RNA polymerase II (RNAPII) transcribes protein-coding mRNAs and a subset of non-coding RNAs (ncRNAs). For both, transcription termination is regulated by specific post-transcriptional modifications of C-terminal domain (CTD) of RNAPII. The aforementioned modifications are recognized by RNA processing and transcription termination factors via CTD-interacting domains (CIDs). Termination of ncRNAs is promoted by the NNS (Nrd1p-Nab3p-Sen1p) complex via the CID domain of Nrd1p. Nrd1p CID interconnects RNAPII termination with subsequent trimming by the nuclear exosome via the interaction with Trf4p subunit of the TRAMP (Trf4p-Air2p-Mtr4p) complex, an activator of the nuclear exosome. The CID of Nrd1p recognizes the C-terminal sequence of Trf4p that mimics phosphorylated CTD diheptad (Nrd1p-interacting motif; NIM) [1].
In this work, we found that Trf4p NIM interacts with mRNA termination factor, Rtt103p, via its CID. Interestingly, we found that Rtt103p interacts with an additional region of Trf4p termed RIM (for Rtt103p-interacting motif). By using NMR studies, we demonstrate that Rtt103p CID utilizes the same binding pocket to interact with CTD and both Trf4p sequences in a mutually exclusive manner. Furthermore, we show Rtt103p dimerization is crucial for binding with both interacting motifs of Trf4p.
In vivo, trf4 deletion strains show accumulation of extended, improperly terminated mRNAs. This phenotype is rescued by wild type, but not Trf4p defective in interaction with Rtt103p CID. RNAPII ChIP analysis on selected mRNAs did not reveal any substantial read-through phenotype. Rtt103p – Trf4p interaction, therefore, appears to be important for recruitment of the TRAMP complex and potentially exosome to proofread and remove improperly terminated mRNAs.