Poster abstracts
Poster number 2 submitted by Clare Austin
Understanding segmentation clock oscillatory gene regulation and function
Clare Austin (Molecular Genetics), Thomas Gallagher, Sharon Amacher
Abstract:
A genetic oscillatory network, the segmentation clock, controls somite formation during vertebrate development. In zebrafish, segmentation clock genes oscillate with 30-minute periodicity in the presomitic mesoderm (PSM), the region that gives rise to mesodermal segments called somites. Maintenance of ultradian oscillators like the segmentation clock requires that synthesis and decay of oscillatory RNAs and proteins are rapid. We have shown that the decay adaptor protein Pnrc2 promotes rapid decay of half the known segmentation clock oscillatory transcripts in zebrafish. In human cells, Pnrc2 does not appear to directly bind RNA but does interact with transcript decay-associated and P-body-associated proteins. To understand the mechanism by which Pnrc2 promotes rapid RNA decay of oscillatory transcripts during segmentation, we are endogenously tagging Pnrc2 with a 15 amino-acid ALFA epitope, a short peptide with high affinity for an ALFA-specific nanobody. After recovering a functional tagged allele, we will use ALFA nanobody variants fused to fluorophores, halo tags, and degrons to analyze tissue-specific and subcellular Pnrc2 localization, identify Pnrc2-interacting proteins, and characterize phenotypes induced by rapid, acute loss of Pnrc2 function, respectively.
We performed RNA-sequencing to identify additional Pnrc2-regulated transcripts. We show that transcripts of the rhov gene, which encodes a highly active atypical Rho family GTPase, are among the most highly upregulated transcripts in pnrc2 mutants. In a previous study, rhov transcripts were shown to oscillate in the zebrafish PSM; we now show that rhov transcripts are specifically expressed in PSM cells and are overexpressed in Pnrc2 mutants. Rho family GTPases are known to regulate cell polarity, cell adhesion, and cytoskeletal rearrangements, and some function during mammalian segmentation. To understand the role of rhov during zebrafish development, we are generating rhov alleles via CRISPR and characterizing F0 crispant and mutant phenotypes. We will report our recent progress.
References:
Boswell, Curtis W., et al. "Genetically encoded affinity reagents (GEARs): A toolkit for visualizing and manipulating endogenous protein function in vivo." bioRxiv (2023): 2023-11.
Gallagher, Thomas L., et al. "P-body factors Ddx6 and Ddx61 support development in mRNA-decay deficient pnrc2 mutants." bioRxiv (2025): 2025-05.
Krol, Aurélie J., et al. "Evolutionary plasticity of segmentation clock networks." Development 138.13 (2011): 2783-2792.
Nakaya, Yukiko, et al. "Mesenchymal-epithelial transition during somitic segmentation is regulated by differential roles of Cdc42 and Rac1." Developmental cell 7.3 (2004): 425-438.
Keywords: RNA decay