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When one is better than two: RNA with dual functions

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Abstract

The central dogma of biology, until not long ago, held that genetic information stored on DNA molecules was translated into the final protein products through RNA as intermediate molecules. Then, an additional level of complexity in the regulation of genome expression was added, implicating new classes of RNA molecules called non-coding RNA (ncRNA). These ncRNA are also often referred to as functional RNA in that, although they do not contain the capacity to encode proteins, do have a function as RNA molecules. They have been thus far considered as truly non-coding RNA since no ORF long enough to be considered, nor protein, have been associated with them. However, the recent identification and characterization of bifunctional RNA, i.e. RNA for which both coding capacity and activity as functional RNA have been reported, suggests that a definite categorization of some RNA molecules is far from being straightforward. Indeed, several RNA primarily classified as non-protein-coding RNA has been showed to hold coding capacities and associated peptides. Conversely, mRNA, usually regarded as strictly protein-coding, may act as functional RNA molecules. Here, we describe several examples of these bifunctional RNA that have been already characterized from bacteria to mammals. We also extend this concept to fortuitous acquisition of dual function in pathological conditions and to the recently highlighted duality between information carried by a gene and its pseudogenes counterparts.

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... However, difficulties in genomic annotation are also complicated by mounting evidence that multifunctionality can also apply to transcripts themselves. Indeed, certain precursor RNAs can release more than one class of transcripts, i.e. coding and non-coding or two non-coding RNAs, and more strikingly, that some mature transcripts can perform more than one function [reviewed in [12] and Figure 1]. The first example of an RNA with dual function was probably the Steroid Receptor RNA activator (SRA) [13,14], and the term 'bifunctional' was then coined by Marcel Dinger and John Mattick [15]. ...
... We will go over bifunctional precursor RNAs (pre-RNA) that can release coding and noncoding, or two non-coding, functions depending on posttranscriptional maturation processes. We will also mention single RNA molecules that can operate at least two functions [for reviews see [12,15,21,22]]. This includes messenger RNAs (mRNA) shown to operate as functional RNAs, and certain lncRNAs initially classified as non-coding but shown to release small peptides or re-annotated as coding RNAs. Following the logic, any functional ncRNA that serves as a precursor to a smaller regulatory RNA should be considered as bifunctional (Figure 1). ...
... We already mentioned cases where the dual coding/noncoding function is carried by the products of a gene and its related pseudogene (reviewed in [12,23]), yet it potentially involves thousands of pairs of gene/pseudogene transcripts that remain to be characterized. In a more remarkable way, this duality of functions can also be released by the same transcription unit, i.e. carried by the same pre-RNA, after steps of post-transcriptional maturation. ...
Article
The findings that an RNA is not necessarily either coding or non-coding, or that a precursor RNA can produce different types of mature RNAs, whether coding or non-coding, long or short, have challenged the dichotomous view of the RNA world almost 15 years ago. Since then, and despite an increasing number of studies, the diversity of information that can be conveyed by RNAs is rarely searched for, and when it is known, it remains largely overlooked in further functional studies. Here, we provide an update with prominent examples of multiple functions that are carried by the same RNA or are produced by the same precursor RNA, to emphasize their biological relevance in most living organisms. An important consequence is that the overall function of their locus of origin results from the balance between various RNA species with distinct functions and fates. The consideration of the molecular basis of this multiplicity of information is obviously crucial for downstream functional studies when the targeted functional molecule is often not the one that is believed.
... When we refer to bifRNAs, we assume that the same molecule is able to perform both functions, namely encode for a protein and possess its own regulatory function, but this term is often misused ( Figure 1A). In fact, this is the case for a subset of them, such as SRA (Steroid Receptor RNA Activator), the pioneer member of this family [for review see (Ulveling et al., 2011b)]. Other examples have been described; SgrS (SuGar transport-Related sRNA) RNA partially inhibits glucose transporters mRNAs through base-pairing, and encodes a small polypeptide that prevents glucose transport (Maki et al., 2010;Vanderpool et al., 2011;Wadler and Vanderpool, 2007). ...
... Thus, many mRNAs may act as regulatory RNAs whereas more and more regulatory RNAs, first classified as non-coding, are shown to hide small coding sequences i.e. less than the 300nt/100aa limit defined so far (Dinger et al., 2008;Ulveling et al., 2011b). A strict discrimination between these two classes of molecules appears even less realistic since, in eukaryotes, some ncRNAs have features comparable to that of mRNAs, such as polymerase II-dependant transcription and addition of a cap and a polyadenylated tail for instance (Dinger et al., 2008;Kondo et al., 2010;Ulveling et al., 2011b), and even their presence in polysome fractions (Ingolia et al., 2011). ...
... Thus, many mRNAs may act as regulatory RNAs whereas more and more regulatory RNAs, first classified as non-coding, are shown to hide small coding sequences i.e. less than the 300nt/100aa limit defined so far (Dinger et al., 2008;Ulveling et al., 2011b). A strict discrimination between these two classes of molecules appears even less realistic since, in eukaryotes, some ncRNAs have features comparable to that of mRNAs, such as polymerase II-dependant transcription and addition of a cap and a polyadenylated tail for instance (Dinger et al., 2008;Kondo et al., 2010;Ulveling et al., 2011b), and even their presence in polysome fractions (Ingolia et al., 2011). In various mammals, from mice to humans, many of these long ncRNAs contain coding sequences that may be expressed in specific contexts. ...
... Here, we introduce RBM14, Gle1, HuR, RBM8A, and MAGOH, the functions of which in the centrosome have been recently described. Although these proteins are likely associated with mRNA, it is possible that they also bind to ncRNA to regulate centrosome biogenesis or their associated mRNAs perform a dual role as functional RNA molecules [40,41]. ...
... In this chapter, the role of RNA and RNA-binding proteins in microtubules and mitotic spindles are introduced (Figure 4). Given that treating human cells with transcription inhibitors [74] or RNase, but not translation inhibitors [17], destabilizes the mitotic spindle structure, it can be assumed that functional ncRNAs or mRNAs [40,41] act as regulators or structural components of the mitotic spindle. One example is the regulation of aurora-kinase-B and MCAK. ...
Article
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Mounting experimental evidence shows that non-coding RNAs (ncRNAs) serve a wide variety of biological functions. Recent studies suggest that a part of ncRNAs are critically important for supporting the structure of subcellular architectures. Here, we summarize the current literature demonstrating the role of ncRNAs and RNA-binding proteins in regulating the assembly of mitotic apparatus, especially focusing on centrosomes, kinetochores, and mitotic spindles.
... On investigating the functions of lncRNAs, a few studies confirmed that some lncRNAs indeed had small open reading frames (sORF, length <300 nt) that could code for a short peptide with key biological functions [52][53][54][55][56][57][58][59][60][61][62][63]. The presence of functional small peptides coded by the lncRNAs suggests that these lncRNAs could play dual roles, with both RNA and peptides, and therefore should be reclassified as bifunctional RNAs [64][65][66]. This review provides a brief overview of computational and combinatorial approaches for the classification of coding/noncoding RNAs and for the systematic identification of small peptides coded by these transcripts and summarizes functional small peptides encoded by invertebrate and vertebrate lncRNAs. ...
... The extent to which lncRNAs can produce small peptides is still debatable; however, it is now widely accepted that some lncRNAs can be translated [64,66]. Although computational approaches that do not use Ribo-seq and/or MS data have been successful in detecting coding potential in RNAs, the majority of them lack the capability to detect sORFs that could encode small peptides. ...
Article
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Long noncoding RNAs (lncRNAs) are a group of transcripts that are longer than 200 nucleotides (nt) without coding potential. Over the past decade, tens of thousands of novel lncRNAs have been annotated in animal and plant genomes because of advanced high-throughput RNA sequencing technologies and with the aid of coding transcript classifiers. Further, a considerable number of reports have revealed the existence of stable, functional small peptides (also known as micropeptides), translated from lncRNAs. In this review, we discuss the methods of lncRNA classification, the investigations regarding their coding potential and the functional significance of the peptides they encode.
... While the phenomenon of "translated ncRNAs" is highly discussed for eukaryotes [70,71,[84][85][86][87][88][89], this observation has, to our knowledge, only rarely been reported for bacteria, i.e. SgrS/SgrT or the "ncRNA" C0343 ( [90]; see below, [91]). In any case, the ribosomal "coverage" of tRNAs (median RCV 0.03), taken as background in this study, is far below the high ribosomal coverage of some ncRNAs. ...
... A few ncRNAs which are also translated have been suggested to exist in bacteria and are termed coding non-coding RNAs (cncRNAs) [24]. sgrS/sgrT is the only known example for E. coli K12 [90]. In EHEC EDL933, the ATG start codon used by E. coli K12 is mutated to ATT. ...
Article
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Background While NGS allows rapid global detection of transcripts, it remains difficult to distinguish ncRNAs from short mRNAs. To detect potentially translated RNAs, we developed an improved protocol for bacterial ribosomal footprinting (RIBOseq). This allowed distinguishing ncRNA from mRNA in EHEC. A high ratio of ribosomal footprints per transcript (ribosomal coverage value, RCV) is expected to indicate a translated RNA, while a low RCV should point to a non-translated RNA. Results Based on their low RCV, 150 novel non-translated EHEC transcripts were identified as putative ncRNAs, representing both antisense and intergenic transcripts, 74 of which had expressed homologs in E. coli MG1655. Bioinformatics analysis predicted statistically significant target regulons for 15 of the intergenic transcripts; experimental analysis revealed 4-fold or higher differential expression of 46 novel ncRNA in different growth media. Out of 329 annotated EHEC ncRNAs, 52 showed an RCV similar to protein-coding genes, of those, 16 had RIBOseq patterns matching annotated genes in other enterobacteriaceae, and 11 seem to possess a Shine-Dalgarno sequence, suggesting that such ncRNAs may encode small proteins instead of being solely non-coding. To support that the RIBOseq signals are reflecting translation, we tested the ribosomal-footprint covered ORF of ryhB and found a phenotype for the encoded peptide in iron-limiting condition. Conclusion Determination of the RCV is a useful approach for a rapid first-step differentiation between bacterial ncRNAs and small mRNAs. Further, many known ncRNAs may encode proteins as well.
... This possibility can be supported by several lines of evidence: (i) lincRNAs can be translated but in a non-canonical mode (17)(18)(19)(20); (ii) the encoded products are generally unstable and rapidly degraded (34,58); and (iii) at least some products encoded by sORFs, if not all, can exist in stable functional micro-peptides (21,22). It has also been proposed that these lincRNAs may act as bifunctional RNAs that are generally non-coding, but under specific circumstances, enclosed sORFs can be translated (54,(59)(60)(61). Therefore, even though some lincR-NAs appear translated in ribosome profiling data, they are usually largely invisible in mass spectrometry (18,20,(62)(63)(64)(65). ...
