title: Cotranscriptional association of mRNA export factor Yra1 with C-terminal domain of RNA polymerase II.
abstract: The unique C-terminal domain (CTD) of RNA polymerase II, composed of tandem heptad repeats of the consensus sequence YSPTSPS, is subject to differential phosphorylation throughout the transcription cycle. Several RNA processing factors have been shown to bind the phosphorylated CTD and use it to localize to nascent pre-mRNA during transcription. In Saccharomyces cerevisiae, the mRNA export protein Yra1 (ALY/RNA export factor in metazoa) cotranscriptionally associates with mRNA and delivers it to the nuclear pore complex for export to the cytoplasm. Here we report that Yra1 directly binds in vitro the hyperphosphorylated form of the CTD characteristic of elongating RNA polymerase II and contains a phospho-CTD-interacting domain within amino acids 18-184, which also include an "RNA recognition motif" (RRM) (residues 77-184). Using UV cross-linking, we showed that the RRM alone binds RNA, although a larger segment extending to the C terminus (amino acids 77-226) displayed stronger RNA binding activity. Although the RRM is implicated in both RNA and CTD binding, RRM point mutations separated these two functions. Both functions are important in vivo as RNA binding-defective or CTD binding-defective versions of Yra1 engendered growth and mRNA export defects. We also report the construction and characterization of a useful new temperature-sensitive YRA1 allele (R107A/F126A). Using ChIP, we demonstrated that removing the N-terminal 76 amino acids of Yra1 (all of the phospho-CTD-interacting domain up to the RRM) results in a 10-fold decrease in Yra1 recruitment to genes during elongation. These results indicate that the phospho-CTD is likely involved directly in the cotranscriptional recruitment of Yra1.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: April L
authors.initials: AL
authors.lastname: MacKellar
authors.forename: Arno L
authors.initials: AL
authors.lastname: Greenleaf
mesh_terms: D020816:Amino Acid Motifs; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D009687:Nuclear Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C105887:YRA1 protein, S cerevisiae; D012319:RNA Polymerase II