Research

Animal embryos receive approximately equal amounts of DNA from each of their parents, but nearly all of their cytoplasm is inherited from Mom. This cytoplasm contains information in the form of maternal proteins and mRNAs that are used by the embryo prior to zygotic gene activation, the point at which maternal mRNA is cleared and the embryo becomes reliant upon its own genome. This maternal load of mRNA is critical to successful sexual reproduction in animals. Maternal mRNAs provide instructions to the embryo to guide early patterning and cell fate specification events prior to the onset of zygotic transcription. Much remains to be learned about how maternal mRNAs are produced, silenced, and reactivated after fertilization. The wiring diagram, the mechanisms at play, and the overall functional significance of individual regulatory circuits within the network of maternal RNA regulation remain to be described.

My lab uses Caenorhabditis elegans to investigate maternal RNA regulation. Forward genetic screens identified numerous genes that when mutated give maternal effect lethal or sterile phenotypes caused by failures in embryogenesis and/or gametogenesis. Many encode RNA-binding proteins and/or their target mRNAs. My lab has systematically mapped the motifs recognized by several maternal RNA-binding proteins and used this information to build a predictive interaction database based upon quantitative binding criteria. Our work enabled preliminary mapping of post-transcriptional regulatory circuits in the embryo. We then tested the functional relevance of key motifs in live animals using integrated fluorescent reporter strains. The results of our studies revealed several themes:

• Most germline RNA-binding proteins recognize short, partially degenerate, linear sequence motifs that are abundant in the genome.
• Not all binding sites are equally functional. Most high affinity sites do not confer regulation to a reporter on their own, even within the natural context of an entire 3´UTR. Binding sites are necessary for regulation, but few are sufficient.
• The binding motifs we have mapped, by the very nature of their sequence, often overlap with adjacent motifs. The pattern of binding sites and their proximity appears to be important to regulation.

Despite these advances, there remained a major limitation. It was not possible to make targeted gene deletions or conversions in the worm, so the physiological relevance of each new regulatory interaction could not be assessed in its endogenous context. The CRISPR-Cas9 genome editing revolution has swept through the C. elegans research community and removed this roadblock. We can now make precision engineered mutations to assess the physiological impact of each new regulatory event that we map. We are using this approach to make alleles within the 3´ untranslated region (UTR) of the most important cell-fate determinants conserved from worms to humans.