Article
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Advances in transcriptomics have led to the discovery of a large number of long intergenic non-coding RNAs (lincRNAs), which are now recognized as important regulators of diverse cellular processes. Although originally thought to be non-coding, recent studies have revealed that many lincRNAs are bound by ribosomes, with a few lincRNAs even having ability to generate micropeptides. The question arises: how widespread the translation of lincRNAs may be and whether such translation is likely to be functional. To better understand biological relevance of lincRNA translation, we systematically characterized lincRNAs with ribosome occupancy by the expression, structural, sequence, evolutionary and functional features for eight human cell lines, revealed that lincRNAs with ribosome occupancy have remarkably distinctive properties compared with those without ribosome occupancy, indicating that translation has important biological implication in categorizing and annotating lincRNAs. Further analysis revealed lincRNAs exhibit remarkable cell-type specificity with differential translational repertoires and substantial discordance in functionality. Collectively, our analyses provide the first attempt to characterize global and cell-type specific properties of translation of lincRNAs in human cells, highlighting that translation of lincRNAs has clear molecular, evolutionary and functional implications. This study will facilitate better understanding of the diverse functions of lincRNAs.
... RNA is no longer considered to be merely an intermediary between genes and proteins. It has become increasingly apparent that noncoding RNAs are crucial players in a variety of cellular and physiological functions [1][2][3][4]. ...
Article
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In recent functional genomics studies, a large number of non-coding RNAs have been identified. It has become increasingly apparent that noncoding RNAs are crucial players in a wide range of cellular and physiological functions. They have been shown to modulate gene expression on different levels, including transcription, post-transcriptional processing, and translation. This review aims to highlight the diverse mechanisms of the regulation of gene expression by small noncoding RNAs in different conditions and different types of human cells. For this purpose, various cellular functions of microRNAs (miRNAs), circular RNAs (circRNAs), snoRNA-derived small RNAs (sdRNAs) and tRNA-derived fragments (tRFs) will be exemplified, with particular emphasis on the diversity of their occurrence and on the effects on gene expression in different stress conditions and diseased cell types. The synthesis and effect on gene expression of these noncoding RNAs varies in different cell types and may depend on environmental conditions such as different stresses. Moreover, noncoding RNAs play important roles in many diseases, including cancer, neurodegenerative disorders, and viral infections.
... However, the role of mRNAs is not limited to being the source of protein production. An emerging body of evidence suggests that coding mRNAs can have independent functions [8]. In zebrafish, Squint (Sqt), a Nodal-related signaling molecule belonging to the transforming growth factor b (TGF-b) superfamily, is involved in mesoderm induction and left-right axis specification. ...
Article
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How cells position their proteins is a key problem in cell biology. Targeting mRNAs to distinct regions of the cytoplasm contributes to protein localization by providing local control over translation. Here, we reveal that an interdependence of a protein and cognate mRNA maintains asymmetric protein distribution in mitotic Drosophila neural stem cells. We tagged endogenous mRNA or protein products of the gene miranda that is required for fate determination with GFP. We find that the mRNA localizes like the protein it encodes in a basal crescent in mitosis. We then used GFP-specific nanobodies fused to localization domains to alter the subcellular distribution of the GFP-tagged mRNA or protein. Altering the localization of the mRNA resulted in mislocalization of the protein and vice versa. Protein localization defects caused by mislocalization of the cognate mRNA were rescued by introducing untagged mRNA coding for mutant non-localizable protein. Therefore, by combining the MS2 system and subcellular nanobody expression, we uncovered that maintenance of Mira asymmetric localization requires interaction with the cognate mRNA.
... More recently, advances in high throughput transcriptome analysis highlighted the pervasive nature of transcription of mammalian genomes and revealed an ever-growing number of non protein-coding yet functional transcripts, which amount parallels that of genomes complexity (6,56). Furthermore, the recent characterization of RNAs for which both coding capacity and activity as functional RNAs have been reported, adds an additional degree of complexity (30,31,57,58). We recently proposed that intron retention contributes to the diversification of the information carried by genes by producing functional RNA instead of a protein product (58). ...
Article
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Introns represent almost half of the human genome, although they are eliminated from transcripts through RNA splicing. Yet, different classes of non-canonical miRNAs have been proposed to originate directly from intron splicing. Here, we considered the alternative splicing of introns as an interesting source of miRNAs, compatible with a developmental switch. We report computational prediction of new Short Intron-Derived ncRNAs (SID), defined as precursors of smaller ncRNAs like miRNAs and snoRNAs produced directly by splicing, and tested their dependence on each key factor in canonical or alternative miRNAs biogenesis (Drosha, DGCR8, DBR1, snRNP70, U2AF65, PRP8, Dicer, Ago2). We found that about half of predicted SID rely on debranching of the excised intron-lariat by the enzyme DBR1, as proposed for mirtrons. However, we identified new classes of SID for which miRNAs biogenesis may rely on intermingling between canonical and alternative pathways. We validated selected SID as putative miRNAs precursors and identified new endogenous miRNAs produced by non-canonical pathways, including one hosted in the first intron of SRA (Steroid Receptor RNA activator). Consistent with increased SRA intron retention during myogenic differentiation, release of SRA intron and its associated mature miRNA decreased in cells from healthy subjects but not from myotonic dystrophy patients with splicing defects.
... Most miRNAs are encoded by independent transcription units [8], located in intergenic regions or hosted within introns of protein-coding or non-coding genes [9][10][11][12][13]. Beside these intronic miRNAs that are potentially transcribed independently of the host genes, novel classes of intronic miRNAs precursors have been reported recently by which production is initiated by splicing of the intron, independently of the microprocessor [10,11,14]. ...
... Regions encoding lncRNAs include intergenic regions, the sense and antisense orientations of protein-coding genes with transcription start sites located in introns, exons, promoter regions, enhancers, or the 3 -and 5 -UTRs [41]. LncRNAs usually lack an open reading frame, although a few studies have shown their ability to encode proteins [42]. LncRNAs are synthesized by the RNA polymerase II (RNAP II), III complex and spRNAP IV (single-polypeptide nuclear RNA polymerase IV). ...
Article
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Pituitary neuroendocrine tumors (PitNET) are common intracranial neoplasms. While in case of hormone secreting tumors pituitary hormone measurements can be used for monitoring the disease, in non-functional tumors there is a need to discover non-invasive biomarkers. Non-coding RNAs (ncRNAs) are popular biomarker candidates due to their stability and tissue specificity. Among ncRNAs, miRNAs, lncRNAs and circRNAs have been investigated the most in pituitary tumor tissues and in circulation. However, it is still not known whether ncRNAs are originated from the pituitary, or whether they are casually involved in the pathophysiology. Additionally, there is strong diversity among different studies reporting ncRNAs in PitNET. Therefore, to provide an overview of the discrepancies between published studies and to uncover the reasons why despite encouraging experimental data application of ncRNAs in clinical routine has not yet taken hold, in this review available data are summarized on circulating ncRNAs in PitNET. The data on circulating miRNAs, lncRNAs and circRNAs are organized according to different PitNET subtypes. Biological (physiological and pathophysiological) factors behind intra- and interindividual variability and technical aspects of detecting these markers, including preanalytical and analytical parameters, sample acquisition (venipuncture) and type, storage, nucleic acid extraction, quantification and normalization, which reveal the two sides of the same coin are discussed.
... However, this gene also encodes a long non-coding RNA (lncRNA, 680 bases) with exons overlapping the intron sequences of RAD18 (Supplemental Figure 4). CAV3 may belong to the growing class of "bifunctional genes" encoding not only proteins but also lncRNA isoforms, which often have a function antagonistic to that of the protein (47). CAV3 mRNA is also detected in liver tissues (GeneAtlas U133A, gcrma) and this gene has been reported to be associated with hepatic insulin signaling (48). ...
Article
Background: There is growing evidence that human genetic variants contribute to liver fibrosis in subjects with hepatitis C virus (HCV) mono-infection, but this aspect has been little investigated in patients co-infected with HCV and human immunodeficiency virus (HIV). We performed the first genome-wide association study (GWAS) of liver fibrosis progression in patients co-infected with HCV and HIV, using the well-characterized French ANRS CO13 HEPAVIH cohort. Liver fibrosis was assessed by elastography (FibroScan®) providing a quantitative fibrosis score. After quality control, GWAS was conducted on 289 Caucasian patients, for a total of 8,426,597 genotyped (Illumina Omni2.5 BeadChip) or reliably imputed SNPs. SNPs with p-values < 10(-6) were investigated in two independent replication cohorts of European patients infected with HCV alone. Results: Two signals of genome-wide significance (p-value < 5x10(-8) ) were obtained. The first, on chromosome 3p25 and corresponding to rs61183828 (p-value = 3.8x10(-9) ), was replicated in the two independent cohorts of patients with HCV mono-infection. The cluster of SNPs in linkage disequilibrium with rs61183828 was located close to two genes involved in mechanisms affecting both cell signaling and cell structure (CAV3) or HCV replication (RAD18). The second signal, obtained with rs11790131 (p-value = 9.3x10(-9) ) on chromosome region 9p22, was not replicated. Conclusion: Our GWAS identified a new locus associated with liver fibrosis severity in patients with HIV/HCV co-infection, on chromosome 3p25. This finding was replicated in patients with HCV mono-infection. These results provide new relevant hypotheses for the pathogenesis of liver fibrosis in patients with HIV/HCV co-infection that may help define new targets for drug development or new prognostic tests, to improve patient care. This article is protected by copyright. All rights reserved.
... SRA was found to be dysregulated and showing high-expression patterns in different types of cancers including breast, uterus, and ovarian cancers. 82,83 A lncRNA that acts as oncogenes and aids in tumor development and progression is prostate cancer gene expression marker 1 (PCGEM1) and was found upregulated in prostate cancer, contributing to initiation and progression of prostate cancer. Furthermore, PCGEM1 is a target of miR-145, an miRNA that plays a tumor suppressive role in prostate cancer. ...
