An end in sight? Xrn2 and transcriptional termination by RNA polymerase II

Eaton, Joshua D.; West, Steven

doi:10.1080/21541264.2018.1498708

Cited by 17 publications

(14 citation statements)

References 31 publications

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“…However, RNAPII moves further downstream and elongates the post-termination uncapped 3′ RNA, which is promptly degraded by the Xrn2 exonuclease. Running after polymerase for several hundreds of nucleotides, Xrn2 eventually prompts RNAPII arrest and unload from its chromatin template (Eaton and West 2018). Post-termination RNA is rapidly degraded, hence difficult to recover, but provides unique insight into the mechanisms and protein complexes that oversee termination.…”

Section: Resultsmentioning

confidence: 99%

Rapid and scalable profiling of nascent RNA with fastGRO

Barbieri

Hill

Quesnel-Vallières

et al. 2020

Preprint

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Genome-wide profiling of nascent RNA has become a fundamental tool to study transcription regulation. Over the past decade, next-generation sequencing has fostered development of a handful of techniques (i.e. GRO-seq, PRO-seq, TT-seq and NET-seq) that map unprocessed transcripts originating from both the coding and the noncoding portion of the genome. Unlike steady-state RNA sequencing, nascent RNA profiling mirrors the real-time activity of RNA Polymerases and provides an accurate readout of transcriptome-wide variations that occur during short time frames (i.e. response to external stimuli or rapid metabolic changes). Some species of nuclear RNAs, albeit functional, have a short half-life and can only be accurately gauged by nascent RNA techniques (i.e. lincRNAs and eRNAs). Furthermore, these techniques capture uncapped post-cleavage RNA at termination sites or promoter-associated antisense RNAs, providing a unique insight into RNAPII dynamics and processivity.Here we present a run-on assay with 4s-UTP labelling, followed by reversible biotinylation and affinity purification via streptavidin. Our protocol allows streamlined sample preparation within less than 3 days. We named the technique fastGRO (fast Global Run-On). We show that fastGRO is highly reproducible and yields a more complete and extensive coverage of nascent RNA than comparable techniques. Importantly, we demonstrate that fastGRO is scalable and can be performed with as few as 0.5x10^6 cells.

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Section: Resultsmentioning

confidence: 99%

Rapid and scalable profiling of nascent RNA with fastGRO

Barbieri

Hill

Quesnel-Vallières

et al. 2020

Preprint

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“…The peaks were ~1 kbp downstream of the polyadenylation site (Figure b; primer sets 7, 8 and 9). The peaks of these factors overlap with that of RNAP II, indicating the region where these factors accumulated is the RNAP II pause site of HSPA1A gene (Anamika et al, ; Eaton & West, ; Glover‐Cutter et al, ; Gromak et al, ; Swinburne et al, ; West et al, ; Figure a, b). Our data also indicate that at the pause site of the HSPA1A gene, preparations for transcription termination and transcript release, such as the recruitment of termination/CPA factors, reorganization of the transcription machinery and pretermination cleavage of nascent transcript, may occur (Anamika et al, ; Eaton & West, ; Glover‐Cutter et al, ; Gromak et al, ; Richard & Manley, ; Swinburne et al, ; West et al, ).…”

Section: Resultsmentioning

confidence: 99%

“…The molecular details of recruitment and subsequent formation of CPA and termination machinery are becoming more well understood (Anamika et al, ; Eaton & West, ; Glover‐Cutter et al, ; Gromak et al, ; Hsin & Manley, ; Lian et al, ; Richard & Manley, ; Swinburne et al, ; West et al, ). In contrast, the mechanism of the disengagement of intricate protein complexes from active loci after completing their functions is only poorly understood.…”

