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DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts

ABSTRACT: The timely termination of RNA polymerase II (Pol II) transcription is essential for recycling of this enzyme and preventing transcriptional interference. While 5'-3' exonuclease Xrn2 is known to degrade nascent RNA after poly(A) site cleavage to promote Pol II release, its molecular mechanism remains not fully understood. Spt5 is one of highly conserved transcription factors that controls and regulates multiple aspects of Pol II transcription. Here, using mass spectrometry analysis of immunoprecipitated Flag-tagged Spt5-WT and Spt5-mutant complexes from fission yeast cells, we show that Xrn2 is highly enriched in these samples. Through in vitro BS3 crosslinking experiments with purified components, we further demonstrated that Xrn2 associates with transcription machinery and we probed interfaces between different subunits of transcription machinery

INSTRUMENT(S): Q Exactive

ORGANISM(S): Schizosaccharomyces Pombe

SUBMITTER: Vaishnavi Ravikumar

LAB HEAD: Lidia Vasilieva

PROVIDER: PXD058168 | Pride | 2024-11-22

REPOSITORIES: Pride

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			Action	DRS
	KK_APMS_SPT5_GLY_FDR0.01.xlsx	Xlsx
	KK_FlagSPTWT_T1A_T1E_FDR0.01_Uniprot.xlsx	Xlsx
	QEX01_MF_210720_KK_XL_145.raw	Raw
	QEX02_MF_210420_KK1_173.raw	Raw
	QEX02_MF_210420_KK2_175.raw	Raw

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Similar Datasets

DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts

Project description:RNA polymerase II (Pol II) termination is a crucial step in the transcriptional cycle, ensuring the recycling of Pol II and preventing interference with neighboring gene transcription. Xrn2 is an enzyme with 5'-3'- exonuclease activity, responsible for degrading RNA that is still being transcribed by Pol II after cleavage at the poly(A) site, ultimately leading to Pol II termination. We have confirmed that Xrn2's catalytic activity is essential for timely transcription termination. Furthermore, we have discovered a link between Xrn2 and the conserved, multifunctional transcription factor Spt5. Importantly, our research demonstrates that Xrn2 interacts with Pol II/Spt5 complexes. Additionally, when Spt5 is depleted, it not only impairs global transcription but also results in defective transcription termination. On the other hand, absence of Spt5 leads to the derepression of non-coding transcription and premature termination/attenuation at the 5'-end of coding genes. We propose that Spt5 acts as a "licensing" factor, ensuring that Pol II complexes are properly assembled for efficient transcription and co-transcriptional pre-mRNA processing.

2024-09-30 | GSE244546 | GEO

DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts.

Project description:RNA polymerase II (Pol II) termination is a crucial step in the transcriptional cycle, ensuring the recycling of Pol II and preventing interference with neighbouring gene transcription. Xrn2 is an enzyme with 5'-3'- exonuclease activity, responsible for degrading RNA that is still being transcribed by Pol II after cleavage at the poly(A) site, ultimately leading to Pol II termination. We have confirmed that Xrn2's catalytic activity is essential for timely transcription termination. Furthermore, we have discovered a link between Xrn2 and the conserved, multifunctional transcription factor Spt5. Importantly, our research demonstrates that Xrn2 interacts with Pol II/Spt5 complexes. Additionally, when Spt5 is depleted, it not only impairs global transcription but also results in defective transcription termination. On the other hand, absence of Spt5 leads to the derepression of non-coding transcription and premature termination/attenuation at the 5'-end of coding genes. We propose that Spt5 acts as a "licensing" factor, ensuring that Pol II complexes are properly assembled for efficient transcription and co-transcriptional pre-mRNA processing.

2024-09-30 | GSE273510 | GEO

mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription

Project description:We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a and Dcp2 and the termination factor TTF2 co-immunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease torpedo that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2 and TTF2 localize near transcription start sites (TSSs) by ChIP-Seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors, Xrn2 and TTF2, shifted polymerase away from the TSS toward upstream and downstream distal positions. This re-distribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the torpedo mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated co-transcriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation. RNA pol II (GSE30895: GSM766171), Xrn2, TTF2 and Dcp1a were localized by ChIP-Seq in HeLa cells. RNA pol II was localized in control HEK293 cells and cells infected with lentiviruses expressing a scrambled control shRNA (scr), and shRNAs targeting the following proteins: Xrn2, TTF2, Xrn2+TTF2, Edc3, Dcp1a, and Dcp2.

2012-04-04 | E-GEOD-36185 | biostudies-arrayexpress

Control of RNA pol II speed by PNUTS-PP1 and Spt5 dephosphorylation facilitates termination by a “sitting duck torpedo” mechanism

Project description:Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA pol II elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, pol II decelerates from >2kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a new model for termination, the “sitting duck torpedo” mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homologue NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.

