Publications by authors named "Nicholas J Proudfoot"

35 Publications

Enhancers predominantly regulate gene expression during differentiation via transcription initiation.

Mol Cell 2021 03 3;81(5):983-997.e7. Epub 2021 Feb 3.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK; MRC WIMM Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK. Electronic address:

Gene transcription occurs via a cycle of linked events, including initiation, promoter-proximal pausing, and elongation of RNA polymerase II (Pol II). A key question is how transcriptional enhancers influence these events to control gene expression. Here, we present an approach that evaluates the level and change in promoter-proximal transcription (initiation and pausing) in the context of differential gene expression, genome-wide. This combinatorial approach shows that in primary cells, control of gene expression during differentiation is achieved predominantly via changes in transcription initiation rather than via release of Pol II pausing. Using genetically engineered mouse models, deleted for functionally validated enhancers of the α- and β-globin loci, we confirm that these elements regulate Pol II recruitment and/or initiation to modulate gene expression. Together, our data show that gene expression during differentiation is regulated predominantly at the level of initiation and that enhancers are key effectors of this process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2021.01.002DOI Listing
March 2021

SCAF4 and SCAF8, mRNA Anti-Terminator Proteins.

Cell 2019 06 16;177(7):1797-1813.e18. Epub 2019 May 16.

Mechanisms of Transcription Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

Accurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative polyadenylation (polyA) sites. The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) phosphorylated on both Ser2 and Ser5 and are detected at early, alternative polyA sites. Concomitant knockout of human SCAF4 and SCAF8 results in altered polyA selection and subsequent early termination, leading to expression of truncated mRNAs and proteins lacking functional domains and is cell lethal. While SCAF4 and SCAF8 work redundantly to suppress early mRNA termination, they also have independent, non-essential functions. SCAF8 is an RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical, distal transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.04.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579486PMC
June 2019

R-loop formation during S phase is restricted by PrimPol-mediated repriming.

EMBO J 2019 02 26;38(3). Epub 2018 Nov 26.

MRC Laboratory of Molecular Biology, Cambridge, UK

During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three-stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single-stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here, we show how R-loop formation drives a short purine-rich repeat, (GAA), to become a replication impediment that engages the repriming activity of the primase-polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R-loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R-loops promote the creation of replication blocks at susceptible structure-forming sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201899793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356060PMC
February 2019

RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing.

Mol Cell 2018 10;72(2):369-379.e4

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal. Electronic address:

The highly intronic nature of protein coding genes in mammals necessitates a co-transcriptional splicing mechanism as revealed by mNET-seq analysis. Immunoprecipitation of MNase-digested chromatin with antibodies against RNA polymerase II (Pol II) shows that active spliceosomes (both snRNA and proteins) are complexed to Pol II S5P CTD during elongation and co-transcriptional splicing. Notably, elongating Pol II-spliceosome complexes form strong interactions with nascent transcripts, resulting in footprints of approximately 60 nucleotides. Also, splicing intermediates formed by cleavage at the 5' splice site are associated with nearly all spliced exons. These spliceosome-bound intermediates are frequently ligated to upstream exons, implying a sequential, constitutive, and U12-dependent splicing process. Finally, lack of detectable spliced products connected to the Pol II active site in human HeLa or murine lymphoid cells suggests that splicing does not occur immediately following 3' splice site synthesis. Our results imply that most mammalian splicing requires exon definition for completion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2018.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201815PMC
October 2018

Mitochondrial double-stranded RNA triggers antiviral signalling in humans.

Nature 2018 08 25;560(7717):238-242. Epub 2018 Jul 25.

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

Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0363-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570621PMC
August 2018

RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination.

Mol Cell 2018 05 3;70(4):650-662.e8. Epub 2018 May 3.

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

Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2018.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971202PMC
May 2018

Nuclear fate of yeast snoRNA is determined by co-transcriptional Rnt1 cleavage.

Nat Commun 2018 05 3;9(1):1783. Epub 2018 May 3.

