Structural basis for transcription termination coupled to RNA processing/degradation

Tomasz K. Kabzinski1, Agnieszka Tudek2, Jana Lalakova1, Karel Kubicek1, Odil Porrua-Fuerte2, Andrea Jesenska1, Stepanka Vanacova1, Patrick Cramer3, Domenico Libri2 and Richard Stefl1

1CEITEC - Central European Institute of Technology, Masaryk University; Brno, Czech Republic

2Centre de Genetique Moleculaire – CNRS; Gif-sur-Yvette, France

3Department of Biochemistry, Ludwig-Maximilians-Universitat; Munich, Germany


In Saccharomyces cerevisiae, production of both, protein-coding (mRNAs) and non-coding (ncRNAs) RNA, is performed by an enzyme termed RNA polymerase II (RNAPII). RNAPII consists of the core where the process of transcription occurs, and a flexible C-terminal domain (CTD) that is connected with the RNAPII core by a short linker. Termination of transcription is highly dependent on the size of RNA molecule to be terminated, and it is divided into two pathways, Nrd1p-dependent and Rtt103p-dependent for ncRNAs and mRNAs, respectively. For each pathway, the interaction between transcription termination machinery and CTD of RNAPII is crucial for the proper termination of transcription. The interaction with CTD is achieved by the CTD-interacting domain (CID) of either Nrd1p or Rtt103p.

We demonstrate that transcription termination of ncRNAs is physically coupled to RNA degradation/processing performed by the nuclear exosome. The coupling is achieved by the CID of Nrd1p that recognizes a mimic of CTD (Nrd1p-interactin motif, NIM) within the TRAMP (Trf4p-Air2p-Mtr4p) complex, a cofactor of the nuclear exosome. We report the structure of Nrd1p CID bound to the Trf4p NIM. Binding assays show that Nrd1p binds Trf4p NIM with a KD of ~1μM, whereas the binding affinity for CTD has a KD of ~100μM. Mutational analyses of the binding surface of both Nrd1p CID and Trf4p NIM confirm our structural findings. What is more, competition experiments show that Nrd1p CID binds NIM and CTD in a mutually exclusive manner.

Since the interaction between CID, of either Nrd1p or Rtt103p, and CTD of RNAPII is crucial for the proper transcription termination, we have hypothesized that transcription termination of mRNAs may also be coupled to RNA processing/degradation performed by the nuclear exosome/TRAMP. Pull-down experiments show that Rtt103p CID also recognizes the NIM, and another region of Trf4p specific only to Rtt103p, the RIM (Rtt103-interacting motif). Specifically, we report the structure of Rtt103p CID bound to Trf4p NIM, and initial structural studies of the complex between Rtt103p CID and Trf4p RIM. We show that Rtt103p CID utilizes the same pocket to bind both CTD and the NIM. Overall, our results unveil a novel role of CID, of Nrd1p and Rtt103p, in not only transcription termination but also in RNA processing/degradation by the nuclear exosome.