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.

• Keywords
• CTD, RNA polymerase II, Spt6, NMR structure, phosphorylation,
• MeSH
• Epitopes genetics   MeSH
• Phosphorylation genetics   MeSH
• Transcription, Genetic * MeSH
• Histone Chaperones genetics   MeSH
• RNA Polymerase II genetics   MeSH
• Saccharomyces cerevisiae Proteins genetics   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Amino Acid Sequence genetics   MeSH
• src Homology Domains genetics   MeSH
• Transcriptional Elongation Factors genetics   MeSH
• Protein Binding genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epitopes MeSH
• Histone Chaperones MeSH
• RNA Polymerase II MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SPT6 protein, S cerevisiae MeSH Browser
• Transcriptional Elongation Factors 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.

• Keywords
• RNA polymerase II CTD, Sen1 helicase, non-coding transcription, pervasive transcription, transcription termination,
• MeSH
• DNA Helicases chemistry   metabolism   MeSH
• RNA, Fungal metabolism   MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• RNA, Untranslated metabolism   MeSH
• Protein Domains MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• Gene Expression Regulation, Fungal MeSH
• RNA Helicases chemistry   metabolism   MeSH
• RNA Polymerase II chemistry   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Transcription Termination, Genetic MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Helicases MeSH
• RNA, Fungal MeSH
• RNA, Untranslated MeSH
• NRD1 protein, S cerevisiae MeSH Browser
• RNA-Binding Proteins MeSH
• RNA Helicases MeSH
• RNA Polymerase II MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SEN1 protein, S cerevisiae MeSH Browser

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
• Amino Acid Motifs MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Humans MeSH
• Oligoribonucleotides metabolism   MeSH
• Protein Subunits chemistry   metabolism   MeSH
• RNA-Binding Proteins analysis   chemistry   metabolism   MeSH
• RNA, Small Nuclear chemistry   metabolism   MeSH
• Base Sequence MeSH
• Uracil Nucleotides metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• oligo(U) MeSH Browser
• Oligoribonucleotides MeSH
• Protein Subunits MeSH
• RNA-Binding Proteins MeSH
• RBM7 protein, human MeSH Browser
• RNA, Small Nuclear MeSH
• Uracil Nucleotides MeSH