The Role of mRNA Degradation in Embryonic Cell Fate Specification

项目总结

Project Summary During development, cells undergo dynamic changes in gene expression that are required for appropriate cell fate specification. Although developmental gene expression is best studied in terms of transcriptional regulation, the regulation of mRNA degradation may also have important contributions to these expression patterns. Defects in mRNA decay machinery have been linked to diseases with distinct phenotypes, such as osteosarcoma and neurodegenerative diseases. In addition, the widespread degradation of maternal mRNAs in all animals during early embryogenesis is critical for the control of development to switch from maternally provided to zygotically encoded products. Studies of maternal and zygotic mRNA decay dynamics have established that transcript stability is largely regulated by the binding of protein or RNA factors to cis-regulatory elements within the 3’ untranslated region (3’ UTR) of transcripts. Codon usage is another major determinant of mRNA stability, as translation can affect mRNA stability in a codon-dependent manner. Considering the great diversity of RNA- binding proteins and small RNAs in eukaryotes, along with alternative splicing and polyadenylation, the regulation of mRNA degradation has the potential to be highly complex. This complexity may shape precise gene expression patterns during development, though the extent of developmentally regulated zygotic mRNA degradation is unclear. To explore this, I am studying zygotic mRNA degradation in Caenorhabditis elegans throughout embryonic development. In Aim 1, I will generate a transcriptome-wide map of mRNA decay rates throughout embryogenesis with spatial and temporal resolution. Transcript half-lives will be determined using single cell RNA-sequencing to measure mRNA abundance in embryonic cells treated with a transcription inhibitor. To validate half-lives measured by this transcription inhibition approach, I will use metabolic labeling and degradation of RNA polymerase II as two orthogonal methods to measure decay rates. Mechanisms of differential mRNA degradation, namely genes with different rates of decay in different cell types, will be explored using a transgene approach. In Aim 2, I will establish the roles of the major 5′ to 3′ and 3′ to 5′ mRNA decay pathways in development. I will identify mRNA targets of both pathways through RNA-sequencing of staged embryos depleted of the cognate exoribonuclease. Genes that are significantly upregulated compared to control embryos will be treated as putative targets. Additionally, I will determine the roles of both pathways in cell fate specification by analyzing cell fate marker expression in exoribonuclease-depleted embryos using live imaging. By characterizing mRNA decay rates across cell types and developmental stages and establishing mechanisms of differential mRNA degradation, I will begin to uncover the role of zygotic mRNA turnover in embryonic cell fate specification. Such findings will provide a more comprehensive understanding of regulatory strategies used during embryogenesis to mediate developmental transitions and patterning.