Spt6 levels are modulated by PAAF1 and proteasome to regulate the HIV-1 LTR

<p>Abstract</p> <p>Background</p> <p>Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR.</p> <p>Results</p> <p>Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1.</p> <p>Conclusion</p> <p>These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.</p

Microprocessor, Setx, Xrn2, and Rrp6 Co-operate to Induce Premature Termination of Transcription by RNAPII

SummaryTranscription elongation is increasingly recognized as an important mechanism of gene regulation. Here, we show that microprocessor controls gene expression in an RNAi-independent manner. Microprocessor orchestrates the recruitment of termination factors Setx and Xrn2, and the 3′–5′ exoribonuclease, Rrp6, to initiate RNAPII pausing and premature termination at the HIV-1 promoter through cleavage of the stem-loop RNA, TAR. Rrp6 further processes the cleavage product, which generates a small RNA that is required to mediate potent transcriptional repression and chromatin remodeling at the HIV-1 promoter. Using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq), we identified cellular gene targets whose transcription is modulated by microprocessor. Our study reveals RNAPII pausing and premature termination mediated by the co-operative activity of ribonucleases, Drosha/Dgcr8, Xrn2, and Rrp6, as a regulatory mechanism of RNAPII-dependent transcription elongation

The Role of Rrp6 in Gene Expression

L'objectif de mon travail est de comprendre le rôle de Rrp6, une exoribonuclease 3'-5', dans l'expression des gènes. Dans ce but, j'ai utilisé le promoteur du virus de l'immunodéficience humaine (VIH-1) comme modèle d'étude de la régulation des gènes chez les mammifères. En utilisant ce modèle dans le chapitre 1 des résultats, nous avons montré l'existence d'un nouveau mécanisme de répression de l'expression des gènes dépendant de l'ARN qui requiert les actions combinées de Rrp6 et du microprocesseur. A la suite de ce travail, nous avons caractérisé les complexes de protéines associés à Rrp6 qui contribuent à cette répression de la transcription (résultats - chapitre 2). Ces deux études suggèrent un rôle de Rrp6 dans la répression de la transcription au niveau du promoteur du VIH-1 mais aussi sur certains gènes cellulaires. Au cours des études présentées dans le chapitre 1, nous avons observé une forte diminution de l'expression de la protéine Dicer dans les cellules déplétées de Rrp6. Dicer est un élément central de la régulation de la maturation des microARN (miRNA) et donc joue un rôle important dans tous les processus cellulaires qui sont régulés par les miRNA, incluant de nombreux processus biologiques et physiologiques. Ainsi, il est important de connaitre les voies de régulation de Dicer. Dans le chapitre 3 des résultats, nous décrivons un nouveau mécanisme de régulation de Dicer par Rrp6. En effet, nos résultats montrent que Rrp6 est nécessaire pour un epissage efficace de l'ARNm de Dicer. Nos travaux décrivent un nouveau role de Rrp6 dans des processus cellulaires distincts: transcription et splicingThe objective of my doctoral work was to understand the role of a 3' to 5' exoribonuclease, Rrp6, in gene expression. I used the Human Immunodeficiency Virus (HIV-1) promoter as a model to study gene regulation in mammalian cells. Using this model, in Result-chapter 1, we demonstrated a novel mechanism of RNA-dependent transcriptional gene silencing that depends on the cooperative activities of Rrp6 and microprocessor. Following this study, we characterized the Rrp6-containing complex that contributes to the transcriptional silencing at HIV-1 promoter (Result-chapter 2). These two studies suggest a role for Rrp6 in transcriptional repression at the HIV-1 promoter and also at a subset of cellular genes. During the course of our studies presented in chapter 1, we observed a dramatic decrease of Dicer protein level in the cells depleted of Rrp6. Dicer is a central regulator of microRNA (miRNA) maturation and therefore exerts an important role in all cellular processes that are regulated by miRNAs, including diverse biological and physiological processes. Thus, it is important to know how Human Dicer1 is regulated. In Result-chapter 3, we describe a new regulatory mechanism of Dicer1 expression by Rrp6. Indeed, our results demonstrate that Rrp6 is required for efficient splicing of Dicer1 mRNA. Our work describes a novel role for Rrp6 in distinct cellular processes: transcription and splicing.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

The Polyvalent Role of NF90 in RNA Biology

Double-stranded RNA-binding proteins (dsRBPs) are major players in the regulation of gene expression patterns. Among them, Nuclear Factor 90 (NF90) has a plethora of well-known functions in viral infection, transcription, and translation as well as RNA stability and degradation. In addition, NF90 has been identified as a regulator of microRNA (miRNA) maturation by competing with Microprocessor for the binding of pri-miRNAs in the nucleus. NF90 was recently shown to control the biogenesis of a subset of human miRNAs, which ultimately influences, not only the abundance, but also the expression of the host gene and the fate of the mRNA target repertoire. Moreover, recent evidence suggests that NF90 is also involved in RNA-Induced Silencing Complex (RISC)-mediated silencing by binding to target mRNAs and controlling their translation and degradation. Here, we review the many, and growing, functions of NF90 in RNA biology, with a focus on the miRNA pathway and RISC-mediated gene silencing

Des complexes protéiques impliqués dans la surveillance de l’ARN nucléaire inhibent la transcription du VIH-1

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PAF1 facilitates RNA polymerase II ubiquitination by the Elongin A complex through phosphorylation by CDK12

The conserved Polymerase-Associated Factor 1 complex (PAF1C) regulates all stages of the RNA polymerase II (RNAPII) transcription cycle from the promoter to the 3’ end formation site of mRNA encoding genes and has been linked to numerous transcription related processes. Here, we show that PAF1 interacts with Elongin A, a transcription elongation factor as well as a component of a cullin-RING ligase that targets stalled RNAPII for ubiquitination and proteasome-dependent degradation in response to DNA damage or other stresses. We show that, in absence of any induced stress, PAF1 physically interacts with the E3 ubiquitin ligase form of the Elongin A complex and facilitates ubiquitination of RNAPII. We demonstrate that this ubiquitination is dependent of the Ser2 phosphorylation of the RNAPII carboxy-terminal domain (CTD) by CDK12. Our findings highlight a novel unexpected role of PAF1-CDK12 in RNAPII transcription cycle, raising the possibility that the Elongin A ubiquitin ligase plays a role in normal transcription process, and suggest a transcription surveillance mechanism ready to degrade RNAPII if needed