Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation

Abstract

The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

We thank S. Buratowski, G. Prelich, and P. San Segundo for yeast strains. O.C. thanks R. Jiménez for laboratory facilities and support and María Gómez for technical support on qPCR.

This work was supported by grants number BFU 2006-09041 and BFU 2009-07179 from the Spanish Ministerio de Ciencia e Innovación and SA012A08 from the Junta de Castilla y León to O.C. and a grant from the NIH to J.L.M. A.G. was supported by a fellowship from the Junta de Castilla y León.