Article
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Hepatocellular carcinoma (HCC) is a major malignancy in the liver and has emerged as one of the main cancers in the world with a high mortality rate. However, the molecular mechanisms of HCC are still poorly understood. Long noncoding RNAs (lncRNAs) have recently come to the forefront as functional non–protein-coding RNAs that are involved in a variety of cellular processes ranging from maintaining the structural integrity of chromosomes to gene expression regulation in a spatiotemporal manner. Many recent studies have reported the involvement of lncRNAs in HCC which has led to a better understanding of the underlying molecular mechanisms operating in HCC. Long noncoding RNAs have been shown to regulate development and progression of HCC, and thus, lncRNAs have both diagnostic and therapeutic potentials. In this review, we present an overview of the lncRNAs involved in different stages of HCC and their potential in clinical applications which have been studied so far.
... Conceptually, coding a peptide/protein in specific circumstances and functioning as a regulatory RNA molecule in others are not exclusive possibilities for a transcript, whatever it has been primarily annotated as a lncRNA or mRNA. Examples of bifunctional RNAs with dual coding and regulatory functions have been reported (Ulveling et al. 2011). In this respect, in yeast, convergent mRNAs can regulate each other at the RNA level providing additional intriguing cases where mRNAs can switch their initial coding function into regulatory RNAs (Sinturel et al. 2015). ...
Article
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Over the last decade, advances in transcriptomics have revealed that the pervasive transcription of eukaryotic genomes produces plethora of long noncoding RNAs (lncRNAs), which are now recognized as major regulators of multiple cellular processes. Although they have been thought to lack any protein-coding potential, recent ribosome-profiling data indicate that lncRNAs can interact with the translation machinery, leading to the production of functional peptides in some cases. In this perspective, we have explored the idea that translation can be part of the fate of cytoplasmic lncRNAs, raising the possibility for them to work as bifunctional RNAs, endowed with dual coding and regulatory functions.
... Many genomic loci however, are likely to contain a multitude of overlapping and potentially independent func- tions that are transacted at the DNA, RNA and protein levels. Whilst some lncRNAs can in fact encode proteins as either their primary or secondary function [20], messenger RNAs may also have a dual function as a regulatory RNA [21,22]. ...
Article
The human genome sequence is freely available, nearly complete and is providing a foundation of research opportunities that are overturning our current understanding of human biology. The advent of next generation sequencing has revolutionized the way we can interrogate the genome and its transcriptional products and how we analyze, diagnose, monitor and even treat human disease. Personal genetic profiles are increasing dramatically in medical value as researchers accumulate more and more knowledge about the interaction between genetic and environmental factors that contribute to the onset of common disorders. As the cost of sequencing plummets, whole genome sequencing of individuals is becoming a reality and the field of personalized genomic medicine is rapidly developing. Now there is great need for accurate annotation of all functionally important sequences in the human genome and the variations within them that contribute to health and disease. The vast majority of our genome gives rise to RNA transcripts. This extraordinarily versatile molecule not only encodes protein information but also has great structural dynamics and plasticity, capacity for DNA/RNA/protein interactions and catalytic activity. It is a key regulator of biological networks with clear links to human disease and a more comprehensive understanding of its function is needed to maximise its use in medical practice. This review focuses on the complexity of our genome and the impact of sequencing technologies in understanding its many products and functions in health and disease.
... However, studies of many bacteria, animals and plants have revealed an unusual group of RNAs that have both protein coding and non-coding roles. These RNAs have been referred to as 'dual function' or 'bi-functional' RNAs (Dinger et al., 2008;Ulveling et al., 2011b); we refer to them hereafter as 'coding and non-coding RNAs' (cncRNAs) (Kumari and Sampath, 2015). The identification of such cncRNAs raises several interesting questions and poses a challenge to RNA classification. ...
Article
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RNAs are known to regulate diverse biological processes, either as protein-encoding molecules or as non-coding RNAs. However, a third class that comprises RNAs endowed with both protein coding and non-coding functions has recently emerged. Such bi-functional ‘coding and non-coding RNAs’ (cncRNAs) have been shown to play important roles in distinct developmental processes in plants and animals. Here, we discuss key examples of cncRNAs and review their roles, regulation and mechanisms of action during development.
... Biochemistry, the study of chemical processes within and relating to living organisms [1] or chemistry of living matter, deals with the structures, functions and interactions of biological macromolecules. Biochemical indices traverses genetics and molecular biology with emerging novel roles for RNA [2]. Ecology is the branch of biology that deals with relationship among organisms and to their physical surroundings. ...
Article
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Biochemistry, the study of chemical processes within and relating to living organisms or chemistry of living matter, deals with the structures, functions and interactions of biological macromolecules. Biochemical indices traverses genetics and molecular biology with emerging novel roles for RNA.
... [50][51][52][53] The borders between RNA and mRNA are also fuzzy because some bona fide class I RNAs might also encode ORFs, which are occasionally translated 54 (i.e., they are bifunctional f RNAs). 56,57 Likewise, an RNA might be in the process of exaptation as mRNA. 58 An example for a chimera is the bacterial hybrid of a transfer and a mRNA, tmRNA, formerly known as 10Sa RNA, which functions to deblock ribosomes engaged in translating truncated mRNAs devoid of an in-frame stop codon. ...
Article
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Every ribonucleic acid begins its cellular life as a transcript. If the transcript or its processing product has a function it should be regarded an RNA. Nonfunctional transcripts, by-products from processing, degradation intermediates, even from (functional) RNAs, and non-functional products of transcriptional gene regulation accomplished via the act of transcription, as well as stochastic (co)transcripts could be addressed merely as transcripts (class 0). The copious functional RNAs, often maturing after one or more processing steps, can be systematized into ever expanding sub-classifications ranging from micro RNAs to ribosomal RNAs (class I), whereby established sub-classifications addressing a wide functional diversity remain unaffected. Messenger RNAs (class II) are distinct from any other RNA by virtue of their potential to be translated into (poly)peptide(s) on ribosomes. We do not propose a novel RNA classification, but wish to add a basic concept with existing terminology (transcript, RNA, and mRNA) that should serve as an additional framework for carefully delineating RNA function from an avalanche of RNA sequencing data. At the same time, this top level hierarchical model should illuminate important principles of RNA evolution and biology and heighten our awareness that in biology boundaries and categorizations are typically fuzzy.
... One example for such a bi-functional coding mRNA is the Oskar transcript in drosophila, where the 3' UTR is required for oogenesis, independent of the encoded protein [28] which is required for the formation of the posterior pole plasm in the egg. Just a few examples of coding RNAs with noncoding functions were reported so far (reviewed in [29]). Yet, this does not imply that such functions are rare, since effects after invalidation of a coding gene are typically attributed to the protein and most noncoding functions of coding transcripts likely remained undiscovered. ...
Article
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Open reading frames are common in long noncoding RNAs (lncRNAs) and 5’UTRs of protein coding transcripts (uORFs). The question of whether those ORFs are translated was recently addressed by several groups using ribosome profiling. Most of those studies concluded that certain lncRNAs and uORFs are translated, essentially based on computational analysis of ribosome footprints. However, major discrepancies remain on the scope of translation and the translational status of individual ORFs. In consequence, further criteria are required to reliably identify translated ORFs from ribosome profiling data. We examined the effect of the translation inhibitors pateamine A, harringtonine and puromycin on murine ES cell ribosome footprints. We found that pateamine A, a drug that targets eIF4A, allows a far more accurate identification of translated sequences than previously used drugs and computational scoring schemes. Our data show that at least one third but less than two thirds of ES cell lncRNAs are translated. We also identified translated uORFs in hundreds of annotated coding transcripts including key pluripotency transcripts, such as dicer, lin28, trim71, and ctcf. Pateamine A inhibition data clearly increase the precision of the detection of translated ORFs in ribosome profiling experiments. Our data show that translation of lncRNAs and uORFs in murine ES cells is rather common although less pervasive than previously suggested. The observation of translated uORFs in several key pluripotency transcripts suggests that translational regulation by uORFs might be part of the network that defines mammalian stem cell identity.
... Most miRNAs are encoded by independent transcription units [8], located in intergenic regions or hosted within introns of protein-coding or non-coding genes [9][10][11][12][13]. Beside these intronic miRNAs that are potentially transcribed independently of the host genes, novel classes of intronic miRNAs precursors have been reported recently by which production is initiated by splicing of the intron, independently of the microprocessor [10,11,14]. ...
Article
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Currently, the discovery of new small ncRNAs requires high throughput methods even in the case of focused research on the regulation of specific genes or set of genes. We propose herein a simple, rapid, efficient, and cost effective method to clone and sequence single, yet unknown, small ncRNA. This technique that we called “Pocket-sized RNA-Seq” or psRNA-seq is based on in vitro transcription, RNA pull down and adapted RACE-PCR methods that allow its implementation using either available commercial kits or in-house reagents.
... However, the role of mRNAs is not limited to being the source of protein production. An emerging body of evidence suggests that coding mRNAs can have independent functions [8]. In zebrafish, Squint (Sqt), a Nodal-related signaling molecule belonging to the transforming growth factor b (TGF-b) superfamily, is involved in mesoderm induction and left-right axis specification. ...
... Based on transcript size, ncRNAs are categorized into two groups with small ncRNAs, such as microRNAs, snoRNAs, piwiRNAs, etc. being shorter than 200 nts and long ncRNAs (lncRNAs) exceeding 200 nts in length [3]. LncRNAs, normally found as endogenous cellular RNAs, lack an open reading frame of significant length, although few studies have shown their ability to encode proteins [4]. Initially thought to represent transcriptional noise in early RNA-Seq studies [5,6], lncRNAs are now regarded as functional RNA elements, generally expressed at low levels in a tissue-specific and time-restricted manner [5,7], with half-lives varying from less than 2 h to more than 16 h, and a median halflife of 3.5 h [8,9]. ...
Article
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Lung cancer is the leading cause of cancer-related deaths worldwide. Recent advances in whole genome transcriptome analysis have enabled the identification of numerous members of a novel class of non-coding RNAs, i.e., long non-coding RNAs (lncRNAs), which play important roles in a wide range of biological processes and whose deregulation causes human disease, including cancer. Herein we provide a comprehensive survey of lncRNAs associated with lung cancer, with particular focus on the functions that either facilitate or inhibit the progression of lung cancer and the pathways involved. Emerging data on the use of lncRNAs as biomarkers for the diagnosis and prognosis of cancer are also discussed. We cast this information within the wider perspective of lncRNA biogenesis and molecular functions in the cell. Relationships that exist between lncRNAs, genome-wide transcription, and lung cancer are discussed. Deepening our understanding on these processes is critical not only from a mechanistic standpoint, but also for the development of novel biomarkers and effective therapeutic targets for cancer patients. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
... Sometimes the same transcript might act as coding RNA in some circumstances and noncoding in some others [125,126]. Therefore, the co-factors affecting the transcription needs to be studied further to identify the conditions leading to gene expression. A comprehensive integrative and meta-analysis approach is useful in understanding the complex lncRNA mechanisms of action at multiple regulatory levels. ...
