Cotranscriptional Association of mRNA Export Factor Yra1 with C-terminal Domain of RNA Polymerase II

• Published in: The Journal of biological chemistry Vol. 286; no. 42; pp. 36385 - 36395
• Main Authors: MacKellar, April L., Greenleaf, Arno L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.10.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Motifs; CTD; CTD Kinase I; Gene Regulation; Hyperphosphorylated; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; PCID; Phospho-CTD Binding; Phosphorylation; Phosphorylation - physiology; Protein Binding; Protein Structure, Tertiary; RNA Polymerase II; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; RNA Transport; RNA, Fungal - biosynthesis; RNA, Fungal - genetics; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; RNA-binding Proteins; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Transcription; Transcription, Genetic - physiology; Hyperphosphorylated; Phosphorylation; CTD; RNA Transport; Transcription; RNA-binding Proteins; CTD Kinase I; Phospho-CTD Binding; PCID; RNA Polymerase II
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M111.268144

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. Background: Yra1 is an mRNA export factor, but how it is recruited to nascent RNA is not known. Results: We identified a phospho-CTD-interacting domain in Yra1 that binds best to Ser-2,5 doubly phosphorylated CTD repeats as found on elongating RNAPII; truncation of this domain leads to reduced amounts of Yra1 on active genes. Conclusion: The PCTD-interacting domain is involved in recruiting Yra1 to active genes. Significance: A step forward in understanding recruitment of the multicomponent mRNA packaging and export machineries.