Terminating transcription is a highly intricate process for mammalian protein-coding genes. First, the chromatin template slows down transcription at the gene end. Then, the transcript is cleaved at ...the poly(A) signal to release the messenger RNA. The remaining transcript is selectively unraveled and degraded. This induces critical conformational changes in the heart of the enzyme that trigger termination. Termination can also occur at variable positions along the gene and so prevent aberrant transcript formation or intentionally make different transcripts. These may form multiple messenger RNAs with altered regulatory properties or encode different proteins. Finally, termination can be perturbed to achieve particular cellular needs or blocked in cancer or virally infected cells. In such cases, failure to terminate transcription can spell disaster for the cell.
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BFBNIB, NMLJ, NUK, ODKLJ, PNG, SAZU, UL, UM, UPUK
Polyadenylation poly(A) signals (PAS) are a defining feature of eukaryotic protein-coding genes. The central sequence motif AAUAAA was identified in the mid-1970s and subsequently shown to require ...flanking, auxiliary elements for both 3'-end cleavage and polyadenylation of premessenger RNA (pre-mRNA) as well as to promote downstream transcriptional termination. More recent genomic analysis has established the generality of the PAS for eukaryotic mRNA. Evidence for the mechanism of mRNA 3'-end formation is outlined, as is the way this RNA processing reaction communicates with RNA polymerase II to terminate transcription. The widespread phenomenon of alternative poly(A) site usage and how this interrelates with pre-mRNA splicing is then reviewed. This shows that gene expression can be drastically affected by how the message is ended. A central theme of this review is that while genomic analysis provides generality for the importance of PAS selection, detailed mechanistic understanding still requires the direct analysis of specific genes by genetic and biochemical approaches.
The concept of early termination as an important means of transcriptional control has long been established. Even so, its role in metazoan gene expression is underappreciated. Recent technological ...advances provide novel insights into premature transcription termination (PTT). This process is frequent, widespread, and can occur close to the transcription start site (TSS), or within the gene body. Stable prematurely terminated transcripts contribute to the transcriptome as instances of alternative polyadenylation (APA). Independently of transcript stability and function, premature termination opposes the formation of full-length transcripts, thereby negatively regulating gene expression, especially of transcriptional regulators. Premature termination can be beneficial or harmful, depending on its context. As a result, multiple factors have evolved to control this process.
PTT is widespread in metazoans. It can occur close to the TSS or further downstream in the gene body.PTT generates transcripts that, depending on the circumstances, are either rapidly degraded, or are stabilised by polyadenylation, thus contributing to transcriptome diversification.Stable premature transcripts can have independent functions as noncoding (nc)RNA or mRNA encoding proteins with different properties compared with those generated by the full-length transcript.PTT can negatively regulate expression of the full-length transcript and especially controls genes encoding transcriptional regulators.Factors triggering PTT include not only canonical RNA 3′ processing and termination factors, but also other players. Many metazoan factors oppose PTT, thus limiting its damaging potential.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Widespread antisense long noncoding RNA (lncRNA) overlap with many protein-coding genes in mammals and emanate from gene promoter, enhancer, and termination regions. However, their origin and ...biological purpose remain unclear. We show that these antisense lncRNA can be generated by R-loops that form when nascent transcript invades the DNA duplex behind elongating RNA polymerase II (Pol II). Biochemically, R-loops act as intrinsic Pol II promoters to induce de novo RNA synthesis. Furthermore, their removal across the human genome by RNase H1 overexpression causes the selective reduction of antisense transcription. Consequently, we predict that R-loops act to facilitate the synthesis of many gene proximal antisense lncRNA. Not only are R-loops widely associated with DNA damage and repair, but we now show that they have the capacity to promote de novo transcript synthesis that may have aided the evolution of gene regulation.