2019-10-30 | GSE134198 | GEO

mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription

2012-04-05 | GSE36185 | GEO

siRNA profiling of Zebrafish embryos knocked down for Spt5

Project description:The Spt4-Spt5 complex, and its human homolog DSIF (DRB sensitivity-inducing factor), is unique in its ability to regulate Pol II processivity. Previous studies have shown that Spt5 has the characteristics of a general transcription-elongation factor. However, mutagenesis of Spt5 showed specific phenotypes during development, which were far less severe than those of Pol II defects or TBP deficient embryos. It seems paradoxical that a mutation which alters a general elongation factor can cause rather specific developmental defects. By using Spt5 knockdown zebrafish embryos and microarrays, here we showed that transcript abundance for only a small subset of genes is altered by loss of Spt5. Further investigation of the down-regulated genes showed that the genes most intensely repressed by the knockdown were strongly activated during early development in untreated embryos. Thus, this study shows that gene activation levels may create different requirements for Pol II processivity. Active transcription requires Spt5 for efficient elongation through its stimulatory activity on Pol II processivity. Experiment Overall Design: The expression pattern of Spt5 knockdown embryos was compared to that of the control mopholino oligo injected embryos, both at 21 hours post-fertilization, to show the targets of Spt5. The activation levels of the Spt5-knockdown affected genes were measured by compareing the abundance of transcripts in 21 hours post-fertilization untreated embryos to that in 5 hours post-fertilization untreated embryos.

2008-06-14 | E-GEOD-6127 | biostudies-arrayexpress

Structural basis of human transcription-DNA repair coupling

Project description:Transcription-coupled DNA repair (TCR) efficiently removes bulky DNA lesions from transcribed parts of the genome and constitutes a major defence against DNA damage by UV irradiation. TCR begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CsB, the E3 ubiquitin ligase complex CRL4CsA, and the UV-stimulated scaffold protein A (UVSSA). Here we provide five high-resolution structures of Pol II transcription complexes with bound TCR factors and complementary biochemical data. Together with published work, our results converge on a model for the molecular mechanism of transcription-DNA repair coupling. In this model, Pol II stalling triggers the formation of an alternative transcription elongation complex that we call ECact. In ECact, CsB has replaced the elongation factor DSIF, binds the PAF1 complex, and repositions upstream DNA such that it contacts the elongation factor SPT6. The CsB translocase now pulls on the upstream DNA template, thereby pushing Pol II forward. If the lesion cannot be bypassed, Pol II gets stably stalled. CRL4CsA spans over the Pol II clamp to reach the Pol II jaw and direct ubiquitination of RPB1 residue lysine-1268. This triggers the recruitment of TFIIH to UVSSA, which is located at downstream DNA, and enables nucleotide excision repair. Finally, large-scale conformational changes in CRL4CsA enable ubiquitination of CsB and the release of TCR factors, thereby allowing Pol II to resume elongation and continue transcription over repaired DNA.

2022-02-17 | PXD025328 | Pride

ELOF1 anchors CRL4CSA to RNA pol II to activate transcription-coupled repair

Project description:Transcription-coupled DNA repair (TCR) removes transcription-blocking DNA lesions through the coordinated assembly of repair factors on arrested RNA polymerase II (Pol II). This process requires the site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase. Pol II ubiquitylation is facilitated by ELOF1, a recently identified TCR factor. Using cryogenic electron microscopy, biochemical assays, and cell biology, we reveal that ELOF1 functions as an adaptor to correctly dock CRL4CSA onto Pol II and facilitate the recruitment of UVSSA. The ELOF1-CSA-UVSSA interaction positions CRL4CSA on arrested Pol II, leading to its ubiquitylation upon ligase activation by neddylation. ELOF1-mediated repositioning enables a TFIIS-like element in UVSSA to reach the Pol II active site and the pore region to prevent Pol II re-activation. These findings provide the structural and molecular basis underlying the transition of Pol II from a transcriptionally active to an arrested state, primed for DNA repair.

2023-11-24 | PXD042388 | Pride

ChIP-chip by array of yeast to determine genome-wide occupancy profiles for RNA polymerase (Pol) II, its phosphorylated forms, and transcription factors

Project description:To investigate the chromatin transcription cycle, we determined genome-wide occupancy profiles for RNA polymerase (Pol) II, its phosphorylated forms, and transcription factors in growing yeast. ChIP-chip was performed to identify the genomic binding locations for Rpb3, TFIIB, Tfg1, Kin28, Cet1, Spt4, Spt5, Spt6, Elf1, Spn1, Bur1, Ctk1, Paf1, Spt16, Pcf11, and Rpb1 phosphorylated at serine 2, 5, and 7 residues of the CTD, respectively.

2010-08-23 | E-TABM-1033 | biostudies-arrayexpress

Genome wide localization of Activation induced cytidine deaminase (AID), Spt5, and RNA Polymerase II (PolII) in mouse

Project description:Activation induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes. Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these key transactions little is known about how AID finds its target sites. To examine AID regulation we performed an shRNA screen. We found that Spt5, a factor associated with paused RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for class switch recombination (CSR). Spt5 interacts with AID, it is required for the association of AID and Pol II, and for AID recruitment to switch regions. ChIP-seq experiments reveal that Spt5 co-localizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II pausing is predictive of AID-induced mutation. Our data suggest that Spt5 acts as an adaptor, which brings AID to Pol II on target DNA. PolII, AID, and Spt5 were immunoprecipitated from in vitro activated B cells. For Spt5 and AID, two different antibodies were used in separate, biological replicates

2010-10-05 | E-GEOD-24178 | biostudies-arrayexpress