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 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-dependentní DNA-polymerasy chemie   metabolismus   MeSH
• exozómy metabolismus   MeSH
• fungální RNA metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• nekódující RNA metabolismus   MeSH
• polyadenylace MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• RNA-polymerasa II metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• stabilita RNA MeSH
• terminace genetické transkripce * MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy MeSH
• fungální RNA MeSH
• nekódující RNA MeSH
• NRD1 protein, S cerevisiae MeSH Prohlížeč
• PAP2 protein, S cerevisiae MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny 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
• dimerizace MeSH
• molekulární modely MeSH
• mutace MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• NRD1 protein, S cerevisiae MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA MeSH
• Saccharomyces cerevisiae - proteiny MeSH