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•R-loops formed within plasmids promote antisense transcription in nuclear extracts•TSS of lncRNA and eRNA are often near R-loop structures and sensitive to RNase H1•Preinitiation complexes associated with lncRNA synthesis are R-loop dependent•Many mammalian lncRNA derive from R-loop promoter activity
Tan-Wong et al. demonstrate that R-loop structures, often formed at the promoter, terminator, and enhancer regions of human protein-coding genes, act as promoters to generate antisense lncRNA. In effect, R-loop promoter activity may account for the existence of many lncRNA that are detected across mammalian genomes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Numerous long intervening noncoding RNAs (lincRNAs) are generated from the mammalian genome by RNA polymerase II (Pol II) transcription. Although multiple functions have been ascribed to lincRNAs, ...their synthesis and turnover remain poorly characterized. Here, we define systematic differences in transcription and RNA processing between protein-coding and lincRNA genes in human HeLa cells. This is based on a range of nascent transcriptomic approaches applied to different nuclear fractions, including mammalian native elongating transcript sequencing (mNET-seq). Notably, mNET-seq patterns specific for different Pol II CTD phosphorylation states reveal weak co-transcriptional splicing and poly(A) signal-independent Pol II termination of lincRNAs as compared to pre-mRNAs. In addition, lincRNAs are mostly restricted to chromatin, since they are rapidly degraded by the RNA exosome. We also show that a lincRNA-specific co-transcriptional RNA cleavage mechanism acts to induce premature termination. In effect, functional lincRNAs must escape from this targeted nuclear surveillance process.
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•lincRNAs and pre-mRNAs are transcribed by different Pol II phospho-CTD isoforms•lincRNAs are rarely spliced and mainly non-polyadenylated•lincRNAs are stabilized in the nucleoplasm following exosome inactivation•lincRNAs are co-transcriptionally cleaved
Schlackow and Nojima et al. show that mammalian pre-mRNAs and long intergenic noncoding (linc) RNAs employ radically different transcription and RNA-processing strategies. Pre-mRNAs are transcribed by defined RNA polymerase (Pol) II isoforms reflecting co-transcriptional splicing and polyadenylation. Instead, lincRNAs are mainly transcribed by deregulated Pol II and simultaneously degraded.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The pathway from gene activation in the nucleus to mRNA translation and decay at specific locations in the cytoplasm is both streamlined and highly interconnected. This review discusses how pre-mRNA ...processing, including 5′ cap addition, splicing, and polyadenylation, contributes to both the efficiency and fidelity of gene expression. The connections of pre-mRNA processing to upstream events in transcription and downstream events, including translation and mRNA decay, are elaborate, extensive, and remarkably interwoven.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) ...transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and ...non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.
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•SPT6 promotes the selective distribution of H3K36me3 over protein-coding genes•SPT6 loss leads to formation of extended lncRNAs that are prone to R-loop formation•Deregulated Pol II collides with DNA replisomes on lncRNA genes•Collision between Pol II and DNA replisome leads to cellular senescence
Nojima and colleagues demonstrate that loss of human SPT6 protein elevates levels of noncoding transcription and perturbs its termination. The deregulated noncoding transcription increases R-loop formation with consequent DNA damage, transcription-replication collision, and cellular senescence. This implies that noncoding transcription is restricted to avoid such conflicts in proliferating cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The pathway from gene activation in the nucleus to mRNA translation and decay at specific locations in the cytoplasm is both streamlined and highly interconnected. This review discusses how pre-mRNA ...processing, including 5' cap addition, splicing, and polyadenylation, contributes to both the efficiency and fidelity of gene expression. The connections of pre-mRNA processing to upstream events in transcription and downstream events, including translation and mRNA decay, are elaborate, extensive, and remarkably interwoven.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly ...because of technical limitations in purifying authentic nascent transcripts. We present a new approach to characterize nascent RNA, called polymerase intact nascent transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5′ ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano), and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5′ P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that co-transcriptional splicing either occurs immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.
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•POINT methodology dissects intact nascent RNA processing•Specificity of Xrn2 exonuclease in co-transcriptional RNA degradation•Splicing suppresses Xrn2-dependent premature termination•Different kinetic classes of co-transcriptional splicing in human genes
Sousa-Luís et al. describe tripartite methodology to purify and sequence RNA polymerase II-associated intact nascent transcripts (POINT) from mammalian genomes. POINT-5 distinguishes nascent RNA 5′ end caps from 5′ end cleavage, POINT-seq profiles full-length transcription units, and POINT-nano determines the kinetics of co-transcriptional splicing at a single-molecule level.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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