Global Changes In the 3'UTRome Toggle Responsiveness To Growth Factors
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SUMMARYRNA binding proteins like the Pumilio family (PUFs) exert repression on 3''UTRs, and hencemRNA stability and translation. Alternative polyadenylation (APA) causes 3''UTR length to vary.Coordinated large-scale shortening of 3''UTRs through APA can lead to evasion from 3''UTR-mediated repressive signals, and may be a key regulatory regime during development. Yet mostidentified cases of 3''UTR-mediated PUF repression - and APA to evade repression - were foundad hoc, between interacting gene pairs. Thus a gap exists in our understanding of how theseinteractions are orchestrated at the global level of the 3''UTRome and APA.EGF patterns the C. elegans vulva. Of six equipotent vulval precursor cells (VPCs), the threeclosest to the EGF source are induced to form the vulva, while the distal three remainuninduced. Remarkably, this event occurs with 99.8% fidelity. Three redundant PUFs areexpressed specifically in the uninduced cells, suggesting that distal VPCs enact a PUF- and3''UTR-dependent program to become non-responsive to signal. In the germline, the samePUFs repress ERK/MAP kinase; this same mechanism may be adopted by non-responsiveVPCs. Germline immunoprecipitation (IP) of one PUF identified many potential target 3''UTRs.From this dataset, we identified multiple target mRNAs from genes in each vulval signalingcascade (EGFR→Ras→Raf→MEK→ERK, Notch→CSL, PI3K→PDK→Akt, and EGFR→Ras→RalGEF→Ral). We hypothesize that the redundant PUFs collectively repress mRNAs of all fouridentified signaling cascades to demarcate signal-non-responsive from signal-responsive cells.Our central hypothesis is that switching from proximal (short 3''UTR) to distal (long 3''UTR)polyadenylation sequence (PAS) usage governs switching from signal-responsiveness to non-responsiveness. We will systematically test this hypothesis by joining bottom-up and top-downspecific aims. We propose to: 1) test 3''UTRs from the PUF IP list of vulval signaling genes forability to mediate PUF-dependent reporter repression in non-responsive cells, 2) survey theVPC 3''UTRome and global proximal-to-distal PAS switching, and 3) validate select candidatesby altering endogenous 3''UTRs and polyadenylation signals via CRISPR/Cas9 genome editing,and deleting associated PUFs. We will ascertain the contribution to developmental fidelity byAPA, and the changes in repressive access points in the 3''UTRs caused by APA.
