The Nrd1–Nab3–Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain

Abstract

RNA polymerase II (Pol II) in Saccharomyces cerevisiae can terminate transcription via several pathways. To study how a mechanism is chosen, we analyzed recruitment of Nrd1, which cooperates with Nab3 and Sen1 to terminate small nucleolar RNAs and other short RNAs. Budding yeast contains three C-terminal domain (CTD) interaction domain (CID) proteins, which bind the CTD of the Pol II largest subunit. Rtt103 and Pcf11 act in mRNA termination, and both preferentially interact with CTD phosphorylated at Ser2. The crystal structure of the Nrd1 CID shows a fold similar to that of Pcf11, but Nrd1 preferentially binds to CTD phosphorylated at Ser5, the form found proximal to promoters. This indicates why Nrd1 cross-links near 5′ ends of genes and why the Nrd1–Nab3–Sen1 termination pathway acts specifically at short Pol II–transcribed genes. Nrd1 recruitment to genes involves a combination of interactions with CTD and Nab3.

Yeast RNA-Binding Protein Nab3 Regulates Genes Involved in Nitrogen Metabolism

“…The NNS complex role in NCR may represent part of this pathway intersection. Nrd1 preferentially interacts with Ser5-phosphorylated CTD (21,22). Nrd1 and Nab3 binding to NCR transcripts could normally act together with the appropriate GATA factors to attenuate expression of genes involved in the use of nonpreferred nitrogen sources.…”

“…NNS interacts with Pol II in part through binding of Nrd1 to phosphorylated Ser5 on the C-terminal domain (CTD) (21)(22)(23), a pattern of CTD phosphorylation most prominent in the early stages of the transcription cycle, limiting NNS termination to promoter-proximal transcripts (24)(25)(26)(27)(28)(29). The NNS complex also interacts with the TRAMP (Trf4/Trf5-Air1/Air2-Mtr4 polyadenylation) complex to couple termination to processing by the nuclear exosome (30)(31)(32)(33).…”

Transcription termination by nuclear RNA polymerases

“…The studies of Gudipati et al (2008) and Vasiljeva et al (2008a) provide an explanation of why termination depends on the distance traveled by RNAPII, and how RNAPII chooses between the 39 cleavage and polyadenylation pathway for mRNA termination and the Nrd1 complex for noncoding RNAs, short ORFs and CUT termination. Short transcripts are mostly associated with Ser5 phosphorylation, which recruits the Nrd1 complex.…”

“…They also showed that binding of Nrd1 to the nascent transcript, even if not sufficient, is necessary for efficient termination. Accordingly, Vasiljeva et al (2008a) showed that Nrd1 has a high affinity for the CTD phosphorylated at Ser5 and Ser5/Ser2. Additionally, association of Nrd1 with a snoRNA or mRNA gene is not affected by inhibition of Ser2 phosphorylation.…”

“…Nrd1 (nuclear pre-mRNA down-regulation) is an essential nuclear RNA-binding protein that directs termination of snoRNA and some mRNA transcripts, in association with an interacting protein, the helicase Sen1 (Steinmetz and Brow 1996;Steinmetz et al 2001;Ursic et al 2004;Vasiljeva and Buratowski 2006). Nrd1 contains a CID (Meinhart and Cramer 2004;Meinhart et al 2005) that interacts with CTD phosphorylated at Ser5 (Conrad et al 2000;Gudipati et al 2008;Vasiljeva et al 2008a). Nrd1 also interacts with another essential RNA-binding protein, Nab3 (Yuryev et al 1996).…”