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

Small nucleolar RNA (snoRNA) are conserved and essential non-coding RNA that are transcribed by RNA Polymerase II (Pol II). Two snoRNA classes, formerly distinguished by their structure and ribonucleoprotein composition, act as guide RNA to target RNA such as ribosomal RNA, and thereby introduce specific modifications. We have studied the 5'end processing of individually transcribed snoRNA in S. cerevisiae to define their role in snoRNA biogenesis and functionality. Here we show that pre-snoRNA processing by the endonuclease Rnt1 occurs co-transcriptionally with removal of the mG cap facilitating the formation of box C/D snoRNA. Failure of this process causes aberrant 3'end processing and mislocalization of snoRNA to the cytoplasm. Consequently, Rnt1-dependent 5'end processing of box C/D snoRNA is critical for snoRNA-dependent methylation of ribosomal RNA. Our results reveal that the 5'end processing of box C/D snoRNA defines their distinct pathway of maturation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-04094-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934358PMC
May 2018

Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide.

Nat Protoc 2016 Mar 4;11(3):413-28. Epub 2016 Feb 4.

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

The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide-resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nprot.2016.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5152764PMC
March 2016

Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing.

Cell 2015 Apr;161(3):526-540

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2015.03.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410947PMC
April 2015

BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair.

Mol Cell 2015 Feb;57(4):636-647

Department of Genetics, Harvard Medical School, Boston, MA 02215, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2015.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351672PMC
February 2015

R-loops induce repressive chromatin marks over mammalian gene terminators.

Nature 2014 Dec 5;516(7531):436-9. Epub 2014 Oct 5.

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

The formation of R-loops is a natural consequence of the transcription process, caused by invasion of the DNA duplex by nascent transcripts. These structures have been considered rare transcriptional by-products with potentially harmful effects on genome integrity owing to the fragility of the displaced DNA coding strand. However, R-loops may also possess beneficial effects, as their widespread formation has been detected over CpG island promoters in human genes. Furthermore, we have previously shown that R-loops are particularly enriched over G-rich terminator elements. These facilitate RNA polymerase II (Pol II) pausing before efficient termination. Here we reveal an unanticipated link between R-loops and RNA-interference-dependent H3K9me2 formation over pause-site termination regions in mammalian protein-coding genes. We show that R-loops induce antisense transcription over these pause elements, which in turn leads to the generation of double-stranded RNA and the recruitment of DICER, AGO1, AGO2 and the G9a histone lysine methyltransferase. Consequently, an H3K9me2 repressive mark is formed and heterochromatin protein 1γ (HP1γ) is recruited, which reinforces Pol II pausing before efficient transcriptional termination. We predict that R-loops promote a chromatin architecture that defines the termination region for a substantial subset of mammalian genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature13787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272244PMC
December 2014

A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression.

Genes Dev 2014 Jul;28(13):1384-96

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.242990.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083084PMC
July 2014

Drosha regulates gene expression independently of RNA cleavage function.

Cell Rep 2013 Dec 19;5(6):1499-510. Epub 2013 Dec 19.

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

Drosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription-dependent manner. This binding is not associated with miRNA production or RNA cleavage. Drosha knockdown in HeLa cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mRNA produced from these gene regions. Furthermore, we show that this function of Drosha is dependent on its N-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2013.11.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898267PMC
December 2013

Histone 3 s10 phosphorylation: "caught in the R loop!".

Mol Cell 2013 Nov;52(4):470-2

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

In this issue of Molecular Cell, Castellano-Pozo et al. (2013) describe a connection between R loop structures and histone 3 S10 phosphorylation (H3S10P), a mark of chromatin compaction. Their results constitute a significant advance in our understanding of the role of R loops in genomic instability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2013.11.006DOI Listing
November 2013

Genome-wide analysis of poly(A) site selection in Schizosaccharomyces pombe.

RNA 2013 Dec 23;19(12):1617-31. Epub 2013 Oct 23.

Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is mediated by cis elements collectively called the polyadenylation signal. Genome-wide analysis of such polyadenylation signals was missing in fission yeast, even though it is an important model organism. We demonstrate that the canonical AATAAA motif is the most frequent and functional polyadenylation signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from cells grown under various physiological conditions, we identify 3' UTRs for nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is alternative polyadenylation within and between conditions. We validated the computationally identified sequence elements likely to promote polyadenylation by functional assays, including qRT-PCR and 3'RACE analysis. The biological importance of the AATAAA motif is underlined by functional analysis of the genes containing it. Furthermore, it has been shown that convergent genes require trans elements, like cohesin for efficient transcription termination. Here we show that convergent genes lacking cohesin (on chromosome 2) are generally associated with longer overlapping mRNA transcripts. Our bioinformatic and experimental genome-wide results are summarized and can be accessed and customized in a user-friendly database Pomb(A).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.040675.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3884648PMC
December 2013

R-loop stabilization represses antisense transcription at the Arabidopsis FLC locus.

Science 2013 May;340(6132):619-21

Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

Roles for long noncoding RNAs (lncRNAs) in gene expression are emerging, but regulation of the lncRNA itself is poorly understood. We have identified a homeodomain protein, AtNDX, that regulates COOLAIR, a set of antisense transcripts originating from the 3' end of Arabidopsis FLOWERING LOCUS C (FLC). AtNDX associates with single-stranded DNA rather than double-stranded DNA non-sequence-specifically in vitro, and localizes to a heterochromatic region in the COOLAIR promoter in vivo. Single-stranded DNA was detected in vivo as part of an RNA-DNA hybrid, or R-loop, that covers the COOLAIR promoter. R-loop stabilization mediated by AtNDX inhibits COOLAIR transcription, which in turn modifies FLC expression. Differential stabilization of R-loops could be a general mechanism influencing gene expression in many organisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1234848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5144995PMC
May 2013

Definition of RNA polymerase II CoTC terminator elements in the human genome.

Cell Rep 2013 Apr 4;3(4):1080-92. Epub 2013 Apr 4.

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

Mammalian RNA polymerase II (Pol II) transcription termination is an essential step in protein-coding gene expression that is mediated by pre-mRNA processing activities and DNA-encoded terminator elements. Although much is known about the role of pre-mRNA processing in termination, our understanding of the characteristics and generality of terminator elements is limited. Whereas promoter databases list up to 40,000 known and potential Pol II promoter sequences, fewer than ten Pol II terminator sequences have been described. Using our knowledge of the human β-globin terminator mechanism, we have developed a selection strategy for mapping mammalian Pol II terminator elements. We report the identification of 78 cotranscriptional cleavage (CoTC)-type terminator elements at endogenous gene loci. The results of this analysis pave the way for the full understanding of Pol II termination pathways and their roles in gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2013.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644702PMC
April 2013

Transcription termination between polo and snap, two closely spaced tandem genes of D. melanogaster.

Transcription 2012 Jul-Aug;3(4):198-212

Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.

Transcription termination of RNA polymerase II between closely spaced genes is an important, though poorly understood, mechanism. This is true, in particular, in the Drosophila genome, where approximately 52% of tandem genes are separated by less than 1 kb. We show that a set of Drosophila tandem genes has a negative correlation of gene expression and display several molecular marks indicative of promoter pausing. We find that an intergenic spacing of 168 bp is sufficient for efficient transcription termination between the polo-snap tandem gene pair, by a mechanism that is independent of Pcf11 and Xrn2. In contrast, analysis of a tandem gene pair containing a longer intergenic region reveals that termination occurs farther downstream of the poly(A) signal and is, in this case, dependent on Pcf11 and Xrn2. For polo-snap, displacement of poised polymerase from the snap promoter by depletion of the initiation factor TFIIB results in an increase of polo transcriptional read-through. This suggests that poised polymerase is necessary for transcription termination. Interestingly, we observe that polo forms a TFIIB dependent gene loop between its promoter and terminator regions. Furthermore, in a plasmid containing the polo-snap locus, deletion of the polo promoter causes an increase in snap expression, as does deletion of polo poly(A) signals. Taken together, our results indicate that polo forms a gene loop and polo transcription termination occurs by an Xrn2 and Pcf11 independent mechanism that requires TFIIB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/trns.21967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654770PMC
February 2013

Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination.

Mol Cell 2011 Jun;42(6):794-805

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2011.04.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145960PMC
June 2011

RNA polymerase II kinetics in polo polyadenylation signal selection.

