Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNApolymerase II

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently show n to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain , which binds membrane phospholipids; and three WW domains, which are prote in interaction modules that bind proline-rich Ligands, The carboxyl-termina l half of Rsp5 contains a HECT (homologous to EG-AP carboxyl terminus) doma in that catalytically ligates ubiquitin to proteins and functionally classi fies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are pres umed to act as membrane localization and substrate recognition modules, res pectively. me report that the second (and possibly third) Rsp5 WW domain me diates binding to the carboxyl-terminal domain (CTD) of the RNA pol II larg e subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a P XY core that is critical for interaction with a specific group of WW domain s. An analysis of synthetic peptides revealed a minimal CTD sequence that i s sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and i n yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown th at phosphorylation of this minimal CTD sequence on serine, threonine and ty rosine residues acts as a negative regulator of the Rsp5 WW2-CTD interactio n. In view of the recent data pertaining to phosphorylation-driven interact ions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II de gradation.