The Role of mRNA Degradation in Embryonic Cell Fate Specification

项目摘要 在发育过程中，细胞经历基因表达的动态变化，这些变化是适当的细胞生长所必需的。 命运规格虽然发育基因表达在转录调控方面的研究最好， mRNA降解的调节也可能对这些表达模式有重要贡献。缺陷 在mRNA衰变机制中，已经将具有不同表型的疾病，如骨肉瘤和 神经退行性疾病此外，在所有动物中，母体mRNA的广泛降解， 早期胚胎发生对于控制发育从母体提供到合子提供是至关重要的。 编码产品。对母体和合子mRNA衰变动力学的研究已经确定了该转录本 稳定性在很大程度上受蛋白质或RNA因子与3'端顺式调节元件的结合调节。 转录物的非翻译区（3' UTR）。密码子使用是mRNA稳定性的另一个主要决定因素， 翻译可以以密码子依赖性方式影响mRNA的稳定性。考虑到RNA的巨大多样性- 结合蛋白和小RNA，沿着选择性剪接和多聚腺苷酸化， mRNA降解的调节可能是高度复杂的。这种复杂性可以塑造精确的基因 在发育过程中的表达模式，虽然发育调节合子mRNA的程度， 降解不清楚。为了探索这一点，我正在研究秀丽隐杆线虫合子mRNA的降解 在胚胎发育过程中。在目标1中，我将生成一个mRNA衰减率的转录组范围图， 在整个胚胎发生过程中的空间和时间分辨率。转录本半衰期将使用 单细胞RNA测序以测量用转录抑制剂处理的胚胎细胞中的mRNA丰度。 为了验证通过这种转录抑制方法测量的半衰期，我将使用代谢标记， RNA聚合酶II的降解作为两种正交方法来测量衰减速率。机制 将探索差异mRNA降解，即在不同细胞类型中具有不同降解速率的基因 使用转基因方法。在目标2中，我将建立主要的5′到3′和3′到5′ mRNA衰变的作用， 发展的道路。我将通过RNA测序确定这两种途径的mRNA靶点， 同源核糖核酸外切酶耗尽的胚胎。与对照组相比显著上调的基因 胚胎将被视为假定的靶。此外，我将确定这两种途径在细胞命运中的作用， 通过使用活体成像分析外切核糖核酸酶耗尽的胚胎中的细胞命运标志物表达来鉴定。 通过表征不同细胞类型和发育阶段的mRNA衰减率， 差异mRNA降解，我将开始揭示胚胎细胞命运合子mRNA周转的作用 规范.这些发现将提供对所使用的监管策略的更全面的理解 在胚胎发生期间介导发育转变和模式。

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.