EMBO J 2011 May 20;30(12):2431-44. Epub 2011 May 20.

Cell Activation and Gene Expression Group, IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.

Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, we show that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. Our results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/emboj.2011.156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116286PMC
May 2011

Transcriptional activation of the general amino acid permease gene per1 by the histone deacetylase Clr6 Is regulated by Oca2 kinase.

Mol Cell Biol 2010 Jul 19;30(13):3396-410. Epub 2010 Apr 19.

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

Expression of nitrogen metabolism genes is regulated by the quality of the nitrogen supply. Here, we describe a mechanism for the transcriptional regulation of the general amino acid permease gene per1 in Schizosaccharomyces pombe. We show that when ammonia is used as the nitrogen source, low levels of per1 are transcribed and histones in the coding and surrounding regions of per1 are acetylated. In the presence of proline, per1 transcription is upregulated and initiates from a more upstream site, generating 5'-extended mRNAs. Concomitantly, histones at per1 are deacetylated in a Clr6-dependent manner, suggesting a positive role for Clr6 in transcriptional regulation of per1. Upstream initiation and histone deactylation of per1 are constitutive in cells lacking the serine/threonine kinase oca2, indicating that Oca2 is a repressor of per1. Oca2 interacts with a protein homologous to the Saccharomyces cerevisiae transcriptional activator Cha4 and with Ago1. Loss of Cha4 or Ago1 causes aberrant induction of per1 under noninducing conditions, suggesting that these proteins are also involved in per1 regulation and hence in nitrogen utilization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00971-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897586PMC
July 2010

Coupled RNA processing and transcription of intergenic primary microRNAs.

Mol Cell Biol 2009 Oct 10;29(20):5632-8. Epub 2009 Aug 10.

University of Rome La Sapienza, P.le A. Moro 5, 00185 Rome, Italy.

The first step in microRNA (miRNA) biogenesis occurs in the nucleus and is mediated by the Microprocessor complex containing the RNase III-like enzyme Drosha and its cofactor DGCR8. Here we show that the 5'-->3' exonuclease Xrn2 associates with independently transcribed miRNAs and, in combination with Drosha processing, attenuates transcription in downstream regions. We suggest that, after Drosha cleavage, a torpedo-like mechanism acts on nascent long precursor miRNAs, whereby Xrn2 exonuclease degrades the RNA polymerase II-associated transcripts inducing its release from the template. While involved in primary transcript termination, this attenuation effect does not restrict clustered miRNA expression, which, in the majority of cases, is separated by short spacers. We also show that transcripts originating from a miRNA promoter are retained on the chromatin template and are more efficiently processed than those produced from mRNA or snRNA Pol II-dependent promoters. These data imply that coupling between transcription and processing promotes efficient expression of independently transcribed miRNAs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00664-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2756881PMC
October 2009

Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing.

Science 2009 Jul;325(5936):90-3

Chemistry Research Laboratory and Oxford Centre for Integrative Systems Biology, University of Oxford, 12 Mansfield Road, Oxford, Oxon OX1 3TA, UK.

The finding that the metazoan hypoxic response is regulated by oxygen-dependent posttranslational hydroxylations, which regulate the activity and lifetime of hypoxia-inducible factor (HIF), has raised the question of whether other hydroxylases are involved in the regulation of gene expression. We reveal that the splicing factor U2 small nuclear ribonucleoprotein auxiliary factor 65-kilodalton subunit (U2AF65) undergoes posttranslational lysyl-5-hydroxylation catalyzed by the Fe(II) and 2-oxoglutarate-dependent dioxygenase Jumonji domain-6 protein (Jmjd6). Jmjd6 is a nuclear protein that has an important role in vertebrate development and is a human homolog of the HIF asparaginyl-hydroxylase. Jmjd6 is shown to change alternative RNA splicing of some, but not all, of the endogenous and reporter genes, supporting a specific role for Jmjd6 in the regulation of RNA splicing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1175865DOI Listing
July 2009

Transcriptional termination enhances protein expression in human cells.

