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.
