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.
R loops are three-stranded nucleic acid structures that comprise nascent RNA hybridized with the DNA template, leaving the nontemplate DNA single-stranded. R loops form naturally during transcription ...even though their persistent formation can be a risky outcome with deleterious effects on genome integrity. On the other hand, over the last few years, an increasingly strong case has been built for R loops as potential regulators of gene expression. Therefore, understanding their function and regulation under these opposite situations is essential to fully characterize the mechanisms that control genome integrity and gene expression. Here we review recent findings about these interesting structures that highlight their opposite roles in cellular fitness.
We present a molecular dissection of pause site-dependent transcriptional termination for mammalian RNA polymerase II (Pol II)-transcribed genes. We show that nascent transcripts form RNA/DNA hybrid ...structures (R-loops) behind elongating Pol II and are especially prevalent over G-rich pause sites positioned downstream of gene poly(A) signals. Senataxin, a helicase protein associated with AOA2/ALS4 neurodegenerative disorders, acts to resolve these R-loop structures and by so doing allows access of the 5′–3′ exonuclease Xrn2 at 3′ cleavage poly(A) sites. This affords 3′ transcript degradation and consequent Pol II termination. In effect, R-loops formed over G-rich pause sites, followed by their resolution by senataxin, are key steps in the termination process.
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► Senataxin is required for pause-dependent Pol II termination in human genes ► R-loops accumulate over G-rich pause regions and are required for termination ► R-loops dependent on transcription, functional poly(A) signal, and pause elements ► Senataxin resolves R-loops to allow Xrn2 to mediate Pol II release from the gene
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.
Transcription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide ...resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5'splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.
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.
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.
The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also ...associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites.
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•Endogenous BRCA1 and senataxin (SETX) interact in a BRCA1-driven process•BRCA1/SETX complexes are recruited to R-loop-associated termination regions (TRs)•BRCA1/SETX complexes suppress transcriptional DNA damage arising at nearby R-loops•BRCA1 breast cancers reveal indel mutations near BRCA1 TR binding regions
Transcriptional R-loops represent a potential threat to genome integrity. Hatchi et al. show that BRCA1, in partnership with SETX, is engaged in a DNA repair mechanism that deals with R-loop-associated genomic instability at transcriptional termination pause sites.
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.
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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