Mol Cell 2009 Feb;33(3):354-64

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX13RE, UK.

Transcriptional termination of mammalian RNA polymerase II (Pol II) requires a poly(A) (pA) signal and, often, a downstream terminator sequence. Termination is triggered following recognition of the pA signal by Pol II and subsequent pre-mRNA cleavage, which occurs either at the pA site or in transcripts from terminator elements. Although this process has been extensively studied, it is generally considered inconsequential to the level of gene expression. However, our results demonstrate that termination acts as a driving force for optimal gene expression. We show that this effect is general but most dramatic where weak or noncanonical pA signals are present. We establish that termination of Pol II increases the efficiency of pre-mRNA processing that is completed posttranscriptionally. As such, transcripts escape from nuclear surveillance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2009.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706331PMC
February 2009

An ungracious host for an unwelcome guest.

Cell Host Microbe 2008 Aug;4(2):89-91

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

Persistence of latently infected CD4(+) T cell "reservoirs" presents a major obstacle for current HIV-1 antiretroviral therapies. In Cell Host & Microbe, two separate studies use model systems of latent infection to dissect the influence of host genes on HIV-1 transcription, yielding intriguing, though partially contradictory, conclusions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2008.07.010DOI Listing
August 2008

Dynamic interactions between the promoter and terminator regions of the mammalian BRCA1 gene.

Proc Natl Acad Sci U S A 2008 Apr 28;105(13):5160-5. Epub 2008 Mar 28.

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

The 85-kb breast cancer-associated gene BRCA1 is an established tumor suppressor gene, but its regulation is poorly understood. We demonstrate by gene conformation analysis in both human cell lines and mouse mammary tissue that gene loops are imposed on BRCA1 between the promoter, introns, and terminator region. Significantly, association between the BRCA1 promoter and terminator regions change upon estrogen stimulation and during lactational development. Loop formation is transcription-dependent, suggesting that transcriptional elongation plays an active role in BRCA1 loop formation. We show that the BRCA1 terminator region can suppress estrogen-induced transcription and so may regulate BRCA1 expression. Significantly, BRCA1 promoter and terminator interactions vary in different breast cancer cell lines, indicating that defects in BRCA1 chromatin structure may contribute to dysregulated expression of BRCA1 seen in breast tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0801048105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2278189PMC
April 2008

Molecular dissection of mammalian RNA polymerase II transcriptional termination.

Mol Cell 2008 Mar;29(5):600-10

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

Transcriptional termination of mammalian RNA polymerase II (Pol II) is an essential but little-understood step in protein-coding gene expression. Mechanistically, termination by all DNA-dependent RNA polymerases can be reduced to two steps, namely release of the RNA transcript and release of the DNA template. Using a simple nuclear fractionation procedure, we have monitored transcript and template release in the context of both natural and artificial Pol II terminator sequences. We describe the timing and relationship between these events and in so doing establish the roles of the poly(A) signal, cotranscriptional RNA cleavage events, and 5'-3' exonucleolytic RNA degradation in the mammalian Pol II termination process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2007.12.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2288634PMC
March 2008

Human Pcf11 enhances degradation of RNA polymerase II-associated nascent RNA and transcriptional termination.

Nucleic Acids Res 2008 Feb 17;36(3):905-14. Epub 2007 Dec 17.

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

The poly(A) (pA) signal possesses a dual function in 3' end processing of pre-mRNA and in transcriptional termination of RNA polymerase II (Pol II) for most eukaryotic protein-coding genes. A key protein factor in yeast and Drosophila Pol II transcriptional termination is the 3'-end processing factor, Pcf11. In vitro studies suggest that Pcf11 is capable of promoting the dissociation of Pol II elongation complexes from DNA. Moreover, several mutant alleles of yeast Pcf11 effect termination in vivo. However, functions of human Pcf11 (hPcf11) in Pol II termination have not been explored. Here we show that depletion of hPcf11 from HeLa cells reduces termination efficiency. Furthermore, we provide evidence that hPcf11 is required for the efficient degradation of the 3' product of pA site cleavage. Finally, we show that these functions of hPcf11 require an intact pA signal.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkm1112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2241900PMC
February 2008