The Role of mRNA Degradation in Embryonic Cell Fate Specification

项目摘要 在发育过程中，细胞经历适当细胞所需的基因表达的动态变化 命运规范。虽然从转录调控的角度来研究发育基因的表达是最好的， 对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的衰退机制中，已经被认为与具有不同表型的疾病有关，如骨肉瘤和 神经退行性疾病。此外，母体mRNAs在所有动物中的广泛降解 早期胚胎发育对于控制从母体提供到合子提供的发育至关重要 编码产品。对母体和受精卵mRNA衰变动力学的研究已经建立了转录本 稳定性在很大程度上是由蛋白质或RNA因子与3‘端的顺式调节元件结合而调节的 转录本的非翻译区(3‘UTR)。密码子的使用是mRNA稳定性的另一个主要决定因素，因为 翻译可以以一种密码子依赖的方式影响mRNA的稳定性。考虑到RNA的巨大多样性- 真核生物中的结合蛋白和小RNA，以及选择性剪接和多聚腺苷酸化， 对信使核糖核酸降解的调控可能是高度复杂的。这种复杂性可能会塑造精确的基因 发育过程中的表达模式，尽管发育调节的合子mRNA的程度 退化情况尚不清楚。为了探索这一点，我正在研究秀丽线虫合子mRNA的降解。 整个胚胎发育过程中。在目标1中，我将生成转录组范围的mrna衰减率图。 在整个胚胎发育过程中具有空间和时间分辨率。记录的半衰期将使用以下方法确定 单细胞rna测序以测量经转录抑制物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，我将使用代谢标记和 用两种正交法测定RNA聚合酶II的降解速率。机制： 不同的信使核糖核酸降解，即在不同细胞类型中具有不同衰减率的基因，将被探索。 使用转基因方法。在目标2中，我将确定主要的5‘到3’和3‘到5’信使核糖核酸衰退的作用 发展中的路径。我将通过对分期的RNA进行测序来确定这两条通路的mRNA靶点 没有同源外切核糖核酸酶的胚胎。与对照组相比显著上调的基因 胚胎将被视为假定的目标。此外，我将确定这两条通路在细胞命运中的作用 通过使用实时成像分析外切核糖核酸酶缺失胚胎中细胞命运标记的表达来进行规范。 通过表征不同细胞类型和发育阶段的mRNA衰减率，并建立机制 对于不同的mRNA降解，我将开始揭示合子mRNA周转在胚胎细胞命运中的作用 规格。这些发现将提供对所使用的监管策略的更全面的理解 在胚胎发育期间，以调节发育过渡和图案化。

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.