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

Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code.

• Keywords
• NMR, RNA processing, RNAPII CTD code, structural biology,
• MeSH
• Phosphorylation MeSH
• Transcription, Genetic MeSH
• Protein Kinases metabolism   MeSH
• Proteolysis MeSH
• RNA Polymerase II chemistry   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Serine metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Threonine chemistry   metabolism   MeSH
• Transcription Factors chemistry   metabolism   MeSH
• Tyrosine metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Kinases MeSH
• RNA Polymerase II MeSH
• Rtt103 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Serine MeSH
• Threonine MeSH
• Transcription Factors MeSH
• Tyrosine MeSH

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

The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.

• MeSH
• DNA-Directed DNA Polymerase chemistry   metabolism   MeSH
• Exosomes metabolism   MeSH
• RNA, Fungal metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Magnetic Resonance Spectroscopy MeSH
• Models, Molecular MeSH
• RNA, Untranslated metabolism   MeSH
• Polyadenylation MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• RNA Polymerase II metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• RNA Stability MeSH
• Transcription Termination, Genetic * MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed DNA Polymerase MeSH
• RNA, Fungal MeSH
• RNA, Untranslated MeSH
• NRD1 protein, S cerevisiae MeSH Browser
• PAP2 protein, S cerevisiae MeSH Browser
• RNA-Binding Proteins MeSH
• RNA Polymerase II MeSH
• Saccharomyces cerevisiae Proteins MeSH

In Saccharomyces cerevisiae, the Nrd1-dependent termination and processing pathways play an important role in surveillance and processing of non-coding ribonucleic acids (RNAs). The termination and subsequent processing is dependent on the Nrd1 complex consisting of two RNA-binding proteins Nrd1 and Nab3 and Sen1 helicase. It is established that Nrd1 and Nab3 cooperatively recognize specific termination elements within nascent RNA, GUA[A/G] and UCUU[G], respectively. Interestingly, some transcripts do not require GUA[A/G] motif for transcription termination in vivo and binding in vitro, suggesting the existence of alternative Nrd1-binding motifs. Here we studied the structure and RNA-binding properties of Nrd1 using nuclear magnetic resonance (NMR), fluorescence anisotropy and phenotypic analyses in vivo. We determined the solution structure of a two-domain RNA-binding fragment of Nrd1, formed by an RNA-recognition motif and helix-loop bundle. NMR and fluorescence data show that not only GUA[A/G] but also several other G-rich and AU-rich motifs are able to bind Nrd1 with affinity in a low micromolar range. The broad substrate specificity is achieved by adaptable interaction surfaces of the RNA-recognition motif and helix-loop bundle domains that sandwich the RNA substrates. Our findings have implication for the role of Nrd1 in termination and processing of many non-coding RNAs arising from bidirectional pervasive transcription.

• MeSH
• Dimerization MeSH
• Models, Molecular MeSH
• Mutation MeSH
• RNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• RNA chemistry   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NRD1 protein, S cerevisiae MeSH Browser
• RNA-Binding Proteins MeSH
• RNA MeSH
• Saccharomyces cerevisiae Proteins MeSH

Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.

• MeSH
• Phosphorylation MeSH
• Models, Molecular MeSH
• RNA, Untranslated metabolism   MeSH
• Proline metabolism   MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• RNA Polymerase II metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae cytology   enzymology   genetics   metabolism   MeSH
• Serine metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Cell Survival MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Untranslated MeSH
• NRD1 protein, S cerevisiae MeSH Browser
• Proline MeSH
• RNA-Binding Proteins MeSH
• RNA Polymerase II MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Serine MeSH

Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation.

• MeSH
• Adaptor Proteins, Signal Transducing chemistry   metabolism   MeSH
• DEAD-box RNA Helicases chemistry   metabolism   MeSH
• DNA-Directed DNA Polymerase chemistry   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Protein Subunits chemistry   metabolism   MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• Ribonucleases metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Air2 protein, S cerevisiae MeSH Browser
• DEAD-box RNA Helicases MeSH
• DNA-Directed DNA Polymerase MeSH
• MTR4 protein, S cerevisiae MeSH Browser
• PAP2 protein, S cerevisiae MeSH Browser
• Protein Subunits MeSH
• RNA-Binding Proteins MeSH
• Ribonucleases MeSH
• Saccharomyces cerevisiae Proteins MeSH