Publications by authors named "Nick J Proudfoot"

51 Publications

POINT technology illuminates the processing of polymerase-associated intact nascent transcripts.

Mol Cell 2021 Mar 9. Epub 2021 Mar 9.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK; Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address:

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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http://dx.doi.org/10.1016/j.molcel.2021.02.034DOI Listing
March 2021

Dual RNA 3'-end processing of H2A.X messenger RNA maintains DNA damage repair throughout the cell cycle.

Nat Commun 2021 01 13;12(1):359. Epub 2021 Jan 13.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.

Phosphorylated H2A.X is a critical chromatin marker of DNA damage repair (DDR) in higher eukaryotes. However, H2A.X gene expression remains relatively uncharacterised. Replication-dependent (RD) histone genes generate poly(A)- mRNA encoding new histones to package DNA during replication. In contrast, replication-independent (RI) histone genes synthesise poly(A)+ mRNA throughout the cell cycle, translated into histone variants that confer specific epigenetic patterns on chromatin. Remarkably H2AFX, encoding H2A.X, is a hybrid histone gene, generating both poly(A)+ and poly(A)- mRNA isoforms. Here we report that the selective removal of either mRNA isoform reveals different effects in different cell types. In some cells, RD H2A.X poly(A)- mRNA generates sufficient histone for deposition onto DDR associated chromatin. In contrast, cells making predominantly poly(A)+ mRNA require this isoform for de novo H2A.X synthesis, required for efficient DDR. This highlights the importance of differential H2A.X mRNA 3'-end processing in the maintenance of effective DDR.
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http://dx.doi.org/10.1038/s41467-020-20520-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7807067PMC
January 2021

R-Loops Promote Antisense Transcription across the Mammalian Genome.

Mol Cell 2019 11 31;76(4):600-616.e6. Epub 2019 Oct 31.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address:

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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http://dx.doi.org/10.1016/j.molcel.2019.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868509PMC
November 2019

Transcriptional Control by Premature Termination: A Forgotten Mechanism.

Trends Genet 2019 08 15;35(8):553-564. Epub 2019 Jun 15.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.

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.
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http://dx.doi.org/10.1016/j.tig.2019.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471841PMC
August 2019

Selective Roles of Vertebrate PCF11 in Premature and Full-Length Transcript Termination.

Mol Cell 2019 04 25;74(1):158-172.e9. Epub 2019 Feb 25.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address:

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3' mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
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http://dx.doi.org/10.1016/j.molcel.2019.01.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458999PMC
April 2019

Biosynthesis of histone messenger RNA employs a specific 3' end endonuclease.

Elife 2018 12 3;7. Epub 2018 Dec 3.

Department of Chemistry, University of Oxford, Oxford, United Kingdom.

Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo β-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target.
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http://dx.doi.org/10.7554/eLife.39865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303110PMC
December 2018

Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence.

Mol Cell 2018 12 15;72(6):970-984.e7. Epub 2018 Nov 15.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address:

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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http://dx.doi.org/10.1016/j.molcel.2018.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309921PMC
December 2018

Influenza Virus Mounts a Two-Pronged Attack on Host RNA Polymerase II Transcription.

Cell Rep 2018 05;23(7):2119-2129.e3

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address:

Influenza virus intimately associates with host RNA polymerase II (Pol II) and mRNA processing machinery. Here, we use mammalian native elongating transcript sequencing (mNET-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, viral infection causes decreased Pol II gene occupancy downstream of transcription start sites. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(A) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions but also fundamental aspects of mammalian transcription.
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http://dx.doi.org/10.1016/j.celrep.2018.04.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972227PMC
May 2018

WNK1 kinase and the termination factor PCF11 connect nuclear mRNA export with transcription.

Genes Dev 2017 11 1;31(21):2175-2185. Epub 2017 Dec 1.

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.

Nuclear gene transcription is coordinated with transcript release from the chromatin template and messenger RNA (mRNA) export to the cytoplasm. Here we describe the role of nuclear-localized kinase WNK1 (with no lysine [K] 1) in the mammalian mRNA export pathway even though it was previously established as a critical regulator of ion homeostasis in the cytoplasm. Our data reveal that WNK1 phosphorylates the termination factor PCF11 on its RNA polymerase II (Pol II) C-terminal domain (CTD)-interacting domain (CID). Furthermore, phosphorylation of the PCF11 CID weakens its interaction with Pol II. We predict that WNK1 and the associated phosphorylation of the PCF11 CID act to promote transcript release from chromatin-associated Pol II. This in turn facilitates mRNA export to the cytoplasm.
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http://dx.doi.org/10.1101/gad.303677.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749165PMC
November 2017

Distinctive Patterns of Transcription and RNA Processing for Human lincRNAs.

