Poster abstracts
Poster number 3 submitted by Clare Austin
Investigating the role of P-bodies in Pnrc2-mediated oscillatory transcript decay
Clare C. Austin (Department of Molecular Genetics, Center for RNA Biology), Thomas L. Gallagher (Department of Molecular Genetics, Center for RNA Biology), Monica C. Blatnik (Department of Molecular Genetics, Center for RNA Biology, Interdisciplinary Graduate Program in Molecular, Cellular and Developmental Biology), Kathryn G. Thompson (Department of Molecular Genetics, Center for RNA Biology), Danielle M. Pvirre (Department of Molecular Genetics), Sharon L. Amacher (Department of Molecular Genetics, Center for RNA Biology, Interdisciplinary Graduate Program in Molecular, Cellular and Developmental Biology, Center for Muscle Health and Neuromuscular Disorders)
Abstract:
The segmentation clock is an oscillatory gene network that regulates somitogenesis, the formation of embryonic segments that develop into vertebrae and musculature. Proline-rich nuclear receptor coactivator 2 (Pnrc2), an mRNA decay adaptor, is necessary for rapid decay of oscillatory transcripts in the zebrafish presomitic mesoderm (PSM). When Pnrc2 function is lost, over 1700 transcripts, including known oscillatory transcripts her1 and dlc, are overexpressed. Despite overexpression of many transcripts, pnrc2 mutant embryo development is overtly normal, with developmental delay and reduced viability. Lack of an overt embryonic mutant phenotype is likely because accumulated transcripts are poorly translated. The mechanism by which Pnrc2 regulates rapid decay and translational repression of oscillatory transcripts is unknown. The ddx6 and ddx61genes encode DEAD-box helicases associated with P-bodies and are two of the few known genes that are upregulated at both the transcript and protein level in pnrc2 mutant embryos. Pnrc2 is known to interact with P-body-associated proteins in human cultured cells, consistent with our hypothesis that P-bodies contribute to oscillatory transcript regulation. To characterize Pnrc2 localization and identify Pnrc2 interactors, we are using CRISPR-mediated knock-in to tag endogenous Pnrc2 with an ALFA epitope sequence, which will allow us to detect the endogenous Pnrc2 protein using the ALFA nanobody. Using an ALFA nanobody-GFP fusion, we will observe Pnrc2 localization during somitogenesis in wild-type and pnrc2 mutant embryos and co-localization with Ddx6 and Ddx61. Using an ALFA nanobody-Halo tag fusion, we will use co-immunoprecipitation and mass spectrometry to identify Pnrc2 interactors. I predict Pnrc2 interacts with P-body proteins in the zebrafish PSM and will colocalize with Ddx6 and Ddx61. This study will identify the role of P-bodies in Pnrc2-mediated oscillatory transcript decay during somitogenesis.
Keywords: somitogenesis, oscillatory gene expression, P-bodies