Preprint
Long noncoding RNA (lncRNA) are implicated in various genetic diseases and cancer, attributed to their critical role in gene regulation. RNA sequencing is used to capture their transcripts from certain cell types or conditions. For some studies, lncRNA interactions with other biomolecules have also been captured, which can give clues to their mechanisms of action. Complementary \textit{in silico} methods have been proposed to predict non-coding nature of transcripts and to analyze available RNA interaction data. Here we provide a critical review of such methods and identify associated challenges. Broadly, these can be categorized as reference-based and reference-free or \textit{ab initio}, with the former category of methods requiring a comprehensive annotated reference. The \textit{ab initio} methods can make use of machine learning classifiers that are trained on features extracted from sequences, making them suitable to predict novel transcripts, especially in non-model species. Machine learning approaches such as Logistic Regression, Support Vector Machines, Random Forest, and Deep Learning are commonly used. Initial approaches relied on basic sequential features to train the model, whereas the use of secondary structural features appears to be a promising approach for functional annotation. However, adding secondary features will result in model complexities, thus demanding an algorithm that can handle it and furthermore, considerably increasing the utilization of computation resources. Computational strategies combining identification and functional annotation which can be easily customized are currently lacking. These can be of immense value to accelerate research in this class of RNAs.
... Studies have reported that sRNAs can also have proteincoding potential and encode small open reading frames (sORFs) which typically contain between 30 and 100 amino acids (Ulveling et al., 2011;Andrews and Rothnagel, 2014;Li et al., 2014;Ruiz-Orera et al., 2014;Kumari and Sampath, 2015). The portfolio of organisms with functional sORFs include bacteria, yeast, Drosophila, plants and human (Oyama et al., 2004;Kastenmayer et al., 2006;Hanada et al., 2007;Ladoukakis et al., 2011;Aspden et al., 2014;Ruiz-Orera et al., 2014;Shell et al., 2015;Ma et al., 2016). ...
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Ecto- and endo-mycorrhizal colonization of Populus roots have a positive impact on the overall tree health and growth.A complete molecular understanding of these interactions will have important implications for increasing agricultural or forestry sustainability using plant: microbe-based strategies. These beneficial associations entail extensive morphological changes orchestrated by the genetic reprogramming in both organisms.In this study, we performed a comparative analysis of two Populus species (Populus deltoides and P. trichocarpa) that were colonized by either an arbuscular mycorrhizal fungus (AmF), Rhizophagus irregularis or an ectomycorrhizal fungus (EmF), Laccaria bicolor, to describe the small RNA (sRNA) landscape including small open reading frames (sORFs) and micro RNAs (miRNAs) involved in these mutualistic interactions.We identified differential expression of sRNAs that were, to a large extent, (1) within the genomic regions lacking annotated genes in the Populus genome and (2) distinct for each fungal interaction. These sRNAs may be a source of novel sORFs within a genome, and in this regard, we identified potential sORFs encoded by the sRNAs. We predicted a higher number of differentially-expressed miRNAs in P. trichocarpa (4 times more) than in P. deltoides (conserved and novel). In addition, 44 miRNAs were common in P. trichocarpa between the EmF and AmF treatments, and only 4 miRNAs were common in P. deltoides between the treatments. Root colonization by either fungus was more effective in P. trichocarpa than in P. deltoides, thus the relatively few differentiallyexpressed miRNAs predicted in P. deltoides might reflect the extent of the symbiosis. Finally, we predicted several genes targets for the plant miRNAs identified here, including potential fungal gene targets.Our findings shed light on additional molecular tiers with a role in Populus-fungal mutualistic associations and provides a set of potential molecular targets for future enhancement.
... Meanwhile, other work has led to different conclusions, including that lncRNAs are deprived of functional open reading frames (ORFs) [8,9] or that some lncRNAs are actively translated [6,10,11]. Other research reports that some lncRNAs are capable of coding short functional peptides in mice [12,13], and that lncRNAs may be bifunctional, with coding and non-coding isoforms reacting to cell conditions [14,15]. Unfortunately, the extent to which these ribosome-associated lncRNAs are translated remains unknown. ...
Article
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Background: LncRNAs are long regulatory non-coding RNAs, some of which are arguably predicted to have coding potential. Despite coding potential classifiers that utilize ribosome profiling data successfully detected actively translated regions, they are less sensitive to lncRNAs. Furthermore, lncRNA annotation can be susceptible to false positives obtained from 3' untranslated region (UTR) fragments of mRNAs. Results: To lower these limitations in lncRNA annotation, we present a novel tool TERIUS that provides a two-step filtration process to distinguish between bona fide and false lncRNAs. The first step successfully separates lncRNAs from protein-coding genes showing enhanced sensitivity compared to other methods. To eliminate 3'UTR fragments, the second step takes advantage of the 3'UTR-specific association with regulator of nonsense transcripts 1 (UPF1), leading to refined lncRNA annotation. Importantly, TERIUS enabled the detection of misclassified transcripts in published lncRNA annotations. Conclusions: TERIUS is a robust method for lncRNA annotation, which provides an additional filtration step for 3'UTR fragments. TERIUS was able to successfully re-classify GENCODE and miTranscriptome lncRNA annotations. We believe that TERIUS can benefit construction of extensive and accurate non-coding transcriptome maps in many genomes.
... They are defined as transcripts that do not code for functional peptides and if they have any impact on cellular function, this impact is mediated through the RNA molecule itself. Interestingly, some transcripts both code for functional peptides and have an independent function as an RNA, and thus fall into the category of bifunctional RNAs [23,24]. In terms of their length, lncRNAs range from tens of thousands of nucleotides to around 200 nucleotides, with this lower limit being an arbitrary and functionally inconsequential cutoff proposed merely to help distinguish them from the small cellular non-coding RNAs such as snRNAs and snoRNAs [22,25,26]. ...
Article
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The interferon (IFN) response is a critical and ubiquitous component of the innate immune response to pathogens. Detailed studies in the last decades have elucidated the function of a large number of proteins that mediate the complex signaling pathways and gene expression programs involved in the interferon response. The recent discovery of the long non-coding RNAs (lncRNAs) as a new category of cellular effectors has led to studies aiming to understand the role of these transcripts in the IFN response. Several high throughput studies have shown that a large number of lncRNAs are differentially expressed following IFN stimulation and/or viral infections. In-depth study of a very small fraction of the identified lncRNAs has revealed critical roles for this class of transcripts in the regulation of multiple steps of the IFN response, and pointed to the presence of an extensive RNA-mediated regulatory network during the antiviral response. As the vast majority of the identified potential regulatory lncRNAs remain unstudied, it is highly likely that future studies will reveal a completely new perspective on the regulation of the IFN response, with lncRNA- and protein-mediated regulatory networks coordinating the duration, magnitude, and character of this aspect of the innate immune response. In addition to providing a more complete picture of the IFN response, these studies will likely identify new therapeutic targets that in the long term may impact the therapeutic options available against microbial infections and diseases of the immune system.
... Given the above observations, the class of RNAs endowed with both protein-coding and noncoding functions has been referred to as 'dual-function RNA' (31)(32)(33), 'binary functional RNA (bifunctional RNA)' (8,34) or 'cn-cRNA (this term was proposed as RNA with both coding and noncoding function in 2015)' (35,36). In a sense, the discovery of cncRNA blurred the boundary between 'coding' and 'noncoding' RNAs and led researchers to reconsider the function, evolution and understanding of RNAs (8,25,37,38). ...
Article
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RNA endowed with both protein-coding and noncoding functions is referred to as 'dual-function RNA', 'binary functional RNA (bifunctional RNA)' or 'cncRNA (coding and noncoding RNA)'. Recently, an increasing number of cncRNAs have been identified, including both translated ncRNAs (ncRNAs with coding functions) and untranslated mRNAs (mRNAs with noncoding functions). However, an appropriate database for storing and organizing cncRNAs is still lacking. Here, we developed cncRNAdb, a manually curated database of experimentally supported cncRNAs, which aims to provide a resource for efficient manipulation, browsing and analysis of cncRNAs. The current version of cncRNAdb documents about 2600 manually curated entries of cncRNA functions with experimental evidence, involving more than 2,000 RNAs (including over 1300 translated ncRNAs and over 600 untranslated mRNAs) across over 20 species. In summary, we believe that cncRNAdb will help elucidate the functions and mechanisms of cncRNAs and develop new prediction methods. The database is available at http://www.rna-society.org/cncrnadb/.
... Subsequent evidence of lncRNA encoded micro peptide has also been reported in Human, Mouse, Chicken, Zebrafish, Nematode and other species as well [30][31][32][33]. This raises the question of the true non-coding identity of the lncRNAs or whether they are performing the dual role as bifunctional lncRNAs [34,35]. Small Open Reading Frames (sORFs) which were initially regarded as 'background noise' of proteomics experiments have now been proved to have the potential for coding clinically significant micro peptides [33]. ...
Article
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The recent discovery of long non-coding RNA as a regulatory molecule in the cellular system has altered the concept of the functional aptitude of the genome. Since our publication of the first version of LncRBase in 2014, there has been an enormous increase in the number of annotated lncRNAs of multiple species other than Human and Mouse. LncRBase V.2 hosts information of 549,648 lncRNAs corresponding to six additional species besides Human and Mouse, viz. Rat, Fruitfly, Zebrafish, Chicken, Cow and C.elegans. It provides additional distinct features such as (i) Transcription Factor Binding Site (TFBS) in the lncRNA promoter region, (ii) sub-cellular localization pattern of lncRNAs (iii) lnc-pri-miRNAs (iv) Possible small open reading frames (sORFs) within lncRNA. (v) Manually curated information of interacting target molecules and disease association of lncRNA genes (vi) Distribution of lncRNAs across multiple tissues of all species. Moreover, we have hosted ClinicLSNP within LncRBase V.2. ClinicLSNP has a comprehensive catalogue of lncRNA variants present within breast, ovarian, and cervical cancer inferred from 561 RNA-Seq data corresponding to these cancers. Further, we have checked whether these lncRNA variants overlap with (i)Repeat elements,(ii)CGI, (iii)TFBS within lncRNA loci (iv)SNP localization in trait-associated Linkage Disequilibrium(LD) region, (v)predicted the potentially pathogenic variants and (vi)effect of SNP on lncRNA secondary structure. Overall, LncRBaseV.2 is a user-friendly database to survey, search and retrieve information about multi-species lncRNAs. Further, ClinicLSNP will serve as a useful resource for cancer specific lncRNA variants and their related information. The database is freely accessible and available at http://dibresources.jcbose.ac.in/zhumur/lncrbase2/.
