טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHenry Swaminathan Stephen Richar
SubjectPost-Translational Modifications of Rpb4 Uncover a Novel
Type of Regulation
DepartmentDepartment of Medicine
Supervisor Professor Mordechai Choder


Abstract

The yeast Rpb4/7 heterodimer plays roles in all the major stages of the mRNA life cycle: mRNA synthesis, export, translation and mRNA decay. Previously we reported that Rpb4/7 binds RNA Polymerase II (Pol II) transcripts co-transcriptionally and accompanies the resulting mRNAs throughout their life. By virtue of its capacity to interact with key regulators (e.g., eIF3, Pat1) - temporally and spatially, Rpb4/7 regulates each of these stages. We proposed that Rpb4/7 integrates all stages into a system, thus functioning as an "mRNA coordinator" (Harel-Sharvit et al., 2010). Using 2-dimensional gel electrophoresis and mass spectrometry, here we show that Rpb4/7 carries multiple combinations of post-translational modifications (PTMs). Most PTMs occur on Rpb4, while Rpb4/7 is engaged in post-transcriptional stages. These modifications are biologically significant as they are responsive to the environment and are required for proliferation under stress. Remarkably, the PTMs repertoire of Rpb4 changes as the mRNP progresses from one stage to the next.  Thus, each stage is characterized by unique combination of PTMs. We mutated a number of residues that underwent PTMs and found that specific mutations affected efficient transcription and efficient mRNA decay. Our preliminary results highlighted PTMs that are involved in translation. Interestingly, some of our mutants uncouple mRNA synthesis from degradation, indicating that Rpb4 residues that undergo PTMs play a key role in the linkage between mRNA synthesis and decay. This function argues against a prevailing model whereby mRNA synthesis and decay are linked by default. Analyses of interacting partners of either Rpb4 or its mutant derivatives highlighted a novel type of interactions that could be discovered only with mutants that mimic constitutive modifications. For example, some specific, but transient, PTMs are required for recruiting some proteasome subunits (Pre2, Pre7 and Ecm29), raising a possibility that Rpb4 recruits the proteasome to either Pol II or the mRNA thus linking mRNA biogenesis and turnover with protein turnover. Moreover, our results suggest that transient Rpb4 PTMs regulate its interactions with key regulators of gene expression.  Taken together, we propose that numerous transient Rpb4/7 PTMs are involved in the cross talks among the various stages of the mRNA life, in agreement with our previously proposed role of Rpb4/7 as an mRNA coordinator.