Mol Cell 2017 Jan 22;65(1):25-38. Epub 2016 Dec 22.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address:

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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http://dx.doi.org/10.1016/j.molcel.2016.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222723PMC
January 2017

Transcriptional termination in mammals: Stopping the RNA polymerase II juggernaut.

Authors:
Nick J Proudfoot

Science 2016 Jun;352(6291):aad9926

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

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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http://dx.doi.org/10.1126/science.aad9926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5144996PMC
June 2016

Pcf11 orchestrates transcription termination pathways in yeast.

Genes Dev 2015 Apr 15;29(8):849-61. Epub 2015 Apr 15.

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom;

In Saccharomyces cerevisiae, short noncoding RNA (ncRNA) generated by RNA polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3, and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is also generally required for NRD-dependent transcription termination through the action of its C-terminal domain (CTD)-interacting domain (CID). Pcf11 localizes downstream from Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore, mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA, and restricted Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar, as both accumulate RNA:DNA hybrids and display Pol II pausing downstream from NRD terminators. We predict a mechanism by which the exchange of Nrd1 and Pcf11 on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo.
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http://dx.doi.org/10.1101/gad.251470.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403260PMC
April 2015

Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs.

Nat Struct Mol Biol 2015 Apr 2;22(4):319-27. Epub 2015 Mar 2.

School of Pharmacy, University of Nottingham, Nottingham, UK.

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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http://dx.doi.org/10.1038/nsmb.2982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492989PMC
April 2015

Terminate and make a loop: regulation of transcriptional directionality.

Trends Biochem Sci 2014 Jul 10;39(7):319-27. Epub 2014 Jun 10.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address:

Bidirectional promoters are a common feature of many eukaryotic organisms from yeast to humans. RNA Polymerase II that is recruited to this type of promoter can start transcribing in either direction using alternative DNA strands as the template. Such promiscuous transcription can lead to the synthesis of unwanted transcripts that may have negative effects on gene expression. Recent studies have identified transcription termination and gene looping as critical players in the enforcement of promoter directionality. Interestingly, both mechanisms share key components. Here, we focus on recent findings relating to the transcriptional output of bidirectional promoters.
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http://dx.doi.org/10.1016/j.tibs.2014.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085477PMC
July 2014

Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA.

Nat Struct Mol Biol 2014 Jun 11;21(6):552-9. Epub 2014 May 11.

Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

Dicer is a central enzymatic player in RNA-interference pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, its nuclear function remains obscure. Here we show that Dicer is present in both the nucleus and cytoplasm, and its nuclear levels are tightly regulated. Dicer interacts with RNA polymerase II (Pol II) at actively transcribed gene loci. Loss of Dicer causes the appearance of endogenous double-stranded RNA (dsRNA), which in turn leads to induction of the interferon-response pathway and consequent cell death. Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping noncoding-RNA transcription units. Failure to do so has catastrophic effects on cell function.
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http://dx.doi.org/10.1038/nsmb.2827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4129937PMC
June 2014

Rad51, friend or foe?

Elife 2013 Jun 11;2:e00914. Epub 2013 Jun 11.

is at the Sir William Dunn School of Pathology , University of Oxford , Oxford , United Kingdom

A protein long recognized for its role in DNA repair has now paradoxically been implicated in DNA damage.
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http://dx.doi.org/10.7554/eLife.00914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679523PMC
June 2013

Feed backwards model for microRNA processing and splicing in plants.

EMBO Rep 2013 Jul 14;14(7):581-2. Epub 2013 Jun 14.

Sir William Dunn School of Pathology, University of Oxford, UK.

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http://dx.doi.org/10.1038/embor.2013.77DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701244PMC
July 2013

AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination.

Nucleic Acids Res 2013 Feb 20;41(3):1797-806. Epub 2012 Dec 20.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

RNA Polymerase II (Pol II) termination is dependent on RNA processing signals as well as specific terminator elements located downstream of the poly(A) site. One of the two major terminator classes described so far is the Co-Transcriptional Cleavage (CoTC) element. We show that homopolymer A/T tracts within the human β-globin CoTC-mediated terminator element play a critical role in Pol II termination. These short A/T tracts, dispersed within seemingly random sequences, are strong terminator elements, and bioinformatics analysis confirms the presence of such sequences in 70% of the putative terminator regions (PTRs) genome-wide.
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http://dx.doi.org/10.1093/nar/gks1335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561976PMC
February 2013

Disengaging polymerase: terminating RNA polymerase II transcription in budding yeast.

Biochim Biophys Acta 2013 Jan 17;1829(1):174-85. Epub 2012 Oct 17.

