Poster abstracts
Poster number 89 submitted by Lauren Levesque
Identification of mediators of NOTCH-dependent mRNA degradation during vertebrate segmentation
Lauren Levesque (Molecular Genetics, The Ohio State University), Kara Braunreiter (Molecular Genetics, The Ohio State University), Susan Cole (Molecular Genetics, The Ohio State University)
Abstract:
Somitogenesis produces the embryonic precursors of the ribs and vertebrae. Oscillation of the transcriptional repressor HES7 in the presomitic mesoderm (PSM), is central to regulating somitogenesis in mammals. Notch signaling activates Hes7 transcription, and the HES7 protein then represses its own expression, creating a negative feedback loop necessary for cyclic expression. To maintain proper HES7 oscillation, the production and degradation of the mRNA must be tightly regulated1,2. In Notch-deficient PSMs, there is a reduction in Hes7 transcription coupled with stabilization of Hes7 mRNA, indicating that in addition to activating Hes7 transcription, Notch promotes decay of Hes7 through an unknown mechanism3,4.
The mouse PSM produces three Hes7 isoforms that differ in the length of their 3′ untranslated region (3′UTR). RNA ISH revealed that all Hes7 isoforms exhibit oscillatory expression in the PSM. Analysis by qRT-PCR shows all three isoforms are stabilized in Notch-deficient PSMs, but it is difficult to quantify the effect as Notch influences both transcription and mRNA stability. To uncouple transcription from Notch, I will use cell-culture based reporter assays to determine which isoforms have Notch-dependent decay, and which cis-region confers this dependency.
To identify Notch-dependent trans-acting factors that may promote mRNA decay, I analyzed publicly available transcriptomic and ChIP-seq data5–9. I searched for genes involved in mRNA processing that had a binding site in the Hes7 3’UTR, exhibit cyclic expression in mouse PSMs, and/or are potential Notch targets. The analysis yielded potential candidates such as: Hnrnpl, Pum2, and Rbm4. Future directions involve qRT-PCR, RNA in-situ hybridization, and cell-culture assays to characterize candidate expression patterns and test their ability to promote Hes7-3′UTR reporter decay.
References:
References
1. Wahi, K., Bochter, M.S., and Cole, S.E. (2016). The many roles of Notch signaling during vertebrate somitogenesis. Semin. Cell Dev. Biol. 49, 68–75. https://doi.org/10.1016/j.semcdb.2014.11.010.
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Keywords: mRNA degradation, development, NOTCH