RNA binding proteins like the Pumilio family (PUFs) exert repression on 3'UTRs, and hence mRNA 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 most identified cases of 3'UTR-mediated PUF repression - and APA to evade repression - were found ad hoc, between interacting gene pairs. Thus a gap exists in our understanding of how these interactions 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 three closest to the EGF source are induced to form the vulva, while the distal three remain uninduced. Remarkably, this event occurs with 99.8% fidelity. Three redundant PUFs are expressed specifically in the uninduced cells, suggesting that distal VPCs enact a PUF- and 3'UTR-dependent program to become non-responsive to signal. In the germline, the same PUFs repress ERK/MAP kinase; this same mechanism may be adopted by non-responsive VPCs. 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 signaling cascade (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 four identified 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-down specific aims. We propose to: 1) test 3'UTRs from the PUF IP list of vulval signaling genes for ability to mediate PUF-dependent reporter repression in non-responsive cells, 2) survey the VPC 3'UTRome and global proximal-to-distal PAS switching, and 3) validate select candidates by altering endogenous 3'UTRs and polyadenylation signals via CRISPR/Cas9 genome editing, and deleting associated PUFs. We will ascertain the contribution to developmental fidelity by APA, and the changes in repressive access points in the 3'UTRs caused by APA.
Our work will provide in-depth understanding of 3?UTR-mediated regulatory networks by which cells that do not respond to growth factor signaling are demarcated from cells that respond. These discoveries will lead to understanding of perturbations that lead to birth defects and tumorigenesis.
| Rasmussen, Neal R; Dickinson, Daniel J; Reiner, David J (2018) Ras-Dependent Cell Fate Decisions Are Reinforced by the RAP-1 Small GTPase in Caenorhabditiselegans. Genetics 210:1339-1354 |
