Integrator terminates promoter-proximal Pol II to generate C. elegans piRNA precursors

AbstractPiwi-interacting RNAs (piRNAs) play key roles in germline development and genome defence in metazoans. In C. elegans, piRNAs are transcribed from >15000 discrete genomic loci by RNA polymerase II, resulting in 28 nt short-capped piRNA precursors. Here we investigate transcription termination at piRNA loci. We show that the Integrator complex, which terminates snRNA transcription, is recruited to piRNA loci. We show that the catalytic activity of Integrator cleaves nascent capped piRNA precursors associated with promoter-proximal Pol II, resulting in termination of transcription. Loss of Integrator activity, however, does not result in transcriptional readthrough at the majority of piRNA loci. Our results draw new parallels between snRNA and piRNA biogenesis in nematodes, and provide evidence of a role for the Integrator complex as a terminator of promoter-proximal RNA polymerase II.Highlights- Integrator localises to sites of piRNA biogenesis in nematodes- Integrator cleaves nascent RNAs associated with promoter-proximal Pol II at piRNA loci to release short capped piRNA precursors from chromatin- Repression of Pol II elongation at the majority of piRNA loci is independent of Integrator

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The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation

Nucleic Acids Research ◽

10.1093/nar/gkz615 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9024-9036 ◽

Cited By ~ 4

Author(s):

Jered M Wendte ◽

Jeremy R Haag ◽

Olga M Pontes ◽

Jasleen Singh ◽

Sara Metcalf ◽

...

Keyword(s):

Dna Methylation ◽

Catalytic Activity ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cytosine Methylation ◽

Transcriptional Gene Silencing ◽

Rna Dependent Rna Polymerase ◽

Pol Ii ◽

Pol Iv ◽

Non Coding Rnas

Abstract In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.

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CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation.

10.1101/2021.06.21.449289 ◽

2021 ◽

Author(s):

Michael Tellier ◽

Justyna Zaborowska ◽

Jonathan Neve ◽

Takayuki Nojima ◽

Svenja Hester ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Premature Termination ◽

Transcription Termination ◽

Elongation Factor ◽

Protein Coding ◽

Pol Ii ◽

Working Together ◽

Rna Maturation ◽

Nascent Transcripts

CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allows the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an ATP analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.

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Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1700128114 ◽

2017 ◽

Vol 114 (20) ◽

pp. E3944-E3953 ◽

Cited By ~ 13

Author(s):

Corey M. Nemec ◽

Fan Yang ◽

Joshua M. Gilmore ◽

Corinna Hintermair ◽

Yi-Hsuan Ho ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Protein Complexes ◽

Transcription Termination ◽

Structural Data ◽

Side Chain ◽

Protein Coding ◽

Pol Ii ◽

Carboxyl Terminal ◽

Nucleolar Rna

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific “readers.” Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phospho-Ser2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engages two distinct CTD modifications that are differentially enriched at noncoding genes, we relied on NMR analysis of Rtt103 in complex with phospho-Thr4– or phospho-Ser2–bearing CTD peptides. The structural data reveal that Rtt103 interacts with phospho-Thr4 in a manner analogous to its interaction with phospho-Ser2–modified CTD. The same set of hydrogen bonds involving either the oxygen on phospho-Thr4 and the hydroxyl on Ser2, or the phosphate on Ser2 and the Thr4 hydroxyl, can be formed by rotation of an arginine side chain, leaving the intermolecular interface otherwise unperturbed. This economy of design enables Rtt103 to engage Pol II at distinct sets of genes with differentially enriched CTD marks.

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Hyperosmotic stress induces downstream-of-gene transcription and alters the RNA Polymerase II interactome despite widespread transcriptional repression

10.1101/2020.06.30.178103 ◽

2020 ◽

Author(s):

Nicolle A. Rosa-Mercado ◽

Joshua T. Zimmer ◽

Maria Apostolidi ◽

Jesse Rinehart ◽

Matthew D. Simon ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Transcriptional Response ◽