... ciRNAs, circular intronic RNAs; circRNAs, circular RNAs; EcircRNAs, exonic circRNAs; IcircRNAs, intronic circRNAs; EIcircRNAs, exonintron circRNAs. circRNAs may possess the ability to act both as coding and ncRNAs, as it has already been reported in the case of the so-called bifunctional RNAs (19). ...
... Both SRA and SRAP have biological functions (32) although, compared to SRA, the physiological function of SRAP has been less studied. Nevertheless, in vitro data reveals that SRAP interacts with some transcription factors and may co-activate or repress their activity depending on the cellular context (33)(34)(35). ...
Article
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Long non-coding RNAs (lncRNA) are emerging as new genetic/epigenetic regulators that can impact almost all physiological functions. Here, we focus on the long non-coding steroid receptor RNA activator (SRA), including new insights into its effects on gene expression, the cell cycle, and differentiation; how these relate to physiology and disease; and the mechanisms underlying these effects. We discuss how SRA acts as an RNA coactivator in nuclear receptor signaling; its effects on steroidogenesis, adipogenesis, and myocyte differentiation; the impact on breast and prostate cancer tumorigenesis; and, finally, its ability to modulate hepatic steatosis through several signaling pathways. Genome-wide analysis reveals that SRA regulates hundreds of target genes in adipocytes and breast cancer cells and binds to thousands of genomic sites in human pluripotent stem cells. Recent studies indicate that SRA acts as a molecular scaffold and forms networks with numerous coregulators and chromatin-modifying regulators in both activating and repressive complexes. We discuss how modifications to SRA's unique stem-loop secondary structure are important for SRA function, and highlight the various SRA isoforms and mutations that have clinical implications. Finally, we discuss the future directions for better understanding the molecular mechanisms of SRA action and how this might lead to new diagnostic and therapeutic approaches.
... Analyzing the ability of ribosomes to associate with lncRNA molecules [80] researcher concluded that some lncRNAs can be translated [36,81] (Figure 2I). Although ribosome profiling supports the interaction of lncRNA with ribosomes the effective presence of small peptides is not guaranteed. ...
Article
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In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts.
... A large fraction of studied lncRNAs lack significant protein-coding capacity, and many are strictly nuclear, precluding a protein-mediated function (4,6,20,21). Another subset of lncRNAs are translated into functional peptides; however, the RNA transcript itself affects cellular processes in a manner that is not dependent on its protein product (22)(23)(24). LncRNAs are highly heterogeneous in their length, ranging from tens of thousands of nucleotides to only a couple of hundred nucleotides or even fewer, and are defined as functional RNA molecules which are generally longer than and functionally distinct from small noncoding RNA classes such as tRNAs, snRNA, miRNAs, and snoRNAs. ...
Article
Genome-wide analyses in the last decade have uncovered the presence of a large number of long non-protein-coding transcripts which show highly tissue- and state-specific expression patterns. High throughput sequencing analyses in diverse subsets of immune cells have revealed a complex and dynamic expression pattern for these long non-coding RNAs (lncRNAs) which correlate with the functional states of immune cells. While the vast majority of lncRNAs expressed in immune cells remain unstudied, functional studies performed on a small subset have indicated that their state-specific expression pattern frequently has a regulatory impact on the function of immune cells. In vivo and in vitro studies have pointed to the involvement of lncRNAs in a wide variety of cellular processes including both the innate and adaptive immune response through mechanisms ranging from epigenetic and transcriptional regulation to sequestration of functional molecules in subcellular compartments. This review will mainly focus on the role of lncRNAs in CD4+ and CD8+ T cells, which play pivotal roles in adaptive immunity. While lncRNAs play important physiological roles in lymphocytic response to antigenic stimulation, differentiation into effector cells and secretion of cytokines, their dysregulated expression can promote or sustain pathological states such as autoimmunity, chronic inflammation, cancer and viremia. This, together with their highly cell type-specific expression patterns, makes lncRNAs ideal therapeutic targets and underscores the need for additional studies into the role of these understudied transcripts in adaptive immune response.
Chapter
The increasing diversity of biological pathways in which RNA molecules play a role requires different approaches to RNA studies. Among other methods, RNA studies on a single-cell level offer the unique possibility not only to measure transcription kinetics but also to obtain information on time- and space-determined RNA dynamics. The past decade has witnessed significant progress in the development of new methods allowing RNA studies on the level of single cells. These methods, and the results obtained, are discussed in this chapter. Keywords: fluorescence in-situ hybridization (FISH); proximity ligation assay (PLA); molecular beacons (MBs); aptamers; protein complementation
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Long non-coding RNAs (lncRNAs) are a diverse class of RNAs that engage in numerous biological processes across every branch of life. Although initially discovered as mRNA-like transcripts that do not encode proteins, recent studies have revealed features of lncRNAs that further distinguish them from mRNAs. In this Review, we describe special events in the lifetimes of lncRNAs - before, during and after transcription - and discuss how these events ultimately shape the unique characteristics and functional roles of lncRNAs.
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Cancer metastasis is the most critical determinant of overall survival in cancer patients. With the significant advancement in next-generation sequencing technologies, our understanding of mechanism of cancer metastasis has been considerably changed especially after the discovery of noncoding RNAs (ncRNAs). The ncRNAs play critical roles in a variety of cellular processes, including development, differentiation, and tumorigenesis. Remarkably, discovery of a class of ncRNAs named as miRNAs led to a paradigm change in our understanding of gene regulation and function. Moreover, long noncoding RNAs (lncRNAs), another type of ncRNAs, have recently depicted their role as drivers of tumorigenesis by regulating tumor suppressive and oncogenic pathways. In this chapter, we describe metastasis and introduce ncRNAs with major focus on lncRNAs and outline a detailed description of few cancer metastasis-associated lncRNAs (MALAT-1, HOTAIR, BC200, and SRA RNA). Overall, a complete understanding of the genomic landscape of metastasis will generate new opportunities in early detection and therapeutic intervention. © 2013 Springer Science+Business Media New York. All rights are reserved.
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Recent advances in genome-wide analysis have revealed that 66% of the genome is actively transcribed into noncoding RNAs (ncRNAs) while less than 2% of the sequences encode proteins. Among ncRNAs, high-resolution microarray and massively parallel sequencing technologies have identified long ncRNAs (>200 nucleotides) that lack coding protein function. LncRNAs abundance, nuclear location, and diversity allow them to create in association with protein interactome, a complex regulatory network orchestrating cellular phenotypic plasticity via modulation of all levels of protein-coding gene expression. Whereas lncRNAs biological functions and mechanisms of action are still not fully understood, accumulating data suggest that lncRNAs deregulation is pivotal in cancer initiation and progression and metastatic spread through various mechanisms, including epigenetic effectors, alternative splicing, and microRNA-like molecules. Mounting data suggest that several lncRNAs expression profiles in malignant tumors are associated with prognosis and they can be detected in biological fluids. In this review, we will briefly discuss characteristics and functions of lncRNAs, their role in carcinogenesis, and their potential usefulness as diagnosis and prognosis biomarkers and novel therapeutic targets.
Chapter
Introduction Cystic fibrosis transmembrane conductance regulator Mutations in CFTR Why is cystic fibrosis so common? Animal models of Cystic fibrosis Pharmacotherapy Gene therapy Conclusion References
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The unique characteristics of chicken primordial germ cells (PGCs) provide potential strategies for transgenic animal generation; however, insufficient PGC availability has limited their application. Regulation of bone morphogenic protein 4 (BMP4), a crucial factor for PGCs formation, may provide new strategies for PGC generation. We here identify a long noncoding RNA (lncRNA) that targets BMP4 (LncBMP4). LncBMP4 has similar functions as BMP4, in that it facilitates the formation and migration of PGCs. LncBMP4 promotes BMP4 expression by adsorbing the miRNA gga-mir-12211, thus reducing its inhibitory effect on BMP4 expression. In addition, the small peptide EPC5 encoded by LncBMP4 promotes the transcription of BMP4. The competing endogenous RNA (ceRNA) effect of LncBMP4 requires N6-methyladenosine (m6A) modification, in a dose-dependent manner, and high levels of m6A modification hinder EPC5 translation. Understanding the molecular mechanisms through which LncBMP4 promotes BMP4 expression during PGC formation may provide new avenues for efficient PGC generation.
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There are recent reports of the important functions of long noncoding RNAs (lncRNAs) in female reproductive and ovarian development. Studies in which there was characterization of lncRNAs in the ovaries of laying compared with nesting poultry, however, are limited. In this study, RNA libraries were constructed by obtaining sequencing data of ovarian tissues from laying and nesting Muscovy ducks. In the ovarian tissues of Muscovy ducks, a total of 334 differentially abundant mRNA transcripts (DEGs) and 36 differentially abundant lncRNA transcripts were identified in the nesting period, when compared with during the laying period. These results were subsequently validated by qRT-PCR using nine randomly-selected lncRNAs and six randomly-selected DAMTs. Furthermore, the cis- and trans-regulatory target genes of differentially abundant lncRNA transcripts were identified, and lncRNA-gene interaction networks of 34 differentially abundant lncRNAs and 263 DEGs were constructed. A total of 7601 lncRNAs neighboring 10,542 protein-coding genes were identified and found to be enriched in the Wnt signaling pathway and oocyte meiosis pathways associated with follicular development. Overall, only 11 cis-targets and 57 mRNA-mRNA except trans-targets were involved in the lncRNA-gene interaction networks. Based on the interaction networks, nine DEGs were trans-regulated by differentially abundant lncRNAs and 20 differentially abundant lncRNAs were hypothesized to have important functions in the regulation of broodiness in Muscovy ducks. In this study, a predicted interaction network of differentially abundant lncRNAs and DEGs in Muscovy ducks was constructed for the first time leading to an enhanced understanding of lncRNA and gene interactions regulating broodiness.
Chapter
Over the last two decades it has become clear that RNA is much more than just a boring intermediate in protein expression. Ancient RNAs still appear in the core information metabolism and comprise a surprisingly large component in bacterial gene regulation. A common theme with these types of mostly small RNAs is their reliance of conserved secondary structures. Large scale sequencing projects, on the other hand, have profoundly changed our understanding of eukaryotic genomes. Pervasively transcribed, they give rise to a plethora of large and evolutionarily extremely flexible noncoding RNAs that exert a vastly diverse array of molecule functions. In this chapter we provide a—necessarily incomplete—overview of the current state of comparative analysis of noncoding RNAs, emphasizing computational approaches as a means to gain a global picture of the modern RNA world.
