POST-TRANSLATIONAL MODIFICATIONS OF RPB4/7 UNCOVER A NOVEL TYPE OF REGULATION

The Yeast proteins Rpb4 and Rpb7 form a dissociable heterodimer. Rpb4/7 plays roles in all major stages of the mRNA life cycle: regulation of transcription, mRNA export, translation and mRNA decay. Rpb4/7 binds Pol II transcripts co-transcriptionally and then escorts the mRNA throughout its life. Previously, we found that it modulates each step that the RNA undergoes by its capacity to interact with key regulators temporally and spatially. We proposed that it can integrate all stages into a system (the "mRNA coordination" concept). Using 2-dimensional gel electrophoresis, here we show that Rpb4 exists in various proteoforms. Using Mass Spectrometry, we found that these proteoforms contain post-translational modifications (PTMs) like N-terminal acetylation, phosphorylation, methylation, and ubiquitination or neddylation. This provoked us to examine whether these PTMs are involved in its capacity to integrate all stages into a system. We found that the PTMs change as Rpb4/7 progresses from one stage to the other. Specifically, in the context of transcribing Pol II, Rpb4/7 seems to be less modified. Most modifications are dependent on prior binding to Pol II. These modifications are biologically significant because they are responsive to environmental conditions, such as heat shock and required for optimal growth at non-permissive temperatures. We show that certain Rpb4 PTMs affect the transcription induction, steady-state mRNA level, and mRNA deadenylation & degradation rates of some genes. Also, in the absence of some Rpb4 PTMs, cells exhibit differential sensitivity to translational inhibitors. Lack of certain Rpb4 PTMs compromise Nonsense-mediated mRNA decay as well. We propose that PTMs play roles in quality controlling the various stages of gene expression and in the capacity of Rpb4/7 to function as a mRNA coordinator.