Hyperosmotic Stress ◽

Transcriptional Level ◽

List Type ◽

Upstream Gene ◽

Pol Ii ◽

Transcription Profiles

SummaryStress-induced readthrough transcription results in the synthesis of thousands of downstream-of-gene (DoG) containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse-seq revealed that hyperosmotic stress induces widespread transcriptional repression. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP-seq confirmed that the stress-induced redistribution of RNA Polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While subunits of the cleavage and polyadenylation machinery remained Pol II-associated, Integrator complex subunits dissociated from Pol II under stress conditions. Depleting the catalytic subunit of the Integrator complex, Int11, using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.In briefRosa-Mercado et al. report that hyperosmotic stress causes widespread transcriptional repression in human cells, yet DoGs arise regardless of the transcriptional response of their upstream genes. They find that the interaction between Pol II and Integrator is disrupted by hypertonicity and that knocking down the Integrator nuclease leads to DoG production.HighlightsHyperosmotic stress triggers transcriptional repression of many genes.DoG RNAs arise independent of the transcriptional level of their upstream gene.The interaction between Pol II and Integrator subunits decreases after salt stress.Depletion of the Int11 nuclease subunit induces the production of hundreds of DoGs.

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Condensin controls recruitment of RNA polymerase II to achieve nematode X-chromosome dosage compensation

eLife ◽

10.7554/elife.00808 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 108

Author(s):

William S Kruesi ◽

Leighton J Core ◽

Colin T Waters ◽

John T Lis ◽

Barbara J Meyer

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

X Chromosome ◽

Dosage Compensation ◽

Pol Ii ◽

Transcription Start Sites ◽

C Elegans ◽

Mapping Strategy ◽

Genome Wide ◽

Mature Mrnas

The X-chromosome gene regulatory process called dosage compensation ensures that males (1X) and females (2X) express equal levels of X-chromosome transcripts. The mechanism in Caenorhabditis elegans has been elusive due to improperly annotated transcription start sites (TSSs). Here we define TSSs and the distribution of transcriptionally engaged RNA polymerase II (Pol II) genome-wide in wild-type and dosage-compensation-defective animals to dissect this regulatory mechanism. Our TSS-mapping strategy integrates GRO-seq, which tracks nascent transcription, with a new derivative of this method, called GRO-cap, which recovers nascent RNAs with 5′ caps prior to their removal by co-transcriptional processing. Our analyses reveal that promoter-proximal pausing is rare, unlike in other metazoans, and promoters are unexpectedly far upstream from the 5′ ends of mature mRNAs. We find that C. elegans equalizes X-chromosome expression between the sexes, to a level equivalent to autosomes, by reducing Pol II recruitment to promoters of hermaphrodite X-linked genes using a chromosome-restructuring condensin complex.

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Structure of the p53/RNA polymerase II assembly

Communications Biology ◽

10.1038/s42003-021-01934-4 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Shu-Hao Liou ◽

Sameer K. Singh ◽

Robert H. Singer ◽

Robert A. Coleman ◽

Wei-Li Liu

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Conformational Changes ◽

P53 Protein ◽

Binding Activity ◽

Transactivation Domain ◽

Pol Ii ◽

Dna Binding Activity ◽

Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

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RecQL5 Promotes Genome Stabilization through Two Parallel Mechanisms—Interacting with RNA Polymerase II and Acting as a Helicase

Molecular and Cellular Biology ◽

10.1128/mcb.01583-09 ◽

2010 ◽

Vol 30 (10) ◽

pp. 2460-2472 ◽

Cited By ~ 47

Author(s):

M. Nurul Islam ◽

David Fox ◽

Rong Guo ◽

Takemi Enomoto ◽

Weidong Wang

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Genome Stability ◽

Transcriptional Regulators ◽

Helicase Activity ◽

Parallel Mechanisms ◽

Recq Helicase ◽

Pol Ii ◽

Kix Domain ◽

Genome Stabilization

ABSTRACT The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

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Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

Journal of Biological Chemistry ◽

10.1074/jbc.m009634200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 12266-12273 ◽

Cited By ~ 31

Author(s):

Wenxiang Wei ◽

Dorjbal Dorjsuren ◽

Yong Lin ◽

Weiping Qin ◽

Takahiro Nomura ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Middle Part ◽

Wild Type ◽

Binding Region ◽

Pol Ii ◽

Transcription Factor Iif ◽

B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

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Transcription of Hepatitis Delta Virus RNA by RNA Polymerase II

Journal of Virology ◽

10.1128/jvi.01758-07 ◽

2007 ◽

Vol 82 (3) ◽

pp. 1118-1127 ◽

Cited By ~ 56

Author(s):

Jinhong Chang ◽

Xingcao Nie ◽

Ho Eun Chang ◽

Ziying Han ◽

John Taylor

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Hepatitis Delta Virus ◽

Cell System ◽

Hepatitis Delta ◽

Pol Ii ◽

Delta Antigen ◽

Nuclear Extracts ◽

Virus Rna ◽

Delta Virus

ABSTRACT Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

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