Cancer Research UK London Research Institute, Blanche Lane South Mimms, Herts, UK.

Termination of transcription by RNA polymerase II requires two distinct processes: The formation of a defined 3' end of the transcribed RNA, as well as the disengagement of RNA polymerase from its DNA template. Both processes are intimately connected and equally pivotal in the process of functional messenger RNA production. However, research in recent years has elaborated how both processes can additionally be employed to control gene expression in qualitative and quantitative ways. This review embraces these new findings and attempts to paint a broader picture of how this final step in the transcription cycle is of critical importance to many aspects of gene regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
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http://dx.doi.org/10.1016/j.bbagrm.2012.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793857PMC
January 2013

Convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells.

Nat Struct Mol Biol 2012 Nov 30;19(11):1193-201. Epub 2012 Sep 30.

Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

We show that convergent transcription induces transcriptional gene silencing (TGS) in trans for both fission yeast and mammalian cells. This method has advantages over existing strategies to induce gene silencing. Previous studies in fission yeast have characterized TGS as a cis-specific process involving RNA interference that maintains heterochromatic regions such as centromeres. In contrast, in mammalian cells, gene silencing is known to occur through a post-transcriptional mechanism that uses exogenous short interfering RNAs or endogenous microRNAs to inactivate mRNA. We now show that the introduction of convergent transcription plasmids into either Schizosaccharomyces pombe or mammalian cells allows the production of double-stranded RNA from inserted gene fragments, resulting in TGS of endogenous genes. We predict that using convergent transcription to induce gene silencing will be a generally useful strategy and allow for a fuller molecular understanding of the biology of TGS.
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http://dx.doi.org/10.1038/nsmb.2392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504457PMC
November 2012

Gene loops enhance transcriptional directionality.

Science 2012 Nov 27;338(6107):671-5. Epub 2012 Sep 27.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Eukaryotic genomes are extensively transcribed, forming both messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). ncRNAs made by RNA polymerase II often initiate from bidirectional promoters (nucleosome-depleted chromatin) that synthesize mRNA and ncRNA in opposite directions. We demonstrate that, by adopting a gene-loop conformation, actively transcribed mRNA encoding genes restrict divergent transcription of ncRNAs. Because gene-loop formation depends on a protein factor (Ssu72) that coassociates with both the promoter and the terminator, the inactivation of Ssu72 leads to increased synthesis of promoter-associated divergent ncRNAs, referred to as Ssu72-restricted transcripts (SRTs). Similarly, inactivation of individual gene loops by gene mutation enhances SRT synthesis. We demonstrate that gene-loop conformation enforces transcriptional directionality on otherwise bidirectional promoters.
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http://dx.doi.org/10.1126/science.1224350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563069PMC
November 2012

Ending the message: poly(A) signals then and now.

Authors:
Nick J Proudfoot

Genes Dev 2011 Sep;25(17):1770-82

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.

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.
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http://dx.doi.org/10.1101/gad.17268411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3175714PMC
September 2011

Autoregulation of convergent RNAi genes in fission yeast.

Genes Dev 2011 Mar 28;25(6):556-68. Epub 2011 Feb 28.

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.

RNAi plays a central role in the regulation of eukaryotic genes. In Schizosaccharomyces pombe fission yeast, RNAi involves the formation of siRNA from dsRNA that acts to establish and maintain heterochromatin over centromeres, telomeres, and mating loci. We showed previously that transient heterochromatin also forms over S. pombe convergent genes (CGs). Remarkably, most RNAi genes are themselves convergent. We demonstrate here that transient heterochromatin formed by the RNAi pathway over RNAi CGs leads to their autoregulation in G1-S. Furthermore, the switching of RNAi gene orientation from convergent to tandem causes loss of their G1-S down-regulation. Surprisingly, yeast mutants with tandemized dcr1, ago1, or clr4 genes display aberrant centromeric heterochromatin, which results in abnormal cell morphology. Our results emphasize the significance of gene orientation for correct RNAi gene expression, and suggest a role for cell cycle-dependent formation of RNAi CG heterochromatin in cellular integrity.
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http://dx.doi.org/10.1101/gad.618611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059830PMC
March 2011

Yeast Sen1 helicase protects the genome from transcription-associated instability.

Mol Cell 2011 Jan;41(1):21-32

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.
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http://dx.doi.org/10.1016/j.molcel.2010.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314950PMC
January 2011

Co-transcriptional RNA cleavage provides a failsafe termination mechanism for yeast RNA polymerase I.

Nucleic Acids Res 2011 Mar 23;39(4):1439-48. Epub 2010 Oct 23.

Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK.

