Poster abstracts

Poster number 52 submitted by Monica Mannings

Pnrc2-dependent mRNA decay and translational control mechanisms promote oscillatory gene expression during vertebrate segmentation

Monica Mannings (The Ohio State University Molecular Genetics Department), Thomas Gallagher (The Ohio State University Molecular Genetics Department), Kiel Tietz (The University of Minnesota), Sharon Amacher (The Ohio State University Molecular Genetics Department)

Abstract:
During early vertebrate embryogenesis, muscle and skeletal stem cells are grouped into reiterated segments, called somites, in a process called somitogenesis. Sequential somite formation is established by a genetic oscillator called the segmentation clock, comprised of a network of genes expressed cyclically in the presomitic mesoderm. Precise control of segmentation clock oscillations is driven by robust temporal regulation of mRNA production, translation, and mRNA decay, and our work explores post-transcriptional mechanisms that regulate oscillatory expression. Previously, we demonstrated that Proline-rich nuclear receptor coactivator 2 (Pnrc2) regulates oscillatory mRNA decay in zebrafish and loss of Pnrc2 results in stabilization and accumulation of cyclic gene transcripts. Surprisingly, pnrc2 mutant embryos exhibit normal protein oscillations and somite patterning is not disrupted. We are currently addressing this discrepancy between mRNA and protein levels using polysome profiling, which allows us to infer the translation status of oscillatory gene transcripts in wild-type and pnrc2 mutant embryos. Using this technique, we found segmentation clock gene transcripts her1, her7, dlc, and rhov are inefficiently translated in both genotypes; namely, all four transcripts are significantly increased in the ribosome-unbound (non-translating) state in pnrc2 mutants, indicating that transcripts that are stabilized by loss of Pnrc2 are translationally repressed. To further dissect the mechanism of cyclic gene transcript regulation, we carried out in vivo reporter analyses that revealed two sequence-specific binding motifs, a Pumilio Response Element (PRE) and AU-rich Element (ARE), in the her1 and dlc 3’UTRs. Pumilio and ARE-binding proteins can regulate both mRNA stability and translation, and mutation of the PRE and ARE in the her1 3’UTR in our reporter system markedly increased transcript half-life and polysome association, compared to the unmodified her1 3’UTR. Our future work will utilize biochemical and genetic approaches to investigate the contribution of these motifs and their respective RNA binding proteins on post-transcriptional regulation of segmentation clock oscillations

Keywords: segmentation, translation, oscillations