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

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Roy Matkovic ◽  
Marina Morel ◽  
Sophie Lanciano ◽  
Pauline Larrous ◽  
Benjamin Martin ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Integration Site ◽  
Underlying Mechanism ◽  
Rna Degradation ◽  
Degradation Pathway ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Methyl Transferase ◽  
Heterochromatin Formation

AbstractThe Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin recruits the histone methyl-transferase SETDB1 to spread H3K9me3 repressive marks across genes and transgenes in an integration site-dependent manner. The deposition of these repressive marks leads to heterochromatin formation and inhibits gene expression, but the underlying mechanism is not fully understood. Here, we show that TASOR silencing or HIV-2 Vpx expression, which induces TASOR degradation, increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. Furthermore, using a yeast 2-hybrid screen, we identify new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 synergistically repress HIV expression from its LTR. Similar to the RNA-induced transcriptional silencing complex found in fission yeast, we show that TASOR interacts with the RNA exosome and RNA Polymerase II, predominantly under its elongating state. Finally, we show that TASOR facilitates the association of RNA degradation proteins with RNA polymerase II and is detected at transcriptional centers. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral expression.

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

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.

scholarly journals RMP, a Novel RNA Polymerase II Subunit 5-Interacting Protein, Counteracts Transactivation by Hepatitis B Virus X Protein

1998 ◽  
Vol 18 (12) ◽  
pp. 7546-7555 ◽  
Author(s):  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Wenxiang Wei ◽  
Tatsuya Yamashita ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Host Protein ◽  
Dependent Manner ◽  
Binding Region ◽  
X Protein ◽  
B Virus

ABSTRACT To modulate transcription, regulatory factors communicate with basal transcription factors and/or RNA polymerases in a variety of ways. Previously, it has been reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of hepatitis B virus X protein (HBx) and that both HBx and RPB5 specifically interact with general transcription factor IIB (TFIIB), implying that RPB5 is one of the communicating subunits of RNA polymerase II involved in transcriptional regulation. In this context, we screened for a host protein(s) that interacts with RPB5. By far-Western blot screening, we cloned a novel gene encoding a 508-amino-acid-residue RPB5-binding protein from a HepG2 cDNA library and designated it RPB5-mediating protein (RMP). Expression of RMP mRNA was detected ubiquitously in various tissues. Bacterially expressed recombinant RMP strongly bound RPB5 but neither HBx nor TATA-binding protein in vitro. Endogenous RMP was immunologically detected interacting with assembled RPB5 in RNA polymerase in mammalian cells. The central part of RMP is responsible for RPB5 binding, and the RMP-binding region covers both the TFIIB- and HBx-binding sites of RPB5. Overexpression of RMP, but not mutant RMP lacking the RPB5-binding region, inhibited HBx transactivation of reporters with different HBx-responsive cis elements in transiently transfected cells. The repression by RMP was counteracted by HBx in a dose-dependent manner. Furthermore, RMP has an inhibitory effect on transcriptional activation by VP16 in the absence of HBx. These results suggest that RMP negatively modulates RNA polymerase II function by binding to RPB5 and that HBx counteracts the negative role of RMP on transcription indirectly by interacting with RPB5.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals Sub1 associates with Spt5 and influences RNA polymerase II transcription elongation rate

2012 ◽  
Vol 23 (21) ◽  
pp. 4297-4312 ◽  
Author(s):  
Alicia García ◽  
Alejandro Collin ◽  
Olga Calvo
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Molecular Data ◽  
Elongation Rate ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Mrna Metabolism ◽  
Carboxy Terminal

The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3′-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5–Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5–Rpb1 complex levels and consequently transcription elongation rate.

scholarly journals Structural basis of transcriptional stalling and bypass of abasic DNA lesion by RNA polymerase II

2018 ◽  
Vol 115 (11) ◽  
pp. E2538-E2545 ◽  
Author(s):  
Wei Wang ◽  
Celine Walmacq ◽  
Jenny Chong ◽  
Mikhail Kashlev ◽  
Dong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Lesions ◽  
Structural Motif ◽  
Structural Basis ◽  
Dependent Manner ◽  
Abasic Site ◽  
Structural Explanation ◽  
Pol Ii ◽  
Abasic Sites

Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3′-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.

scholarly journals Human RNA cap1 methyltransferase CMTr1 cooperates with RNA helicase DHX15 to modify RNAs with highly structured 5′ termini

2018 ◽  
Vol 373 (1762) ◽  
pp. 20180161 ◽  
Author(s):  
Diana Toczydlowska-Socha ◽  
Magdalena M. Zielinska ◽  
Malgorzata Kurkowska ◽  
Astha ◽  
Catarina F. Almeida ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Degradation ◽  
Rna Modification ◽  
Structure Characteristic ◽  
Helicase Activity ◽  
Theme Issue ◽  
Rna Capping ◽  
Advantageous Effect

The 5′-cap structure, characteristic for RNA polymerase II-transcribed RNAs, plays important roles in RNA metabolism. In humans, RNA cap formation includes post-transcriptional modification of the first transcribed nucleotide by RNA cap1 methyltransferase (CMTr1). Here, we report that CMTr1 activity is hindered towards RNA substrates with highly structured 5′ termini. We found that CMTr1 binds ATP-dependent RNA DHX15 helicase and that this interaction, mediated by the G-patch domain of CMTr1, has an advantageous effect on CMTr1 activity towards highly structured RNA substrates. The effect of DHX15 helicase activity is consistent with the strength of the secondary structure that has to be removed for CMTr1 to access the 5′-terminal residues in a single-stranded conformation. This is, to our knowledge, the first demonstration of the involvement of DHX15 in post-transcriptional RNA modification, and the first example of a molecular process in which DHX15 directly affects the activity of another enzyme. Our findings suggest a new mechanism underlying the regulatory role of DHX15 in the RNA capping process. RNAs with highly structured 5′ termini constitute a significant fraction of the human transcriptome. Hence, CMTr1–DHX15 cooperation is likely to be important for the metabolism of RNA polymerase II-transcribed RNAs. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

scholarly journals Identification of a Specific Inhibitor of Human Scp1 Phosphatase Using the Phosphorylation Mimic Phage Display Method