Ribosomal RNA, transcribed by RNA polymerase (Pol) I, accounts for most cellular RNA. Since Pol I transcribes rDNA repeats with high processivity and polymerase density, transcription termination is a critical process. Early in vitro studies proposed polymerase pausing by Reb1 and transcript release at the T-rich element T1 determined transcription termination. However recent in vivo studies revealed a 'torpedo' mechanism for Pol I termination: co-transcriptional RNA cleavage by Rnt1 provides an entry site for the 5'-3' exonuclease Rat1 that degrades Pol I-associated transcripts destabilizing the transcription complex. Significantly Rnt1 inactivation in vivo reveals a second co-transcriptional RNA cleavage event at T1 which provides Pol I with an alternative termination pathway. An intact Reb1-binding site is also required for Rnt1-independent termination. Consequently our results reconcile the original Reb1-mediated termination pathway as part of a failsafe mechanism for this essential transcription process.
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http://dx.doi.org/10.1093/nar/gkq894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045592PMC
March 2011

Role of the RNA/DNA kinase Grc3 in transcription termination by RNA polymerase I.

EMBO Rep 2010 Oct 3;11(10):758-64. Epub 2010 Sep 3.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Transcription termination by RNA polymerase I in Saccharomyces cerevisiae is mediated by a 'torpedo' mechanism: co-transcriptional RNA cleavage by Rnt1 at the ribosomal DNA 3'-region generates a 5'-end that is recognized by the 5'-3' exonuclease Rat1; this degrades the downstream transcript and eventually causes termination. In this study, we identify Grc3 as a new factor involved in this process. We demonstrate that GRC3, an essential gene of previously unknown function, encodes a polynucleotide kinase that is required for efficient termination by RNA polymerase I. We propose that it controls the phosphorylation status of the downstream Rnt1 cleavage product and thereby regulates its accessibility to the torpedo Rat1.
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http://dx.doi.org/10.1038/embor.2010.130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948184PMC
October 2010

Silencing in trans: position matters in fission yeast.

EMBO Rep 2010 Mar 12;11(3):145-6. Epub 2010 Feb 12.

Sir William Dunn School of Pathology, University of Oxford, UK.

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http://dx.doi.org/10.1038/embor.2010.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838698PMC
March 2010

Gene loops function to maintain transcriptional memory through interaction with the nuclear pore complex.

Genes Dev 2009 Nov;23(22):2610-24

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.

Inducible genes in yeast retain a "memory" of recent transcriptional activity during periods of short-term repression, allowing them to be reactivated faster when reinduced. This confers a rapid and versatile gene expression response to the environment. We demonstrate that this memory mechanism is associated with gene loop interactions between the promoter and 3' end of the responsive genes HXK1 and GAL1FMP27. The maintenance of these memory gene loops (MGLs) during intervening periods of transcriptional repression is required for faster RNA polymerase II (Pol II) recruitment to the genes upon reinduction, thereby facilitating faster mRNA accumulation. Notably, a sua7-1 mutant or the endogenous INO1 gene that lacks this MGL does not display such faster reinduction. Furthermore, these MGLs interact with the nuclear pore complex through association with myosin-like protein 1 (Mlp1). An mlp1Delta strain does not maintain MGLs, and concomitantly loses transcriptional memory. We predict that gene loop conformations enhance gene expression by facilitating rapid transcriptional response to changing environmental conditions.
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http://dx.doi.org/10.1101/gad.1823209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779764PMC
November 2009

Fail-safe transcriptional termination for protein-coding genes in S. cerevisiae.

Mol Cell 2009 Oct;36(1):88-98

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Transcription termination of RNA polymerase II (Pol II) on protein-coding genes in S. cerevisiae relies on pA site recognition by 3' end processing factors. Here we demonstrate the existence of two alternative termination mechanisms that rescue polymerases failing to disengage from the template at pA sites. One of these fail-safe mechanisms is mediated by the NRD complex, similar to termination of short noncoding genes. The other termination mechanism is mediated by Rnt1 cleavage of the nascent transcript. Both fail-safe termination mechanisms trigger degradation of readthrough transcripts by the exosome. However, Rnt1-mediated termination can also enhance the usage of weak pA signals and thereby generate functional mRNA. We propose that these alternative Pol II termination pathways serve the dual function of avoiding transcription interference and promoting rapid removal of aberrant transcripts.
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http://dx.doi.org/10.1016/j.molcel.2009.07.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779338PMC
October 2009

Pre-mRNA processing reaches back to transcription and ahead to translation.

Cell 2009 Feb;136(4):688-700

Howard Hughes Medical Institute, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.

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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http://dx.doi.org/10.1016/j.cell.2009.02.001DOI Listing
February 2009