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The diversity of long non-coding RNAs (lncRNAs) in the human transcriptome is in stark contrast to the sparse exploration of their functions concomitant with their conservation and evolution. The pervasive transcription of the largely non-coding human genome makes the evolutionary age and conservation patterns of lncRNAs to a topic of interest. Yet it is a fairly unexplored field and not that easy to determine as for protein-coding genes. Although there are a few experimentally studied cases, which are conserved at the sequence level, most lncRNAs exhibit weak or untraceable primary sequence conservation. Recent studies shed light on the interspecies conservation of secondary structures among lncRNA homologs by using diverse computational methods. This highlights the importance of structure on functionality of lncRNAs as opposed to the poor impact of primary sequence changes. Further clues in the evolution of lncRNAs are given by selective constraints on non-coding gene structures (e.g., promoters or splice sites) as well as the conservation of prevalent spatio-temporal expression patterns. However, a rapid evolutionary turnover is observable throughout the heterogeneous group of lncRNAs. This still gives rise to questions about its functional meaning. For further resources related to this article, please visit the WIREs website.
Thesis
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Les pucerons sont des hémiptères ravageurs des cultures agronomiques particulièrement adaptés à leur environnement. Acyrthosiphon pisum (le puceron du pois) présente un cycle de vie basé sur l’alternance d’une reproduction sexuée ou asexuée en réponse à la photopériode. Ils présentent ainsi un polyphénisme de reproduction aboutissant à la formation de trois phénotypes distincts : femelles asexuées, femelles sexuées, et mâles. Ces derniers étant obtenus par élimination d’un chromosome X, A. pisum est une espèce hétérogamétique mâle présentant un système chromosomique XX chez les femelles et X0 chez les mâles. Le déséquilibre du nombre de chromosome X entre mâles et femelles engendré par cette hétérogamétie nécessite chez certains organismes d’être corrigé par des mécanismes de compensation de dose. Les polyphénismes et compensation de dose impliquent chez d’autres organismes des régulations transcriptionnelles notamment régulées par l’accessibilité de la chromatine.Ma thèse vise ainsi à étudier le polyphénisme de reproduction et la compensation de dose des pucerons sous l’angle d’analyses bio-informatiques de données d’expression des gènes (RNA-seq) et d’accessibilité de la chromatine (FAIRE-seq) dans le but de caractériser l’impact des mécanismes épigénétiques dans ces deux processus biologiques fondamentaux du cycle de vie des pucerons. Les résultats développés dans ma thèse ont permis de montrer d’une part la présence d’une compensation de dose chez le puceron du pois au niveau transcriptomique, supportée par une accessibilité accrue de la chromatine de l’unique X des
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Pervasive transcription of the human genome results in a heterogeneous mix of coding RNAs and long noncoding RNAs (lncRNAs). Only a small fraction of lncRNAs have demonstrated regulatory functions, thus making functional lncRNAs difficult to distinguish from nonfunctional transcriptional byproducts. This difficulty has resulted in numerous competing human lncRNA classifications that are complicated by a steady increase in the number of annotated lncRNAs. To address these challenges, we quantitatively examined transcription, splicing, degradation, localization and translation for coding and noncoding human genes. We observed that annotated lncRNAs had lower synthesis and higher degradation rates than mRNAs and discovered mechanistic differences explaining slower lncRNA splicing. We grouped genes into classes with similar RNA metabolism profiles, containing both mRNAs and lncRNAs to varying extents. These classes exhibited distinct RNA metabolism, different evolutionary patterns and differential sensitivity to cellular RNA-regulatory pathways. Our classification provides an alternative to genomic context-driven annotations of lncRNAs.
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In present study, the next‐generation sequencing technology was employed to development a transcriptome database of gonad and liver from 3‐year‐old male and female Amur sturgeon (Acipenser schrenckii). A total 139,406 unigenes were generated after the Illumina Hiseq. 2500 sequence and assembled by Trinity. The differential expression analysis between male and female obtained 5,199 differentially expressed genes (DEGs) in gonad and 457 DEGs in liver. GO enrich analysis showed that the specific DEGs of gonad play a dominant role in reproductive processes. Although the specific DEGs of liver indicated their primary responsibility for energy metabolism, the DEGs of liver and gonad co‐own enriched in terms associated with reproduction suggested that liver also plays role in sex‐related differences in Amur sturgeon. Furthermore, genes related to sex‐related differences were selected to validate among the four different tissues by real‐time quantitative PCR (qRT‐PCR). In addition, by trans‐acting analysis, a total of 5,206 putative lncRNAs and 3,490 target genes of lncRNAs were predicted from gonad and liver. Moreover, several lncRNAs that targeting Mea1, Piwil1, Tdrd1, Nanos2, Ankrd49, and ZP3 may have potential regulatory effect related to gametogenesis and gonadal differentiation were identified and were validated by qRT‐PCR. These results suggested for the first time that lncRNAs might be one of effect factors on regulation the differential expression of mRNAs associated with sex‐related differences in Amur sturgeon. This article is protected by copyright. All rights reserved.
Article
Transcription and chromatin function are regulated by proteins that bind to DNA, nucleosomes or RNA polymerase II, with specific non-coding RNAs (ncRNAs) functioning to modulate their recruitment or activity. Unlike ncRNAs, nascent pre-mRNA was considered to be primarily a passive player in these processes. In this Opinion article, we describe recently identified interactions between nascent pre-mRNAs and regulatory proteins, highlight commonalities between the functions of nascent pre-mRNA and nascent ncRNA, and propose that both types of RNA have an active role in transcription and chromatin regulation.
Chapter
Non-coding RNAs vary greatly in length, shape and function. Growing interest and recent evidence have identified some of them as essential elements for life, as well as for environmental adaptation and development. Since non-coding RNAs by definition do not code for proteins, their ever-growing roles pose a paradigm shift in biology. In this chapter, we will discuss our current knowledge of two distinct plant non-coding RNAs: long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). These two classes of non-coding transcripts are relatively well characterized in plants, and regulate gene expression through distinct modes of action. We thus anticipate that our current mechanistic knowledge of lncRNAs and miRNAs will provide the basis for future studies of non-coding RNAs in plant genetics and epigenetics.
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[ Main text in French, Articles are in English, published article can be found here https://www.researchgate.net/publication/317602184_Dosage_compensation_and_sex-specific_epigenetic_landscape_of_the_X_chromosome_in_the_pea_aphid ] Abstract: Aphids are hemipterous crops pests that are particularly adapted to their environment. Acyrthosiphon pisum (pea aphid) displays a life cycle based on the alternation of sexual or asexual reproduction in response to photoperiod. They thus exhibit a reproductive polyphenism resulting in the formation of three distinct phenotypes: asexual females, sexual females, and males. The latter being obtained by elimination of an X chromosome, A. pisum is a male heterogametic species with a XX chromosomal system in females and X0 in males. The X chromosome number between males and females caused by this heterogamy requires in some organisms to be corrected by dosage compensation mechanisms. Polyphenisms and dosage compensation both involve in other organisms transcriptional regulations that are notably regulated by the chromatin accessibility regulations. My thesis aims to study the reproductive polyphenism and dosage compensation in aphids in the context of bioinformatic analyzes of gene expression data (RNA-seq) and chromatin accessibility (FAIRE-seq) in order to characterize the impact of epigenetic mechanisms in these two fundamental biological processes of the aphid life cycle. The results developed in my thesis have shown, on one hand, the presence of dose compensation in pea aphid at the transcriptomic level, which is supported by increased chromatin accessibility of the males’ single X in somatic cells. On the other hand, specific sites of chromatin opening between sexual and asexual embryos seem to participate in the definition of their reproduction mode by modulating the expression of certain genes and by allowing the fixation of transcription factors. Their analysis shows the involvement of ecdysone as a new hormonal pathway that may trigger sexual reproduction in response to a short photoperiod. This work suggests the importance of chromatin regulation in aphids’ environmental adaptation and in their life cycle.
Article
Peptides encoded by small open reading frames (sORFs, usually < 100 codons) play critical regulatory roles in plant development and environmental responses. Despite their importance, only a small number of these peptides have been identified and characterized. Genomic studies have revealed that many plant genomes contain thousands of possible sORFs, which could potentially encode small peptides. The challenge is to distinguish translated sORFs from non-translated ones. Here we highlight advances in methodologies for identifying these hidden sORFs in plant genomes, including ribosome profiling and proteomics. We also examine the evidence for new peptides arising from sORFs and discuss their functions in plant development, environmental responses and translational control. This article is protected by copyright. All rights reserved
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The steroid receptor RNA activator (SRA) has the unusual property to function as both a non-coding RNA (ncRNA) and a protein SRAP. SRA ncRNA is known to increase the activity of a range of nuclear receptors as well as the master regulator of muscle differentiation MyoD. The contribution of SRA to either a ncRNA or a protein is influenced by alternative splicing of the first intron, the retention of which disrupts the SRAP open reading frame. We reported here that the ratio between non-coding and coding SRA isoforms increased during myogenic differentiation of human satellite cells but not myotonic dystrophy patient satellite cells, in which differentiation capacity is affected. Using constructs that exclusively produce SRA ncRNA or SRAP, we demonstrated that whereas SRA ncRNA was indeed an enhancer of myogenic differentiation and myogenic conversion of non-muscle cells through the co-activation of MyoD activity, SRAP prevented this SRA RNA-dependant co-activation. Interestingly, the SRAP inhibitory effect is mediated through the interaction of SRAP with its RNA counterpart via its RRM-like domain interacting with the functional sub-structure of SRA RNA, STR7. This study thus provides a new model for SRA-mediated regulation of MyoD transcriptional activity in the promotion of normal muscle differentiation, which takes into account the nature of SRA molecules present.
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Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55-200 CGG repeats in the 5'-UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA-binding proteins by the expanded CGG repeats. Sam68 is an RNA-binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA-binding proteins sequentially, first Sam68, then hnRNP-G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing-regulatory function. Consequently, Sam68-responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain-of-function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.
