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The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II.

ABSTRACT: The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in the linker between the catalytic core and the C-terminal domain (CTD) heptad repeats. This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in a distinct pattern of Spt6 accumulation at these sites. Splicing efficiency was also diminished, as was apparent progression through introns that encode snoRNAs. Chromatin-mediated repression was impaired, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the interaction modulates the availability or activity of the core elongation and histone chaperone functions of Spt6, contributing to coordination between RNAPII and its accessory factors as varying local conditions call for dynamic responses.

SUBMITTER: Connell Z

PROVIDER: S-EPMC8789061 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II

Project description:The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in a region linking the catalytic core and the heptad repeats at the C-terminal domain (CTD). This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in the distinct pattern of Spt6 occupancy at these sites. Splicing efficiency was also impacted, as was apparent progression through introns that encode snoRNAs. Expression of genes that are particularly sensitive to efficient restoration of chromatin after transcription were affected, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the Spt6-tSH2:Rpb1 interaction participates in coordinating appropriate responses to the varying local conditions encountered RNAPII at each gene, perhaps by modulating the elongation rate to allow reconfiguration of accessory factor activity or availability.

2021-12-20 | GSE184954 | GEO

The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II [MNase-seq]

Project description:The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in the linker between the catalytic core and the C-terminal domain (CTD) heptad repeats. This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in a distinct pattern of Spt6 accumulation at these sites. Splicing efficiency was also diminished, as was apparent progression through introns that encode snoRNAs. Chromatin-mediated repression was impaired, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the interaction modulates the availability or activity of the core elongation and histone chaperone functions of Spt6, contributing to coordination between RNAPII and its accessory factors as varying local conditions call for dynamic responses.

2021-12-20 | GSE189831 | GEO

The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II

Project description:The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II

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The C-terminal domain of Rpb1 functions on other RNA polymerase II subunits.

Project description:The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNApII), coordinates recruitment of RNA- and chromatin-modifying factors to transcription complexes. It is unclear whether the CTD communicates with the catalytic core region of Rpb1 and thus must be physically connected, or instead can function as an independent domain. To address this question, CTD was transferred to other RNApII subunits. Fusions to Rpb4 or Rpb6, two RNApII subunits located near the original position of CTD, support viability in a strain carrying a truncated Rpb1. In contrast, CTD fusion to Rpb9 on the other side of RNApII does not. Rpb4-CTD and Rpb6-CTD proteins are functional for phosphorylation and recruitment of various factors, albeit with some restrictions and minor defects. Normal CTD functions are not transferred to RNApI or RNApIII by Rbp6-CTD. These results show that, with some spatial constraints, CTD can function even when disconnected from Rpb1.

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The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II [MNase-seq]

Project description:The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II [MNase-seq]

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Yeast Spt6 Reads Multiple Phosphorylation Patterns of RNA Polymerase II C-Terminal Domain In Vitro.

Project description:Transcription elongation factor Spt6 associates with RNA polymerase II (RNAP II) via a tandem SH2 (tSH2) domain. The mechanism and significance of the RNAP II-Spt6 interaction is still unclear. Recently, it was proposed that Spt6-tSH2 is recruited via a newly described phosphorylated linker between the Rpb1 core and its C-terminal domain (CTD). Here, we report binding studies with isolated tSH2 of Spt6 (Spt6-tSH2) and Spt6 lacking the first unstructured 297 residues (Spt6ΔN) with a minimal CTD substrate of two repetitive heptads phosphorylated at different sites. The data demonstrate that Spt6 also binds the phosphorylated CTD, a site that was originally proposed as a recognition epitope. We also show that an extended CTD substrate harboring 13 repetitive heptads of the tyrosine-phosphorylated CTD binds Spt6-tSH2 and Spt6ΔN with tighter affinity than the minimal CTD substrate. The enhanced binding is achieved by avidity originating from multiple phosphorylation marks present in the CTD. Interestingly, we found that the steric effects of additional domains in the Spt6ΔN construct partially obscure the binding of the tSH2 domain to the multivalent ligand. We show that Spt6-tSH2 binds various phosphorylation patterns in the CTD and found that the studied combinations of phospho-CTD marks (1,2; 1,5; 2,4; and 2,7) all facilitate the interaction of CTD with Spt6. Our structural studies reveal a plasticity of the tSH2 binding pockets that enables the accommodation of CTDs with phosphorylation marks in different registers.

| S-EPMC7327521 | biostudies-literature

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.

| S-EPMC2992272 | biostudies-literature

Direct Analysis of Phosphorylation Sites on the Rpb1 C-Terminal Domain of RNA Polymerase II.

Project description:Dynamic interactions between RNA polymerase II and various mRNA-processing and chromatin-modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code."

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Spt6-tSH2 domain and Rpb1

Project description:This SuperSeries is composed of the SubSeries listed below.

2021-12-20 | GSE184955 | GEO

A tandem SH2 domain in transcription elongation factor Spt6 binds the phosphorylated RNA polymerase II C-terminal repeat domain (CTD).

Project description:Spt6 is an essential transcription elongation factor and histone chaperone that binds the C-terminal repeat domain (CTD) of RNA polymerase II. We show here that Spt6 contains a tandem SH2 domain with a novel structure and CTD-binding mode. The tandem SH2 domain binds to a serine 2-phosphorylated CTD peptide in vitro, whereas its N-terminal SH2 subdomain, which we previously characterized, does not. CTD binding requires a positively charged crevice in the C-terminal SH2 subdomain, which lacks the canonical phospho-binding pocket of SH2 domains and had previously escaped detection. The tandem SH2 domain is apparently required for transcription elongation in vivo as its deletion in cells is lethal in the presence of 6-azauracil.

| S-EPMC3009887 | biostudies-literature

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