Post-transcriptional regulation of germline mRNAs in C. elegans

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Project Summary: My lab is interested in defining how the maternal load is established during oogenesis and decoded after fertilization. We know the identity of most important maternal transcripts and maternally supplied RNA- binding proteins. We know that these factors are required for germline development, oocyte maturation, and pattern formation in early embryogenesis. But we do not yet know which regulatory events are most important for reproduction, or what mechanisms coordinate regulation in space and time. We employed a “protein-centric” approach to map the wiring diagram of maternal RNA regulation in the nematode Caenorhabidits elegans. We defined the sequence motifs recognized by a several maternal RNA- binding proteins (RBPs) and identified of functional cis-regulatory elements in 3’UTR reporter genes representing well studied maternal mRNAs. Our work made revealed that binding specificity is not sufficient to explain mRNA targeting in vivo. All proteins studied to date bind to short linear partially degenerate motifs present in at least 30-50% of all mRNAs. In some cases, the motifs have been shown to be necessary but not sufficient to drive regulatory activity. In other cases, the motifs do not lead to regulation. Putting a motif, even in multiple copies, into a transgene does not confer RBP-dependent regulation. Binding is not a great predictor of regulation, revealing that binding site context is also crucial for targeting. Moving forward, we are pursuing three major strategies. In the first, we are using CRISPR-cas9 genome editing to make mutations in the 3’UTRs of two critical maternal transcripts in order to identify which regulatory events are most important to define the pattern of expression and for reproductive health. Genome editing technology has advanced to the point where we can make targeted UTR deletions and substitutions, so now we can assess importance directly. Our second direction is aimed at defining regulatory mechanisms. We are performing AID-degron tagged experiments to define how RBPs and core regulatory machinery control the maternal mRNA expression with temporal resolution in the germline and in the embryo. Finally, we wish to understand how the biochemical properties of an RBP contribute to its mutant phenotypes. Proteins can have multiple activities, and it is not always clear that the most obvious activity is the one that underlies its mutant phenotypes. We are in position to address this question directly. We have expressed and purified several C. elegans RBPs over the course of the past ten years and have begun to dissect their biochemical properties using quantitative in vitro tools. We now have the ability to introduce mutations that effect RNA-binding properties into the endogenous locus in C. elegans to determine phenotype. Our innovative interdisciplinary approach, coupled to the strong atmosphere at UMass Medical School in RNA biology and C. elegans genetics, will ensure rapid progress in defining the maternal effect in embryogenesis at the functional level.