DNA polymerase ε associates with the elongating form of RNA polymerase II and nascent transcripts

In amphibian oocytes, most lateral loops of the lampbrush chromosomes correspond to active transcriptional sites for RNA polymerase II. We show that newly assembled small nuclear ribonucleoprotein (RNP [snRNP]) particles, which are formed upon cytoplasmic injection of fluorescently labeled spliceosomal small nuclear RNAs (snRNAs), target the nascent transcripts of the chromosomal loops. With this new targeting assay, we demonstrate that nonfunctional forms of U1 and U2 snRNAs still associate with the active transcriptional units. In particular, we find that their association with nascent RNP fibrils is independent of their base pairing with pre–messenger RNAs. Additionally, stem loop I of the U1 snRNA is identified as a discrete domain that is both necessary and sufficient for association with nascent transcripts. Finally, in oocytes deficient in splicing, the recruitment of U1, U4, and U5 snRNPs to transcriptional units is not affected. Collectively, these data indicate that the recruitment of snRNPs to nascent transcripts and the assembly of the spliceosome are uncoupled events.

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Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7219-7225.1994 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7219-7225

Author(s):

J Wuarin ◽

U Schibler

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

S1 Nuclease ◽

Nonhistone Proteins ◽

Detailed Structural Analysis ◽

Physical Isolation ◽

Transcriptional Activator Protein ◽

Nascent Rna ◽

Nuclease Mapping ◽

Nascent Transcripts

In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chains transcribed by RNA polymerase II. A procedure that allows fractionation of nuclei into a chromatin pellet containing DNA, histones, and ternary transcription complexes and a supernatant containing the bulk of the nonhistone proteins and RNAs that are released from their DNA templates was developed. The transcripts of the genes encoding DBP, a transcriptional activator protein, and HMG coenzyme A reductase recovered from the chromatin pellet and the supernatant were analyzed by S1 nuclease mapping. The large majority of the RNA molecules from the pellet appeared to be nascent transcripts, since, in contrast to the transcripts present in the supernatant, they were not cleaved at the polyadenylation site but rather contained heterogeneous 3' termini encompassing this site. Splicing intermediates could be detected among nascent and released transcripts, suggesting that splicing occurs both cotranscriptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intron. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields reliable measurements of relative transcription rates.

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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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TASOR epigenetic repressor cooperates with a CNOT1 RNA degradation pathway to repress HIV

10.1101/2020.11.22.386656 ◽

2020 ◽

Author(s):

Roy Matkovic ◽

Marina Morel ◽

Pauline Larrous ◽

Benjamin Martin ◽

Fabienne Bejjani ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Degradation ◽

Degradation Pathway ◽

Transcriptional Level ◽

Heterochromatin Formation ◽

Nascent Transcripts ◽

Ribosomal Dnas

AbstractThe Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin is involved in the spreading of H3K9me3 repressive marks across genes and transgenes such as ZNF encoding genes, ribosomal DNAs, LINE-1, Retrotransposons and Retroelements or the integrated HIV provirus1–5. The deposit of these repressive marks leads to heterochromatin formation and inhibits gene expression. The precise mechanisms of silencing mediated by HUSH is still poorly understood. Here, we show that TASOR depletion increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. By counteracting HUSH, Vpx from HIV-2 mimics TASOR depletion. With the use of a Yeast-Two-Hybrid screen, we identified new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 interact in vivo and synergistically repress HIV expression from its LTR. In fission yeast, the RNA-induced transcriptional silencing (RITS) complex presents structural homology with HUSH. During transcription elongation by RNA polymerase II, RITS recruits a TRAMP-like RNA degradation complex composed of CNOT1 partners, MTR4 and the exosome, to ultimately repress gene expression via H3K9me3 deposit. Similarly, we show that TASOR interacts and cooperates with MTR4 and the exosome, in addition to CNOT1. We also highlight an interaction between TASOR and RNA Polymerase II, predominantly under its elongating state, and between TASOR and some HUSH-targeted nascent transcripts. Furthermore, we show that TASOR overexpression facilitates the association of the aforementioned RNA degradation proteins with RNA polymerase II. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral gene expression.

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Molecular Modeling of RNA Polymerase II Mutations onto DNA Polymerase I

Journal of Molecular Biology ◽

10.1006/jmbi.1994.1700 ◽

1994 ◽

Vol 244 (1) ◽

pp. 13-22 ◽

Cited By ~ 15

Author(s):

Wan Joon Kim ◽

Lillian P. Burke ◽

Mark A. Mortin

Keyword(s):

Molecular Modeling ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Polymerase ◽

Dna Polymerase I ◽

Polymerase I

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Immunoglobulin switch μ sequence causes RNA polymerase II accumulation and reduces dA hypermutation

Journal of Experimental Medicine ◽

10.1084/jem.20082514 ◽

2009 ◽

Vol 206 (6) ◽

pp. 1237-1244 ◽

Cited By ~ 79

Author(s):

Deepa Rajagopal ◽

Robert W. Maul ◽

Amalendu Ghosh ◽

Tirtha Chakraborty ◽

Ahmed Amine Khamlichi ◽

...

