Fine-tuning Rpb4 phosphorylation modulates Rpb4/7 stoichiometry within yeast RNA polymerase II and regulates gene expression

Abstract

RNA polymerase II (RNAPII) consists of 12 subunits, including Rpb4 and Rpb7, which form a heterodimeric stalk domain. In yeast, assembly of Rpb4/7 with the 10-subunit "core" is substoichiometric. Aside from its role in transcription, Rpb4/7 also binds to mRNAs co-transcriptionally (mRNA imprinting), exits the nucleus together with the mRNA, and modulates subsequent cytoplasmic processes like translation and degradation. However, the mechanisms underlying Rpb4/7 functions and its association with RNAPII remain poorly understood. In this study, we identify five phosphorylation sites in Rpb4 that regulate various steps of mRNA and snoRNA biogenesis, including transcription elongation and the polyadenylation step. We also provide evidence that the kinase Hrr25 and the phosphatase Fcp1, known to target the carboxyl-terminal domain (CTD) of Rpb1 and participate in processes at gene 3'-ends, coordinate Rpb4 phosphorylation. Inefficient Rpb4 phosphorylation leads to increased Rpb4 association with RNAPII, particularly at gene 3'-ends, suggesting that Rpb4-P promotes stalk dissociation from RNAPII either before or during transcription termination. Collectively, our findings underscore the pivotal role of dynamic Rpb4 phosphorylation in regulating Rpb4/7 stoichiometry as well as transcription elongation and termination, with an impact on mRNA imprinting.