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Splice-modulation therapy, whereby molecular manipulation of premessenger RNA splicing is engineered to yield genetic correction, is a promising novel therapy for genetic diseases of muscle and nerve-the prototypical example being Duchenne muscular dystrophy. Duchenne muscular dystrophy is the most common childhood genetic disease, affecting one in 3500 newborn boys, causing progressive muscle weakness, heart and respiratory failure and premature death. No cure exists for this disease and a number of promising new molecular therapies are being intensively studied. Duchenne muscular dystrophy arises due to mutations that disrupt the open-reading-frame in the DMD gene leading to the absence of the essential muscle protein dystrophin. Of all novel molecular interventions currently being investigated for Duchenne muscular dystrophy, perhaps the most promising method aiming to restore dystrophin expression to diseased cells is known as 'exon skipping' or splice-modulation, whereby antisense oligonucleotides eliminate the deleterious effects of DMD mutations by modulating dystrophin pre-messenger RNA splicing, such that functional dystrophin protein is produced. Recently this method was shown to be promising and safe in clinical trials both in The Netherlands and the UK. These trials studied direct antisense oligonucleotide injections into single peripheral lower limb muscles, whereas a viable therapy will need antisense oligonucleotides to be delivered systemically to all muscles, most critically to the heart, and ultimately to all other affected tissues including brain. There has also been considerable progress in understanding how such splice-correction methods could be applied to the treatment of related neuromuscular diseases, including spinal muscular atrophy and myotonic dystrophy, where defects of splicing or alternative splicing are closely related to the disease mechanism.
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Recent studies indicate that the majority of the genome is transcribed, however only a fraction of this transcription is annotated as what we traditionally know as genes. Much of this is low level pervasive, and often overlapping transcription, for which the transcript structure and function, has not as yet been determined. Novel RNAs (and novel classes of RNAs), are now being identified by groups using next generation sequencing and cDNA libraries that target specific sub-fractions of the transcriptome (based upon size, modifications, localization and protein interactions). We discuss the state of the art as to measuring and identifying these novel RNAs and speculate on whether a universal approach to transcript isoform and expression level measurement is possible in the near future.
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Genome-wide analyses of the eukaryotic transcriptome have revealed that the majority of the genome is transcribed, producing large numbers of non-protein-coding RNAs (ncRNAs). This surprising observation challenges many assumptions about the genetic programming of higher organisms and how information is stored and organized within the genome. Moreover, the rapid advances in genomics have given little opportunity for biologists to integrate these emerging findings into their intellectual and experimental frameworks. This problem has been compounded by the perception that genome-wide studies often generate more questions than answers, which in turn has led to confusion and controversy. In this article, we address common questions associated with the phenomenon of pervasive transcription and consider the indices that can be used to evaluate the function (or lack thereof) of the resulting ncRNAs. We suggest that many lines of evidence, including expression profiles, conservation signatures, chromatin modification patterns and examination of increasing numbers of individual cases, argue in favour of the widespread functionality of non-coding transcription. We also discuss how informatic and experimental approaches used to analyse protein-coding genes may not be applicable to ncRNAs and how the general perception that protein-coding genes form the main informational output of the genome has resulted in much of the misunderstanding surrounding pervasive transcription and its potential significance. Finally, we present the conceptual implications of the majority of the eukaryotic genome being functional and describe how appreciating this perspective will provide considerable opportunity to further understand the molecular basis of development and complex diseases.
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During the past few years, it has become increasingly evident that the expression of eukaryotic genomes is far more complex than had been previously noted. The idea that the transcriptome is derived exclusively from protein-coding genes and some specific non-coding RNAs--such as snRNAs, snoRNAs, tRNAs or rRNAs--has been swept away by numerous studies indicating that RNA polymerase II can be found at almost any genomic location. Pervasive transcription is widespread and, far from being a futile process, has a crucial role in controlling gene expression and genomic plasticity. Here, we review recent findings that point to cryptic transcription as a fundamental component of the regulation of eukaryotic genomes.
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Differentiation of female murine ES cells triggers silencing of one X chromosome through X-chromosome inactivation (XCI). Immunofluorescence studies showed that soon after Xist RNA coating the inactive X (Xi) undergoes many heterochromatic changes, including the acquisition of H3K27me3. However, the mechanisms that lead to the establishment of heterochromatin remain unclear. We first analyze chromatin changes by ChIP-chip, as well as RNA expression, around the X-inactivation center (Xic) in female and male ES cells, and their day 4 and 10 differentiated derivatives. A dynamic epigenetic landscape is observed within the Xic locus. Tsix repression is accompanied by deposition of H3K27me3 at its promoter during differentiation of both female and male cells. However, only in female cells does an active epigenetic landscape emerge at the Xist locus, concomitant with high Xist expression. Several regions within and around the Xic show unsuspected chromatin changes, and we define a series of unusual loci containing highly enriched H3K27me3. Genome-wide ChIP-seq analyses show a female-specific quantitative increase of H3K27me3 across the X chromosome as XCI proceeds in differentiating female ES cells. Using female ES cells with nonrandom XCI and polymorphic X chromosomes, we demonstrate that this increase is specific to the Xi by allele-specific SNP mapping of the ChIP-seq tags. H3K27me3 becomes evenly associated with the Xi in a chromosome-wide fashion. A selective and robust increase of H3K27me3 and concomitant decrease in H3K4me3 is observed over active genes. This indicates that deposition of H3K27me3 during XCI is tightly associated with the act of silencing of individual genes across the Xi.
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Non-coding RNAs are emerging as key players in many fundamental biological processes, including specification of higher-order chromatin structure. We examined the implication of RNA transcribed from mouse centromeric minor satellite repeats in the formation and function of centromere-associated complexes. Here we show that the levels of minor satellite RNA vary during cell-cycle progression, peaking in G2/M phase, concomitant with accumulation of proteins of the chromosomal passenger complex near the centromere. Consistent with this, we describe that murine minor satellite RNA are components of CENP-A-associated centromeric fractions and associate with proteins of the chromosomal passenger complex Aurora B and Survivin at the onset of mitosis. Interactions of endogenous Aurora B with CENP-A and Survivin are sensitive to RNaseA. Likewise, the kinase activity of Aurora B requires an RNA component. More importantly, Aurora B kinase activity can be potentiated by minor satellite RNA. In addition, decreased Aurora B activity after RNA depletion can be specifically rescued by restitution of these transcripts. Together, our data provide new functional evidence for minor satellite transcripts as key partners and regulators of the mitotic kinase Aurora B.
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Sugar phosphate stress in Escherichia coli is sensed and managed by the transcriptional regulator SgrR and the small RNA (sRNA) SgrS. SgrS is a dual function RNA that performs base pairing-dependent regulation of mRNA targets and encodes a small protein, SgrT. Homologs of SgrR were analyzed for gene synteny and inter-homolog identity to identify those that are likely to be functionally analogous. These 22 SgrR homologs were used to manually locate adjacent sRNAs functionally analogous to SgrS. SgrS homologs shared little sequence identity with E. coli SgrS, but most shared several structural features. The most conserved feature of SgrS homologs was the base pairing region while the most variable feature was the sgrT-coding sequence. Analyses of predicted interactions between SgrS:ptsG mRNA pairs in different organisms revealed interesting differences in the patterns of base pairing interactions. RNA pairs with more interrupted regions of complementarity had a higher proportion of G:C base pairs than those with longer contiguous stretches of complementarity. The identification of this set of homologous sRNAs and their targets sets the stage for future studies to further elucidate the molecular requirements for regulation by SgrS.
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Products of the Steroid Receptor RNA Activator gene (SRA1) have the unusual property to modulate the activity of steroid receptors and other transcription factors both at the RNA (SRA) and the protein (SRAP) level. Balance between these two genetically linked entities is controlled by alternative splicing of intron-1, whose retention alters SRAP reading frame. We have previously found that both fully-spliced SRAP-coding and intron-1-containing non-coding SRA RNAs co-exist in breast cancer cell lines. Herein, we report a significant (Student's t-test, P < 0.003) higher SRA–intron-1 relative expression in breast tumors with higher progesterone receptor contents. Using an antisense oligoribonucleotide, we have successfully reprogrammed endogenous SRA splicing and increased SRA RNA–intron-1 relative level in T5 breast cancer cells. This increase is paralleled by significant changes in the expression of genes such as plasminogen urokinase activator and estrogen receptor beta. Estrogen regulation of other genes, including the anti-metastatic NME1 gene, is also altered. Overall, our results suggest that the balance coding/non-coding SRA transcripts not only characterizes particular tumor phenotypes but might also, through regulating the expression of specific genes, be involved in breast tumorigenesis and tumor progression.
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Controlling protein-coding gene expression can no longer be attributed purely to proteins involved in transcription, RNA processing, and translation. The role that noncoding RNAs (ncRNAs) play as potent and specific regulators of gene expression is now widely recognized in almost all species studied to date. Long ncRNAs can both upregulate and downregulate gene expression in both eukaryotes and prokaryotes and are essential in processes such as dosage compensation, genomic imprinting, developmental patterning and differentiation, and stress response. Small ncRNAs also play essential roles in diverse organisms, although are limited to eukaryotes. Different small RNA classes regulate diverse processes such as transposon and virus suppression, as well as many key developmental processes.
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In mammals, imprinted genes are clustered and at least one gene in each imprinted cluster is a long i.e., macro non-coding (nc) RNA. Most genes in a cluster show concordant parental-specific expression but the ncRNA is the odd one out, and is expressed from the opposite parental chromosome. While reciprocal expression between imprinted macro non-coding RNAs and flanking mRNA genes is indicative of a functional role, only two of three tested macro ncRNAs have been shown to induce imprinted gene expression. The two known functional imprinted macro non-coding RNAs are both RNAPII transcripts with unusual transcriptional properties that may be functionally relevant and their analysis may shed light on the function of non-coding RNAs that have been shown to comprise the majority of the mammalian transcriptome.
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Spinal muscular atrophy (SMA) is a motor neuron disease caused by the loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients, which produces low levels of functional protein. Although the SMN2 coding sequence has the potential to produce normal, full-length SMN, approximately 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein lacking the final coding exon (exon 7). SMN2, however, is an excellent therapeutic target. Previously, we developed bifunctional RNAs that bound SMN exon 7 and modulated SMN2 splicing. To optimize the efficiency of the bifunctional RNAs, a different antisense target was required. To this end, we genetically verified the identity of a putative intronic repressor and developed bifunctional RNAs that target this sequence. Consequently, there is a 2-fold mechanism of SMN induction: inhibition of the intronic repressor and recruitment of SR proteins via the SR recruitment sequence of the bifunctional RNA. The bifunctional RNAs effectively increased SMN in human primary SMA fibroblasts. Lead candidates were synthesized as 2'-O-methyl RNAs and were directly injected in the central nervous system of SMA mice. Single-RNA injections were able to illicit a robust induction of SMN protein in the brain and throughout the spinal column of neonatal SMA mice. In a severe model of SMA, mean life span was extended following the delivery of bifunctional RNAs. This technology has direct implications for the development of an SMA therapy, but also lends itself to a multitude of diseases caused by aberrant pre-mRNA splicing.
