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Spt6 Association with RNA Polymerase II Directs mRNA Turnover During Transcription.

ABSTRACT: Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 also associates with RNA polymerase II (RNAPII) via a tandem Src2 homology domain. However, the significance of Spt6-RNAPII interaction is not well understood. Here, we show that Spt6 recruitment to genes and the nucleosome reassembly functions of Spt6 can still occur in the absence of its association with RNAPII. Surprisingly, we found that Spt6-RNAPII association is required for efficient recruitment of the Ccr4-Not de-adenylation complex to transcribed genes for essential degradation of a range of mRNAs, including mRNAs required for cell-cycle progression. These findings reveal an unexpected control mechanism for mRNA turnover during transcription facilitated by a histone chaperone.

SUBMITTER: Dronamraju R

PROVIDER: S-EPMC6323640 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Spt6 Association with RNA Polymerase II Directs mRNA Turnover During Transcription.

Dronamraju Raghuvar R Hepperla Austin J AJ Shibata Yoichiro Y Adams Alexander T AT Magnuson Terry T Davis Ian J IJ Strahl Brian D BD

Molecular cell 20180601 6

Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 also associates with RNA polymerase II (RNAPII) via a tandem Src2 homology domain. However, the significance of Spt6-RNAPII interaction is not well understood. Here, we show that Spt6 recruitment to genes and the nucleosome reassembly functions of Spt6 can still occur in the absence of its association with RNAPII. Surprisingly, we found that Spt6-RNAPII association is required for efficient ...[more]

PMID: 29932900

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Spt6 association with RNAPII directs mRNA turnover during transcription

Project description:Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 interacts with elongating RNA polymerase II (RNAPII) via a tandem Src2 homology (tSH2) domain, but it is not known whether this particular interaction is required for the nucleosome reassembly activity of Spt6. Here, we show that Spt6 recruitment to genes and its nucleosome reassembly functions are largely independent of association with RNAPII. Instead, the Spt6-RNAPII association is required for post-transcriptional mRNA turnover. Mechanistically, association of Spt6 with RNAPII couples the Ccr4-Not complex to the transcribed regions of genes, which we show regulates the timely deadenylation and degradation of a broad range of mRNAs including those required for cell cycle progression. Thus, our findings reveal an unexpected control mechanism for mRNA turnover facilitated by a histone chaperone during transcription.

2018-04-18 | GSE111815 | GEO

Spt6 association with RNAPII directs mRNA turnover during transcription

Project description:Spt6 association with RNAPII directs mRNA turnover during transcription

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Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro.

Project description:Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.

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Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae.

Project description:Previous characterization of the Saccharomyces cerevisiae Spt4, Spt5, and Spt6 proteins suggested that these proteins act as transcription factors that modify chromatin structure. In this work, we report new genetic and biochemical studies of Spt4, Spt5, and Spt6 that reveal a role for these factors in transcription elongation. We have isolated conditional mutations in SPT5 that can be suppressed in an allele-specific manner by mutations in the two largest subunits of RNA polymerase II (Pol II). Strikingly, one of these RNA Pol II mutants is defective for transcription elongation and the others cause phenotypes consistent with an elongation defect. In addition, we show that spt4, spt5, and spt6 mutants themselves have phenotypes suggesting defects in transcription elongation in vivo. Consistent with these findings, we show that Spt5 is physically associated with RNA Pol II in vivo, and have identified a region of sequence similarity between Spt5 and NusG, an Escherichia coli transcription elongation factor that binds directly to RNA polymerase. Finally, we show that Spt4 and Spt5 are tightly associated in a complex that does not contain Spt6. These results, taken together with the biochemical identification of a human Spt4-Spt5 complex as a transcription elongation factor (Wada et al. 1998), provide strong evidence that these factors are important for transcription elongation in vivo.

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Spt6 enhances the elongation rate of RNA polymerase II in vivo.

Project description:Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from approximately 1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development.

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ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation.

Project description:Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.

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A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription.

Project description:We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.

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Noncanonical tandem SH2 enables interaction of elongation factor Spt6 with RNA polymerase II.

Project description:Src homology 2 (SH2) domains are mostly found in multicellular organisms where they recognize phosphotyrosine-containing signaling proteins. Spt6, a conserved transcription factor and putative histone chaperone, contains a C-terminal SH2 domain conserved from yeast to human. In mammals, this SH2 domain recognizes phosphoserines rather than phosphotyrosines and is essential for the recruitment of Spt6 by elongating RNA polymerase II (RNAPII), enabling Spt6 to participate in the coupling of transcription elongation, chromatin modulation, and mRNA export. We have determined the structure of the entire Spt6 C-terminal region from Antonospora locustae, revealing the presence of two highly conserved tandem SH2 domains rather than a single SH2 domain. Although the first SH2 domain has a canonical organization, the second SH2 domain is highly noncanonical and appears to be unique in the SH2 family. However, both SH2 domains have phosphate-binding determinants. Our biochemical and genetic data demonstrate that the complete tandem, but not the individual SH2 domains, are necessary and sufficient for the interaction of Spt6 with RNAPII and are important for Spt6 function in vivo. Furthermore, our data suggest that binding of RNAPII to the Spt6 tandem SH2 is more extensive than the mere recognition of a doubly phosphorylated C-terminal domain peptide by the tandem SH2. Taken together, our results show that Spt6 interaction with RNAPII via a novel arrangement of canonical and noncanonical SH2 domains is crucial for Spt6 function in vivo.

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Spn1 regulates the recruitment of Spt6 and the Swi/Snf complex during transcriptional activation by RNA polymerase II.

Project description:We investigated the timing of the recruitment of Spn1 and its partner, Spt6, to the CYC1 gene. Like TATA binding protein and RNA polymerase II (RNAPII), Spn1 is constitutively recruited to the CYC1 promoter, although levels of transcription from this gene, which is regulated postrecruitment of RNAPII, are low. In contrast, Spt6 appears only after growth in conditions in which the gene is highly transcribed. Spn1 recruitment is via interaction with RNAPII, since an spn1 mutant defective for interaction with RNAPII is not targeted to the promoter, and Spn1 is necessary for Spt6 recruitment. Through a targeted genetic screen, strong and specific antagonizing interactions between SPN1 and genes encoding Swi/Snf subunits were identified. Like Spt6, Swi/Snf appears at CYC1 only after activation of the gene. However, Spt6 significantly precedes Swi/Snf occupancy at the promoter. In the absence of Spn1 recruitment, Swi/Snf is constitutively found at the promoter. These observations support a model whereby Spn1 negatively regulates RNAPII transcriptional activity by inhibiting recruitment of Swi/Snf to the CYC1 promoter, and this inhibition is abrogated by the Spn1-Spt6 interaction. These findings link Spn1 functions to the transition from an inactive to an actively transcribing RNAPII complex at a postrecruitment-regulated promoter.

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