date, cloning has identified over 200 miRNAs from diverse eukaryotic organisms. Despite their success, such biochemical approaches are skewed toward identifying abundant miRNAs, unlike genome-wide, sequence-based computational predictions. We developed informatic methods to predict miRNAs in the C. elegans genome using sequence conservation and structural similarity to known miRNAs and generated 214 candidates. We confirmed the expression of four new miRNAs by Northern blotting and used a more sensitive PCR approach to verify the expression of ten additional candidates. Based on hypotheses underlying our computational methods, we estimate that the C. elegans genome may encode between 140 and 300 miRNAs and potentially many more.
The general basis of cancer is the loss of cell identity and inappropriate proliferation of cells. Classically, a universal paradigm in oncogenesis is the accumulation of mutations in the open reading frames of protein-encoding oncogenes and tumor suppressors. The identification of new classes of noncoding RNAs (ncRNA) important for development and cell homeostasis will likely change this current paradigm. Recent data suggests that a special class of ncRNAs called microRNAs might be involved in human disease. This review proposes a role for microRNAs in oncogenesis.
MicroRNAs (miRNAs) can play important gene regulatory roles in nematodes, insects, and plants by basepairing to mRNAs to specify posttranscriptional repression of these messages. However, the mRNAs regulated by vertebrate miRNAs are all unknown. Here we predict more than 400 regulatory target genes for the conserved vertebrate miRNAs by identifying mRNAs with conserved pairing to the 5' region of the miRNA and evaluating the number and quality of these complementary sites. Rigorous tests using shuffled miRNA controls supported a majority of these predictions, with the fraction of false positives estimated at 31% for targets identified in human, mouse, and rat and 22% for targets identified in pufferfish as well as mammals. Eleven predicted targets (out of 15 tested) were supported experimentally using a HeLa cell reporter system. The predicted regulatory targets of mammalian miRNAs were enriched for genes involved in transcriptional regulation but also encompassed an unexpectedly broad range of other functions.
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.
Small interfering RNA (siRNA) duplexes are generally produced by Dicer cleavage of double-stranded RNAs of frequently exogenous origin and can induce the cleavage and degradation of mRNAs bearing an identical sequence. In contrast, microRNAs (miRNAs) are encoded within the eukaryotic genome as short RNA hairpin structures. While these pre-miRNAs are also processed by Dicer, mature miRNAs appear to function primarily by inhibiting the translation of mRNAs bearing multiple, partially mismatched target sites. Nevertheless, recent data argue that the posttranscriptional regulatory machinery utilized by siRNAs and miRNAs is largely or entirely identical. In this review, I will discuss recent progress in unraveling the RNA processing pathway utilized for the biosynthesis of mature miRNAs and argue that this pathway offers at least three distinct entry points for the functional expression of artificial siRNAs in vertebrate cells. While each of these entry points offers distinct advantages and disadvantages, they all have the potential to induce the effective knock-down of specific genes either in cell culture or in experimental animals.
There has been a lack of powerful tools for systematic analysis of mammalian gene function, but RNA interference (RNAi) may now provide such a strategy. Stable transcription of RNAi triggers from suitable expression cassettes integrated into the host cell genome by viral gene transfer can induce long-term and heritable gene silencing in mammalian cells. However, the use of RNAi as a genetic tool is limited by difficulties in identifying efficient RNAi triggers, the problem of effective delivery and off-target effects, as well as potential genotoxic side effects of viral gene transfer strategies. Recent insights into the molecular mechanisms of silencing processes mediated by either siRNA or miRNA will allow further optimization of RNAi triggers as genetic tools.
New evidence suggests that abundant small RNAs, microRNAs (miRNAs) act as regulatory vehicles to repress translation or cleave RNA transcripts, depending on their complementarity to the target gene, and result in modulation of gene expression. This review describes that the miRNAs derived from introns can suppress intracellular RNA homologues and regulate the gene function.