Keyword(s):

B Cells ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Polymerase ◽

Dna Sequences ◽

Repetitive Sequence ◽

Ex Vivo ◽

Dna Structure ◽

Switch Region ◽

Activation Induced Deaminase

Repetitive DNA sequences in the immunoglobulin switch μ region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5-kb region spanning the intronic enhancer to the constant μ gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)–deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1-kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung−/− and Aid−/−Ung−/− mice but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung−/− B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, which are incorporated by DNA polymerase η, decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase η during hypermutation.

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Active RNA polymerases are localized within discrete transcription “factories' in human nuclei

Journal of Cell Science ◽

10.1242/jcs.109.6.1427 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1427-1436 ◽

Cited By ~ 9

Author(s):

F.J. Iborra ◽

A. Pombo ◽

D.A. Jackson ◽

P.R. Cook

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Hela Cells ◽

Rna Polymerases ◽

Transcription Factories ◽

Nascent Rna ◽

Nascent Transcripts ◽

Typical Site

Nascent transcripts in permeabilized HeLa cells were elongated by approximately 30–2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in approximately 2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription ‘factories’. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.

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Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing.

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7219 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7219-7225 ◽

Cited By ~ 146

Author(s):

J Wuarin ◽

U Schibler

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

S1 Nuclease ◽

Nonhistone Proteins ◽

Detailed Structural Analysis ◽

Physical Isolation ◽

Transcriptional Activator Protein ◽

Nascent Rna ◽

Nuclease Mapping ◽

Nascent Transcripts

In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chains transcribed by RNA polymerase II. A procedure that allows fractionation of nuclei into a chromatin pellet containing DNA, histones, and ternary transcription complexes and a supernatant containing the bulk of the nonhistone proteins and RNAs that are released from their DNA templates was developed. The transcripts of the genes encoding DBP, a transcriptional activator protein, and HMG coenzyme A reductase recovered from the chromatin pellet and the supernatant were analyzed by S1 nuclease mapping. The large majority of the RNA molecules from the pellet appeared to be nascent transcripts, since, in contrast to the transcripts present in the supernatant, they were not cleaved at the polyadenylation site but rather contained heterogeneous 3' termini encompassing this site. Splicing intermediates could be detected among nascent and released transcripts, suggesting that splicing occurs both cotranscriptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intron. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields reliable measurements of relative transcription rates.

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Evidence that Negative Elongation Factor Represses Transcription Elongation through Binding to a DRB Sensitivity-Inducing Factor/RNA Polymerase II Complex and RNA

Molecular and Cellular Biology ◽

10.1128/mcb.22.9.2918-2927.2002 ◽

2002 ◽

Vol 22 (9) ◽

pp. 2918-2927 ◽

Cited By ~ 125

Author(s):

Yuki Yamaguchi ◽

Naoto Inukai ◽

Takashi Narita ◽

Tadashi Wada ◽

Hiroshi Handa

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Protein Interactions ◽

Rna Binding ◽

Elongation Factor ◽

Protein Protein Interactions ◽

Negative Elongation Factor ◽

Nascent Transcripts

ABSTRACT Negative elongation factor (NELF) is a human transcription factor complex that cooperates with DRB sensitivity-inducing factor (DSIF)/hSpt4-hSpt5 to repress elongation by RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, including NELF-A, a candidate gene product for Wolf-Hirschhorn syndrome, and NELF-E, a putative RNA-binding protein with arginine-aspartic acid (RD) dipeptide repeats. Here we report several important findings regarding the DSIF/NELF-dependent elongation control. First, we have established an effective method for purifying the active NELF complex using an epitope-tagging technique. Second, the five polypeptides each are important and together are sufficient for its function in vitro. Third, NELF does not bind to either DSIF or RNAPII alone but does bind to the preformed DSIF/RNAPII complex. Fourth, NELF-E has a functional RNA-binding domain, whose mutations impair transcription repression without affecting known protein-protein interactions. Taken together, we propose that NELF causes RNAPII pausing through binding to the DSIF/RNAPII complex and to nascent transcripts. These results also have implications for how DSIF and NELF are regulated in a gene-specific manner in vivo.

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Ultrastructural Analysis of Transcription and Splicing in the Cell Nucleus after Bromo-UTP Microinjection

Molecular Biology of the Cell ◽

10.1091/mbc.10.1.211 ◽

1999 ◽

Vol 10 (1) ◽

pp. 211-223 ◽

Cited By ~ 164

Author(s):

Dusan Cmarko ◽

Pernette J. Verschure ◽

Terence E. Martin ◽

Michael E. Dahmus ◽

Sabine Krause ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Ultrastructural Level ◽

Structural Domain ◽

Mrna Transcription ◽

Rna Transcription ◽

Core Proteins ◽

Transcription Sites ◽

Nascent Transcripts

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

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