The Role of Inosine Monophosphate Dehydrogenase in mRNA Regulation: Identification of mRNAs Bound and Functional Consequences - Project Summary/Abstract Inosine Monophosphate Dehydrogenase (IMPDH) catalyzes the rate limiting step in guanine nucleotide synthesis and is thus critical for proper cellular function. Indeed, mutations in human IMPDH (IMPDH1 and IMPDH2) are known to cause diseases, such as autosomal dominant Retinitis Pigmentosa (adRP). This enzyme also has an exciting, unexplored function: it binds mRNA. The long-term goal of the proposed project is to elucidate the functions and mechanisms of uncharacterized mRNA binding proteins to reveal new principles of gene expression and inform research on human disease. The overall objective for this application is to characterize the role of IMPDH in binding and regulation of mRNA, and to identify the impact that adRP causing mutations have on that activity. The central hypothesis is that IMPDH binds to a select group of mRNAs and regulates their translation. This hypothesis has been formulated because: 1) yeast IMPDH enzymes were robustly identified as mRNA binding proteins that bind to multiple mRNAs; 2) published data demonstrated the presence of human IMPDH in translating complexes; and 3) our preliminary data shows effects on protein levels, thus supporting a role in protein synthesis. The rationale behind this proposal is that understanding the connection between nucleotide biosynthesis and gene expression will deepen our understanding of central biological pathways and support new strategies to address diseases including inherited blindness. The central hypothesis will be tested by pursuing three specific aims: 1) Identify mRNAs bound by yeast IMPDH enzymes; 2) Determine how IMPDH regulates mRNA biology; and 3) Characterize disruptions to mRNA regulation caused by adRP causing mutations. Under the first aim, RIP-seq will be performed on all three of the yeast IMPDH enzymes (Imd2, Imd3, and Imd4) to identify bound mRNAs. For the second aim, consequences of IMPDH binding on mRNA stability and translation will be identified using northern blotting and western blotting to analyze regulation of both a reporter mRNA and endogenous mRNA binding partners of IMPDH. The mechanism of mRNA regulation will be investigated by repeating these assays in yeast strains deficient in key regulators of translation and/or mRNA decay. For the third aim, mutations known to cause adRP will be inserted into the yeast genome and consequences on mRNA binding and regulation will be observed using RIP-seq, western blotting, and northern blotting. The proposed research is innovative, in the applicant’s opinion, because it represents a substantive departure from the status quo by focusing on IMPDH as a regulator of mRNA function and investigating the identity of bound mRNAs as well as the consequences of this interaction on gene expression. The proposed research is significant because it will reveal a new mechanism of mRNA regulation and define an unexplored function of the key enzyme IMPDH. This work will be performed by the PI and a team of exclusively undergraduate researchers, providing transformative research experiences for the next generation of scientists.