MicroRNAs (miRNAs) are a growing family of small non-protein-coding regulatory genes that regulate the expression of homologous target-gene transcripts. They have been implicated in the control of cell death and proliferation in flies, haematopoietic lineage differentiation in mammals, neuronal patterning in nematodes and leaf and flower development in plants. miRNAs are processed by the RNA-mediated interference machinery. Drosha is an RNase III enzyme that was recently implicated in miRNA processing. Here we show that human Drosha is a component of two multi-protein complexes. The larger complex contains multiple classes of RNA-associated proteins including RNA helicases, proteins that bind double-stranded RNA, novel heterogeneous nuclear ribonucleoproteins and the Ewing's sarcoma family of proteins. The smaller complex is composed of Drosha and the double-stranded-RNA-binding protein, DGCR8, the product of a gene deleted in DiGeorge syndrome. In vivo knock-down and in vitro reconstitution studies revealed that both components of this smaller complex, termed Microprocessor, are necessary and sufficient in mediating the genesis of miRNAs from the primary miRNA transcript.
MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular mRNAs that contain partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. MiRNA was originally discovered in the intergenic regions of the Caenorhabditis elegans genome as native RNA fragments that modulate a wide range of genetic regulatory pathways during animal development. However, neither RNA promoter nor polymerase responsible for miRNA biogenesis was determined. Recent findings of intron-derived miRNA in C. elegans, mouse, and human have inevitably led to an alternative pathway for miRNA biogenesis, which relies on the coupled interaction of Pol-II-mediated pre-mRNA transcription and intron excision, occurring in certain nuclear regions proximal to genomic perichromatin fibrils.
RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.
We predict regulatory targets of vertebrate microRNAs (miRNAs) by identifying mRNAs with conserved complementarity to the seed (nucleotides 2-7) of the miRNA. An overrepresentation of conserved adenosines flanking the seed complementary sites in mRNAs indicates that primary sequence determinants can supplement base pairing to specify miRNA target recognition. In a four-genome analysis of 3' UTRs, approximately 13,000 regulatory relationships were detected above the estimate of false-positive predictions, thereby implicating as miRNA targets more than 5300 human genes, which represented 30% of our gene set. Targeting was also detected in open reading frames. In sum, well over one third of human genes appear to be conserved miRNA targets.
Translational control plays a major role in early development, differentiation and the cell cycle. In this review, we focus on the four main mechanisms of translational control by 3' untranslated regions: 1. Cytoplasmic polyadenylation and deadenylation; 2. Recruitment of 4E binding proteins; 3. Regulation of ribosomal subunit binding; 4. Post-initiation repression by microRNAs. Proteins with conserved functions in translational control during development include cytoplasmic polyadenylation element binding proteins (CPEB/Orb), Pumilio, Bruno, Fragile X mental retardation protein and RNA helicases. The translational regulation of the mRNAs encoding cyclin B1, Oskar, Nanos, Male specific lethal 2 (Msl-2), lipoxygenase and Lin-14 is discussed.
MicroRNAs (miRNAs) are short endogenous RNAs known to post-transcriptionally repress gene expression in animals and plants. A microarray profiling survey revealed the expression patterns of 175 human miRNAs across 24 different human organs. Our results show that proximal pairs of miRNAs are generally coexpressed. In addition, an abrupt transition in the correlation between pairs of expressed miRNAs occurs at a distance of 50 kb, implying that miRNAs separated by <50 kb typically derive from a common transcript. Some microRNAs are within the introns of host genes. Intronic miRNAs are usually coordinately expressed with their host gene mRNA, implying that they also generally derive from a common transcript, and that in situ analyses of host gene expression can be used to probe the spatial and temporal localization of intronic miRNAs.
MicroRNAs (miRNAs) are a recently discovered set of regulatory genes that constitute up to an estimated 1% of the total number of genes in animal genomes, including Caenorhabditis elegans, Drosophila, mouse, and humans [Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. (2001) Science 294, 853–858; Lai, E. C., Tomancak, P., Williams, R. W. & Rubin, G.M. (2003) Genome Biol. 4, R42; Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. (2001) Science 294, 858–862; Lee, R. C. & Ambros, V. (2001) Science 294, 862-8644; and Lee, R. C., Feinbaum, R. L. & Ambros, V. (1993) Cell 115, 787–798]. In animals, miRNAs regulate genes by attenuating protein translation through imperfect base pair binding to 3′ UTR sequences of target genes. A major challenge in understanding the regulatory role of miRNAs is to accurately predict regulated targets. We have developed an algorithm for predicting targets that does not rely on evolutionary conservation. As one of the features of this algorithm, we incorporate the folded structure of mRNA. By using Drosophila miRNAs as a test case, we have validated our predictions in 10 of 15 genes tested. One of these validated genes is mad as a target for bantam. Furthermore, our computational and experimental data suggest that miRNAs have fewer targets than previously reported.
• mRNA structure
• target prediction