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.

• Klíčová slova
• CTD, RNA polymerase II, Spt6, NMR structure, phosphorylation,
• MeSH
• epitopy genetika   MeSH
• fosforylace genetika   MeSH
• genetická transkripce * MeSH
• histonové chaperony genetika   MeSH
• RNA-polymerasa II genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence aminokyselin genetika   MeSH
• src homologní domény genetika   MeSH
• transkripční elongační faktory genetika   MeSH
• vazba proteinů genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epitopy MeSH
• histonové chaperony MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SPT6 protein, S cerevisiae MeSH Prohlížeč
• transkripční elongační faktory MeSH

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1.

• Klíčová slova
• RNA polymerase II CTD, Sen1 helicase, non-coding transcription, pervasive transcription, transcription termination,
• MeSH
• DNA-helikasy chemie   metabolismus   MeSH
• fungální RNA metabolismus   MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• nekódující RNA metabolismus   MeSH
• proteinové domény MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• RNA-helikasy chemie   metabolismus   MeSH
• RNA-polymerasa II chemie   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• terminace genetické transkripce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-helikasy MeSH
• fungální RNA MeSH
• nekódující RNA MeSH
• NRD1 protein, S cerevisiae MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA-helikasy MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SEN1 protein, S cerevisiae MeSH Prohlížeč

The Nuclear Exosome Targeting (NEXT) complex is a key cofactor of the mammalian nuclear exosome in the removal of Promoter Upstream Transcripts (PROMPTs) and potentially aberrant forms of other noncoding RNAs, such as snRNAs. NEXT is composed of three subunits SKIV2L2, ZCCHC8 and RBM7. We have recently identified the NEXT complex in our screen for oligo(U) RNA-binding factors. Here, we demonstrate that NEXT displays preference for U-rich pyrimidine sequences and this RNA binding is mediated by the RNA recognition motif (RRM) of the RBM7 subunit. We solved the structure of RBM7 RRM and identified two phenylalanine residues that are critical for interaction with RNA. Furthermore, we showed that these residues are required for the NEXT interaction with snRNAs in vivo. Finally, we show that depletion of components of the NEXT complex alone or together with exosome nucleases resulted in the accumulation of mature as well as extended forms of snRNAs. Thus, our data suggest a new scenario in which the NEXT complex is involved in the surveillance of snRNAs and/or biogenesis of snRNPs.

• MeSH
• aminokyselinové motivy MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• lidé MeSH
• oligoribonukleotidy metabolismus   MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteiny vázající RNA analýza   chemie   metabolismus   MeSH
• RNA malá jaderná chemie   metabolismus   MeSH
• sekvence nukleotidů MeSH
• uracilnukleotidy metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oligo(U) MeSH Prohlížeč
• oligoribonukleotidy MeSH
• podjednotky proteinů MeSH
• proteiny vázající RNA MeSH
• RBM7 protein, human MeSH Prohlížeč
• RNA malá jaderná MeSH
• uracilnukleotidy MeSH