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

Nakamura, Michio; Basavarajaiah, Poornima; Rousset, Emilie; Beraud, Cyprien; Latreille, Daniel; Henaoui, Imène-Sarah; Lassot, Irina; Mari, Bernard; Kiernan, Rosemary

doi:10.1186/1742-4690-9-13

Cited by 15 publications

(16 citation statements)

References 36 publications

(71 reference statements)

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“…Target sequences are shown in Extended Experimental Procedures. PCR primer sequences to amplify TAR, early, luc (coding region), luc 3′, and GAPDH-Q have been described elsewhere (Nakamura et al, 2012). Sequences of additional oligonucleotide pairs used are shown in Extended Experimental Procedures.…”

Section: Methodsmentioning

confidence: 99%

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

Wagschal¹,

Rousset²,

Basavarajaiah³

et al. 2012

Cell

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166

162

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SUMMARY Transcription 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.

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Section: Methodsmentioning

confidence: 99%

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

Wagschal¹,

Rousset²,

Basavarajaiah³

et al. 2012

Cell

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166

162

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“…This study and others aimed at understanding the basic mechanisms of Pol I activity are crucial to provide a framework for future therapies. Spt6 is conserved between yeast and humans, and knockdown of hSpt6 alters the expression of many tumor suppressor and proto-oncogenes (75). Interestingly, hSpt6 has also been shown to physically interact with SIRT7 (76), which in turn binds Pol I.…”

Section: Discussionmentioning

confidence: 99%

Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I

Engel

French

Viktorovskaya

et al. 2015

Molecular and Cellular Biology

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b Spt6 (suppressor of Ty6) has many roles in transcription initiation and elongation by RNA polymerase (Pol) II. These effects are mediated through interactions with histones, transcription factors, and the RNA polymerase. Two lines of evidence suggest that Spt6 also plays a role in rRNA synthesis. First, Spt6 physically associates with a Pol I subunit (Rpa43). Second, Spt6 interacts physically and genetically with Spt4/5, which directly affects Pol I transcription. Utilizing a temperature-sensitive allele, spt6-1004, we show that Spt6 is essential for Pol I occupancy of the ribosomal DNA (rDNA) and rRNA synthesis. Our data demonstrate that protein levels of an essential Pol I initiation factor, Rrn3, are reduced when Spt6 is inactivated, leading to low levels of Pol I-Rrn3 complex. Overexpression of RRN3 rescues Pol I-Rrn3 complex formation; however, rRNA synthesis is not restored. These data suggest that Spt6 is involved in either recruiting the Pol I-Rrn3 complex to the rDNA or stabilizing the preinitiation complex. The findings presented here identify an unexpected, essential role for Spt6 in synthesis of rRNA. Three eukaryotic RNA polymerases (Pols), Pol I, Pol II, and Pol III, have critical and yet distinct roles in gene expression. Pol II synthesizes mainly mRNAs, which serve as the templates for translation and give rise to the diverse collection of cellular proteins. Pols I and III are responsible for producing the stable RNA components of the translation machinery, rRNA and tRNA, respectively. In Saccharomyces cerevisiae (yeast), Pol I transcribes the ribosomal DNA (rDNA) to generate the 35S rRNA precursor within a subcompartment of the nucleus called the nucleolus. This precursor is co-and posttranscriptionally processed into the mature 25S, 18S, and 5.8S rRNAs and assembled into ribosomes. Pol III synthesizes all tRNAs and the 5S rRNA. The highly repetitive nature of the rDNA (ϳ200 tandem copies of the rDNA in yeast) helps cells meet the exceptional demand for protein synthesis and, therefore, for ribosomes. However, only about half of the rDNA repeats are actively transcribed at any given time (1, 2).There is an intimate connection between ribosome biogenesis, protein synthesis, and cell growth capacity. It was observed over 100 years ago that cancer cells have enlarged nucleoli compared to those of normal host cells (3). We now know that the rate of rRNA synthesis is increased in many forms of cancer (4, 5). A number of laboratories have recently shown that selective inhibition of Pol I transcription is an effective method for impairing tumor cell growth (6-8). Taken together, both the old insights and the new insights agree that Pol I is emerging as a promising chemotherapeutic target.Despite the central role Pol I plays during cell growth, a detailed understanding of its regulation is lacking. However, a collection of trans-acting factors have been shown to influence Pol I at the initiation or elongation step in transcription. One such complex is suppressors of Ty4 and -5 (Spt4/5), which was prev...

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“…Previous studies have demonstrated that the base, base-like complex, and RP function in transcription, independent of the proteolytic function of the proteasome, implying that the proteasome subcomplexes may have specific roles in cells [43][44][45] . However, our results suggest that the 26S proteasome is a very stable complex, and that free base, RP, and subunits are not present at detectable levels anywhere in the cell, at least under normal culture conditions.…”

Section: Discussionmentioning

confidence: 99%

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

et al. 2014

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The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-a mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.

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

Cited by 15 publications

References 36 publications

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

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

Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

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