The goal of my research program is to understand how multifunctional protein complexes regulate gene expression, especially during stress responses. Our current focus is the yeast Ccr4-Not complex, which has been implicated in virtually all aspects of gene control, including transcription, mRNA decay, translational repression and protein ubiquitylation. The complex is it is highly conserved in all eukaryotes. Our laboratory has been a leader in understanding its role in transcription, especially RNA polymerase II elongation, employing multifaceted approaches including genetics, molecular biology, reconstitution biochemistry and genomics. Characterizing Ccr4-Not requires a flexible strategy and a willingness to move in new directions. This proposal will tackle two disparate processes under its control, namely the role of its ubiquitylation activity in gene expression and the mechanism of the reprogramming of Ccr4-Not mRNA targets during stress responses as a means to balance mRNA synthesis and decay. Protein destruction during transcription and DNA damage responses is an essential process. The Not4 subunit of the complex contains an E3 RING domain and controls ubiquitin-dependent destruction of proteins. We have shown that Ccr4-Not associates with elongation complexes and, recently, that it regulates the destruction of RNAPII after DNA damage. Theme 1 will identify novel targets of Not4 using global proteomics, identify the sites of modification and use molecular genetics and biochemistry to determine the consequences of ubquitylation on the protein?s function. Identifying and characterizing the consequences of Not4 modification of gene regulatory proteins will not only reveal novel functions of the complex, but lead to a greater understanding of the importance of protein ubiquitylation in transcription and DNA repair. Gene expression buffering is a novel conserved phenomenon where reciprocal changes in the rates in mRNA synthesis and degradation occur in response to stimuli to maintain similar levels of mRNAs to ?balance? the response. Ccr4-Not has been implicated in this process because it controls both the synthesis and destruction of mRNAs. The molecular underpinnings of buffering are unknown and the mechanism behind it is under intense debate. We have mapped the mRNAs associated with Ccr4-Not under resting and oxidative stress conditions, which revealed extensive redistribution of Ccr4 from constitutive mRNAs to those induced by oxidative stress, which is likely a key component of buffering. Theme 2 will reveal what accounts for the reprogramming of the decay machinery during stress by identifying the cis- and trans- factors that control this response, explore the interplay between the two cellular deadenylases and identify changes in protein composition and localization of mRNAs undergoing buffering during stress. These studies will identify the determinants of mRNA targeting during stress responses and undercover the molecular mechanism behind gene expression buffering.
Numerous human diseases and syndromes are caused by disturbances in gene expression, and an important component in this process is the ability of cells to complete the production of mRNAs from the genome. The goal of the work described in this proposal is to understand how a multifunctional protein complexes balances gene expression during stress. The complex that is the focus of this proposal have been implicated in DNA damage resistance, cell proliferation and cardiovascular development; thus, the work described here is directly relevant to human health.!