Section: Discussionmentioning

confidence: 99%

“…RNAP II with hyperphosphorylated CTD Ser2 pauses at 1–5 kbp downstream of pA sites of active genes in human cells. The CPA factors CstF and CPSF preferentially bind to the Ser2 hyperphosphorylated CTD; thus, the “pause site” is the gene region where CPA and transcriptional termination processes are initiated and executed (Anamika, Gyenis, & Tora, ; Eaton & West, ; Glover‐Cutter, Kim, Espinosa, & Bentley, ; Gromak, West, & Proudfoot, ; Hsin & Manley, ; Lian et al, ; Richard & Manley, ; Swinburne, Meyer, Liu, Silver, & Brodsky, ; West, Gromak, & Proudfoot, ). An endonuclease, CPSF73, cleaves the pre‐mRNA at 10–30 nucleotides downstream of the pA signal (Eaton et al, ; Mandel et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…The resulting 3′‐end fragment of the pre‐mRNA is processed by the 5′‐3′ exonuclease XRN2. According to the “torpedo” termination model, XRN2 attacks the newly formed 5′‐end of the read‐through transcript, chases up and eventually destabilizes the elongating RNAP II, leading to transcriptional termination (Eaton & West, ; Porrua, Boudvillain, & Libri, ; Proudfoot, ; Richard & Manley, ). Previous studies have revealed that the p54nrb‐PSF complex, which binds to CTD, recruits XRN2 to the CstF complex of the CPA machinery on transcriptionally active loci, thus coupling transcription, CPA and termination (Emili et al, ; Kaneko, Rozenblatt‐Rosen, Meyerson, & Manley, ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Human THO coordinates transcription termination and subsequent transcript release from the HSP70 locus

et al. 2019

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A multiprotein complex, THO/TREX, couples the transcription, 3′‐end formation and nuclear export of mRNAs. In this study, we report that crucial factors for mRNA processing, such as XRN2, DDX5/DDX17 and CstF64, are copurified with human THO (hTHO). Using chromatin immunoprecipitation, we found increased cross‐linking of XRN2 and CstF64 to the RNA polymerase II (RNAP II) pause site of the HSPA1A gene upon down‐regulation of THOC5, a metazoan‐specific component of hTHO. As observed in THOC5‐depleted cells, knockdown of XRN2 blocked HSP70 transcript release and increased the amount of CstF64 at the pause site. In addition, our data indicate that DDX5/DDX17 is also required for HSP70 transcript release. As the degradation of read‐through transcripts, but not cleavage at polyadenylation sites per se, was hindered upon THOC5 or DDX5/DDX17 down‐regulation, these factors appear to influence transcriptional termination. Interestingly, over‐expression of RNase H suppressed the accumulation of HSP70 transcripts in nuclear foci in THOC5‐ or DDX5/DDX17‐depleted cells. Thus, we propose a model in which hTHO, along with DDX5/DDX17, restricts the formation of R‐loops, thereby facilitating the XRN2‐mediated transcriptional termination and release of the mature transcript from the HSPA1A locus.

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Rapid and Scalable Profiling of Nascent RNA with fastGRO

et al. 2020

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SUMMARY Genome-wide profiling of nascent RNA has become a fundamental tool to study transcription regulation. Unlike steady-state RNA-sequencing (RNA-seq), nascent RNA profiling mirrors real-time activity of RNA polymerases and provides an accurate readout of transcriptome-wide variations. Some species of nuclear RNAs (i.e., large intergenic noncoding RNAs [lincRNAs] and eRNAs) have a short half-life and can only be accurately gauged by nascent RNA techniques. Furthermore, nascent RNA-seq detects post-cleavage RNA at termination sites and promoter-associated antisense RNAs, providing insights into RNA polymerase II (RNAPII) dynamics and processivity. Here, we present a run-on assay with 4-thio ribonucleotide (4-S-UTP) labeling, followed by reversible biotinylation and affinity purification via streptavidin. Our protocol allows streamlined sample preparation within less than 3 days. We named the technique fastGRO (fast Global Run-On). We show that fastGRO is highly reproducible and yields a more complete and extensive coverage of nascent RNA than comparable techniques can. Importantly, we demonstrate that fastGRO is scalable and can be performed with as few as 0.5 × 10 6 cells.

show abstract

An end in sight? Xrn2 and transcriptional termination by RNA polymerase II

Cited by 17 publications

References 31 publications

Rapid and scalable profiling of nascent RNA with fastGRO

Rapid and scalable profiling of nascent RNA with fastGRO

Human THO coordinates transcription termination and subsequent transcript release from the HSP70 locus

Rapid and Scalable Profiling of Nascent RNA with fastGRO

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