Catalysts ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 842 ◽  
Author(s):  
Takuya Yoshida ◽  
Kazuki Yamazaki ◽  
Shunta Imai ◽  
Akinori Banno ◽  
Atsushi Kaneko ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Phage Display ◽  
Rna Polymerase Ii ◽  
Active Center ◽  
Protein Phosphatases ◽  
Specific Inhibitor ◽  
Competitive Inhibitor ◽  
Dependent Manner ◽  
Simple Method ◽  
Ctd Phosphatase

Protein phosphatases are divided into tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. While substrate trapping mutants are frequently used to identify substrates of Tyr phosphatases, a rapid and simple method to identify Ser/Thr phosphatase substrates is yet to be developed. The TFIIF-associating component of RNA polymerase II C-terminal domain (CTD) phosphatase/small CTD phosphatase (FCP/SCP) phosphatase family is one of the three types of Ser/Thr protein phosphatases. Defects in these phosphatases are correlated with the occurrence of various diseases such as cancer and neuropathy. Recently, we developed phosphorylation mimic phage display (PMPD) method with AlF4−, a methodology to identify substrates for FCP/SCP type Ser/Thr phosphatase Scp1. Here, we report a PMPD method using BeF3− to identify novel substrate peptides bound to Scp1. After screening peptide phages, we identified peptides that bound to Scp1 in a BeF3−-dependent manner. Synthetic phosphopeptide BeM12-1, the sequence of which was isolated at the highest frequency, directly bound to Scp1. The binding was inhibited by adding BeF3−, indicating that the peptide binds to the active center of catalytic site in Scp1. The phosphorylated BeM12-1 worked as a competitive inhibitor of Scp1. Thus, PMPD method may be applicable for the identification of novel substrates and inhibitors of the FCP/SCP phosphatase family.

scholarly journals “Maturational” globin switching in primary primitive erythroid cells

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1665-1672 ◽  
Author(s):  
Paul D. Kingsley ◽  
Jeffrey Malik ◽  
Rachel L. Emerson ◽  
Timothy P. Bushnell ◽  
Kathleen E. McGrath ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Expression Patterns ◽  
Yolk Sac ◽  
Transcriptional Level ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Transcript Levels ◽  
Chromosomal Arrangement ◽  
Sequential Appearance

Mammals have 2 distinct erythroid lineages. The primitive erythroid lineage originates in the yolk sac and generates a cohort of large erythroblasts that terminally differentiate in the bloodstream. The definitive erythroid lineage generates smaller enucleated erythrocytes that become the predominant cell in fetal and postnatal circulation. These lineages also have distinct globin expression patterns. Our studies in primary murine primitive erythroid cells indicate that βH1 is the predominant β-globin transcript in the early yolk sac. Thus, unlike the human, murine β-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a βH1- to ϵy-globin switch, up-regulate adult β1- and β2-globins, and down-regulate ζ-globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions. Furthermore, the ϵy- and βH1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. These data suggest that this “maturational” βH1- to ϵy-globin switch is dynamically regulated at the transcriptional level. Globin switching during ontogeny is due not only to the sequential appearance of primitive and definitive lineages but also to changes in globin expression as primitive erythroblasts mature in the bloodstream.

scholarly journals Nucleocytoplasmic Shuttling of Polypyrimidine Tract-binding Protein Is Uncoupled from RNA Export

2001 ◽  
Vol 12 (12) ◽  
pp. 3808-3820 ◽  
Author(s):  
Rajesh V. Kamath ◽  
Daniel J. Leary ◽  
Sui Huang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Binding Protein ◽  
Dependent Manner ◽  
Polypyrimidine Tract ◽  
Nucleocytoplasmic Shuttling ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Rna Export

Polypyrimidine tract binding protein, PTB/hnRNP I, is involved in pre-mRNA processing in the nucleus and RNA localization and translation in the cytoplasm. In this report, we demonstrate that PTB shuttles between the nucleus and cytoplasm in an energy-dependent manner. Deletion mutagenesis demonstrated that a minimum of the N terminus and RNA recognition motifs (RRMs) 1 and 2 are necessary for nucleocytoplasmic shuttling. Deletion of RRM3 and 4, domains that are primarily responsible for RNA binding, accelerated the nucleocytoplasmic shuttling of PTB. Inhibition of transcription directed by either RNA polymerase II alone or all RNA polymerases yielded similar results. In contrast, selective inhibition of RNA polymerase I did not influence the shuttling kinetics of PTB. Furthermore, the intranuclear mobility of GFP-PTB, as measured by fluorescence recovery after photobleaching analyses, increased significantly in transcriptionally inactive cells compared with transcriptionally active cells. These observations demonstrate that nuclear RNA transcription and export are not necessary for the shuttling of PTB. In addition, binding to nascent RNAs transcribed by RNA polymerase II and/or III retards both the nuclear export and nucleoplasmic movement of PTB. The uncoupling of PTB shuttling and RNA export suggests that the nucleocytoplasmic shuttling of PTB may also play a regulatory role for its functions in the nucleus and cytoplasm.

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