Role of Nrd1 CTD-Interacting Domain in RNA Polymerase II Termination

Abstract

Abstract

Role of Nrd1 CTD-Interacting Domain in RNA Polymerase II Termination

Jiwon Gong Biophysics and Chemical Biology The Graduate School of Seoul National University

Carboxy-terminal domain (CTD) of RNA polymerase II (RNAP II) consists of multiple hepta-peptide repeats well conserved in eukaryotes. At each step of transcription, phosphorylation pattern of CTD gradually changes and orchestrates binding of various proteins for RNA processing. When transcription ends, RNAP II dissociates and becomes ready for a new transcription event. This "termination" process is aided by two distinct pathways, which have different affinities for CTD phosphorylation states. Rtt103-Rat1-Rai1 dependent pathway is responsible for long mRNA gene termination where serine 2 phosphorylation of CTD (CTD-S2P) dominates at 3'-end of the gene, whereas Nrd1-Sen1-Nab3 dependent pathway takes the role in short gene termination at which CTD serine 5 phosphorylation (CTD-S5P) prevails. This distinction primarily comes from CTD-interacting domain (CID) protein of each complex: Rtt103 and Nrd1. The specific CID-CTD interaction might affect RNAP II in choice of termination pathway. To verify this idea, swapping CID of Nrd1 with that of Rtt103 was carried out in yeast Saccharomyces serevisiae. CID swapping changes the affinity of Nrd1 for CTD phosphorylation states from CTD-S5P to CTD-S2P in vivo. In chromatin immunoprecipitation (ChIP) assay, nrd1[CIDRTT103] successfully binds to 3'-end of both snoRNA and mRNA genes. Under CID swapped condition, snoRNA genes showed mild termination defects and read-through transcripts were shown on Northern blot. On the other hand, mRNA genes were classified into two different categories. ADH1 showed proper termination by nrd1[CIDRTT103] and this result was supported by genome-wide microarray (ChIP-chip) data, which showed that about 30% of mRNA genes could be terminated by nrd1[CIDRTT103]. On the contrary, PMA1, a typical mRNA gene, showed severe termination defect. In this case, recruitment of nrd1[CIDRTT103] at 3-end of gene might result in interruption of proper termination. In ChIP-chip analysis, about 70% of mRNA genes showed defective termination like PMA1. To verify the assumption that CID-CTD interaction was responsible for the termination failure on PMA1, CTD-S2P kinase null mutant and defective RNA-binding mutant of Nrd1-dependent pathway were prepared and restoration of termination was confirmed by ChIP assay. Furthermore, other interactions of Nrd1-dependent pathway were also examined. In co-immunoprecipitation (Co-IP) assay, nrd1[CIDRTT103] still formed complex with Sen1 and Nab3, and interacted with exosome core components Rrp4 and Rrp41, like wild type Nrd1 does. These results show that the specific CID-CTD interaction plays a major role in choice of termination pathway for RNAP II and not in other interactions involved in Nrd1-dependent pathway. ……………………………………… Keywords : Nrd1, Rtt103, CID, CTD, RNAP II, Termination