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mRNA localization, and translation that is regulated spatially and temporally, are key mechanisms in the execution of polarized developmental programs. For over two decades, the Drosophila oocyte has served as a valuable model to study these mechanisms. Genetic and biochemical studies in flies have greatly contributed to the identification and understanding of factors that govern RNA localization and translational control. Embryonic axis formation is mediated through the subcellular localization and precise translational regulation of four key determinant mRNAs during oogenesis encoded by oskar, bicoid, gurken and nanos. In this review we aim to summarize recent insights into the mechanisms governing the asymmetric distribution and translation of these mRNAs.
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The E3 ubiquitin ligase Mdm2 is a focal regulator of p53 tumour suppressor activity. It binds p53, promoting its polyubiquitination and degradation, and also controls p53 synthesis. However, it is not known how this dual function of Mdm2 on p53 synthesis and degradation is achieved. Here we show that the p53 mRNA region encoding the Mdm2-binding site interacts directly with the RING domain of Mdm2. This impairs the E3 ligase activity of Mdm2 and promotes p53 mRNA translation. We also show that introduction of cancer-derived single silent point-mutations in the p53 mRNA weakens its binding to Mdm2 and results in reduced p53 activity. These data are consistent with a mechanism by which changes in silent nucleotides can affect the function of the encoded protein, and indicate that Mdm2-mediated control of p53 synthesis and degradation has evolved in the p53 mRNA sequence and its encoded amino acids.
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The E3 ligase Mdm2 is a key regulator of p53 activity via a complex regulatory feedback system that involves all levels of expression control including transcription, mRNA translation and protein degradation. Best known is the effect of p53 on Mdm2 transcription and the capacity of Mdm2 to target p53 for degradation, but more recently the role of Mdm2 as a positive regulator of p53 activity has also started to emerge. Mdm2 stimulates p53 mRNA translation by binding the p53 mRNA and, interestingly, this interaction also suppresses Mdm2's capacity to promote p53 polyubiquitination and degradation. Another interesting aspect of the p53 mRNA-Mdm2 interaction is that the p53 mRNA sequence encoding the amino acids which bind the N-terminus of Mdm2 is the same that interacts with the Mdm2 RING domain. Indeed, the regulatory elements for controlling Mdm2-dependent expression of p53 are derived from the same p53 genomic sequence. In addition, the RNA binding and the E3 ligase domain of Mdm2 overlap, indicati that the two functions of Mdm2 to control p53 synthesis and degradation have co-evolved in parallel in both p53 and Mdm2. Here we illustrate how the p53-Mdm2 protein-protein and p53 mRNA-Mdm2 interactions affect Mdm2-mediated control of p53 expression using the Phe19Ala p53 mutant. We discuss how the new insights into the regulation of p53 expression levels can help to shed light on the origin of this elegant feedback system and on the function of Mdm2 isoforms.
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The assumption that RNA can be readily classified into either protein-coding or non-protein-coding categories has pervaded biology for close to 50 years. Until recently, discrimination between these two categories was relatively straightforward: most transcripts were clearly identifiable as protein-coding messenger RNAs (mRNAs), and readily distinguished from the small number of well-characterized non-protein-coding RNAs (ncRNAs), such as transfer, ribosomal, and spliceosomal RNAs. Recent genome-wide studies have revealed the existence of thousands of noncoding transcripts, whose function and significance are unclear. The discovery of this hidden transcriptome and the implicit challenge it presents to our understanding of the expression and regulation of genetic information has made the need to distinguish between mRNAs and ncRNAs both more pressing and more complicated. In this Review, we consider the diverse strategies employed to discriminate between protein-coding and noncoding transcripts and the fundamental difficulties that are inherent in what may superficially appear to be a simple problem. Misannotations can also run in both directions: some ncRNAs may actually encode peptides, and some of those currently thought to do so may not. Moreover, recent studies have shown that some RNAs can function both as mRNAs and intrinsically as functional ncRNAs, which may be a relatively widespread phenomenon. We conclude that it is difficult to annotate an RNA unequivocally as protein-coding or noncoding, with overlapping protein-coding and noncoding transcripts further confounding this distinction. In addition, the finding that some transcripts can function both intrinsically at the RNA level and to encode proteins suggests a false dichotomy between mRNAs and ncRNAs. Therefore, the functionality of any transcript at the RNA level should not be discounted.
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The peptide hormone ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, gut motility and proliferation of cancer cells. We previously identified a gene on the opposite strand of the ghrelin gene, ghrelinOS (GHRLOS), which spans the promoter and untranslated regions of the ghrelin gene (GHRL). Here we further characterise GHRLOS. We have described GHRLOS mRNA isoforms that extend over 1.4 kb of the promoter region and 106 nucleotides of exon 4 of the ghrelin gene, GHRL. These GHRLOS transcripts initiate 4.8 kb downstream of the terminal exon 4 of GHRL and are present in the 3' untranslated exon of the adjacent gene TATDN2 (TatD DNase domain containing 2). Interestingly, we have also identified a putative non-coding TATDN2-GHRLOS chimaeric transcript, indicating that GHRLOS RNA biogenesis is extremely complex. Moreover, we have discovered that the 3' region of GHRLOS is also antisense, in a tail-to-tail fashion to a novel terminal exon of the neighbouring SEC13 gene, which is important in protein transport. Sequence analyses revealed that GHRLOS is riddled with stop codons, and that there is little nucleotide and amino-acid sequence conservation of the GHRLOS gene between vertebrates. The gene spans 44 kb on 3p25.3, is extensively spliced and harbours multiple variable exons. We have also investigated the expression of GHRLOS and found evidence of differential tissue expression. It is highly expressed in tissues which are emerging as major sites of non-coding RNA expression (the thymus, brain, and testis), as well as in the ovary and uterus. In contrast, very low levels were found in the stomach where sense, GHRL derived RNAs are highly expressed. GHRLOS RNA transcripts display several distinctive features of non-coding (ncRNA) genes, including 5' capping, polyadenylation, extensive splicing and short open reading frames. The gene is also non-conserved, with differential and tissue-restricted expression. The overlapping genomic arrangement of GHRLOS with the ghrelin gene indicates that it is likely to have interesting regulatory and functional roles in the ghrelin axis.
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The H19/IGFf2 locus belongs to a large imprinted domain located on human chromosome 11p15.5 (homologue to mouse distal chromosome 7). The H19 gene is expressed from the maternal allele, while IGF2 is paternally expressed. Natural antisense transcripts and intergenic transcription have been involved in many aspects of eukaryotic gene expression, including genomic imprinting and RNA interference. However, apart from the identification of some IGF2 antisense transcripts, few data are available on that topic at the H19/IGF2 locus. We identify here a novel transcriptional activity at both the human and the mouse H19/IGF2 imprinted loci. This activity occurs antisense to the H19 gene and has the potential to produce a single 120-kb transcript that we called the 91H RNA. This nuclear and short-lived RNA is not imprinted in mouse but is expressed predominantly from the maternal allele in both mice and humans within the H19 gene region. Moreover, the transcript is stabilized in breast cancer cells and overexpressed in human breast tumors. Finally, knockdown experiments showed that, in humans, 91H, rather than affecting H19 expression, regulates IGF2 expression in trans.
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RNAIII, an RNA molecule shown to encode δ-hemolysin and independently to regulate toxin synthesis in Staphylococcus aureus, is transcribed at the mid-exponential phase of growth, while its target genes are activated 2 h later, at the post-exponential phase of growth. We show here that the translation of RNAIII to the 26-amino acid peptide δ-hemolysin is delayed by 1 h, and that this delay is abolished when the 3′-end of this molecule is deleted. We suggest that structural changes of RNAIII to a translatable form of the molecule precede its regulation of target gene expression.
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Intronless genes can arise by germline retrotransposition of a cDNA originating as mRNA from an intron-containing source gene. Previously, we described several members of a family of intronless mammalian genes encoding a novel class of zinc-finger proteins, including one that shows imprinted expression and one that escapes X-inactivation. We report here the identification and characterization of the Makorin ring finger protein 1 gene (MKRN1), a highly transcribed, intron-containing source for this family of genes. Phylogenetic analyses clearly indicate that the MKRN1 gene is the ancestral founder of this gene family. We have identified MKRN1 orthologs from human, mouse, wallaby, chicken, fruitfly, and nematode, underscoring the age and conservation of this gene. The MKRN gene family encodes putative ribonucleoproteins with a distinctive array of zinc-finger motifs, including two to four C3H zinc-fingers, an unusual Cys/His arrangement that may represent a novel zinc-finger structure, and a highly conserved RING zinc-finger. To date, we have identified nine MKRN family loci distributed throughout the human genome. The human and mouse MKRN1 loci map to a conserved syntenic group near the T-cell receptor β cluster (TCRB) in chromosome 7q34–q35 and chromosome 6A, respectively. MKRN1 is widely transcribed in mammals, with high levels in murine embryonic nervous system and adult testis. The ancient origin of MKRN1, high degree of conservation, and expression pattern suggest important developmental and functional roles for this gene and its expressed family members.
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Activation of p53 can occur in response to a number of cellular stresses, including DNA damage, hypoxia and nucleotide deprivation. Several forms of DNA damage have been shown to activate p53, including those generated by ionising radiation (IR), radio-mimetic drugs, ultraviolet light (UV) and chemicals such as methyl methane sulfonate (MMS). Under normal conditions, p53 levels are maintained at a low state by virtue of the extremely short-half life of the polypeptide. In addition to this, p53 normally exists in an largely inactive state that is relatively inefficient at binding to DNA and activating transcription. Activation of p53 in response to DNA damage is associated with a rapid increase in its levels and with an increased ability of p53 to bind DNA and mediate transcriptional activation. This then leads to the activation of a number of genes whose products trigger cell-cycle arrest, apoptosis, or DNA repair. Recent work has suggested that this regulation is brought about largely through DNA damage triggering a series of phosphorylation, de-phosphorylation and acetylation events on the p53 polypeptide. Here, we discuss the nature of these modifications, the enzymes that bring them about, and how changes in p53 modification lead to p53 activation.
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To study the genome-wide impact of transposable elements (TEs) on the evolution of protein-coding regions, we examined 13 799 human genes and found 533 (∼4%) cases of TEs within protein-coding regions. The majority of these TEs (∼89.5%) reside within ‘introns’ and were recruited into coding regions as novel exons. We found that TE integration often has an effect on gene function. In particular, there were two mouse genes whose coding regions consist largely of TEs, suggesting that TE insertion might create new genes. Thus, there is increasing evidence for an important role of TEs in gene evolution. Because many TEs are taxon-specific, their integration into coding regions could accelerate species divergence.