The Role of mRNA Degradation in Embryonic Cell Fate Specification

mRNA 降解在胚胎细胞命运规范中的作用

• 批准号: 10604512
• 负责人: Felicia Peng
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604512
• 关键词: 3' Untranslated Regions; Address; Affect; Alternative Splicing; Animals; Auxins; Binding; Binding Proteins; Caenorhabditis elegans; Cell Culture System; Cell Lineage; Cells; Codon Nucleotides; Complex; Data; Data Analyses; Defect; Deposition; Development; Developmental Gene; Developmental Process; Disease; Dissociation; Embryo; Embryonic Development; Ensure; Eukaryota; Exoribonucleases; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Half-Life; Health; Human; Image; Invertebrates; Kinetics; Knowledge; Label; Link; Maps; Maternal Messenger RNA; Measurement; Measures; Mediating; Messenger RNA; Metabolic; Methods; Modification; Nematoda; Neurodegenerative Disorders; Organism; Pathway interactions; Pattern; Phenotype; Physiologic pulse; Poly A; Polyadenylation; Post-Transcriptional Regulation; Process; Proteins; RNA; RNA Decay; RNA Degradation; RNA Polymerase II; RNA-Binding Proteins; Regulation; Regulatory Element; Resolution; Role; Shapes; Small RNA; Specific qualifier value; Specificity; System; Testing; Time; Tissues; Transcript; Transcriptional Regulation; Transgenes; Transgenic Organisms; Translations; blastomere structure; cell fate specification; cell type; comparison control; egg; embryo cell; exosome; gene product; inhibitor; innovation; live cell imaging; mRNA Decay; mRNA Stability; mRNA Transcript Degradation; model organism; osteosarcoma; progenitor; programs; single-cell RNA sequencing; spatiotemporal; temporal measurement; transcriptome; transcriptome sequencing

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.

项目概要 在发育过程中，细胞经历基因表达的动态变化，这是适当细胞所需的 尽管发育基因表达最好是在转录调控方面进行研究， mRNA 降解的调节也可能对这些表达模式有重要贡献。 mRNA 衰变机制与具有不同表型的疾病有关，例如骨肉瘤和 此外，在所有动物中，母体 mRNA 普遍降解。 早期胚胎发生对于控制发育从母体提供到合子提供至关重要 对母体和合子 mRNA 衰变动力学的研究已经确定了该转录本。 稳定性很大程度上是通过蛋白质或RNA因子与3’内顺式调控元件的结合来调节的 转录本的非翻译区 (3’ UTR) 密码子使用是 mRNA 稳定性的另一个主要决定因素。 考虑到 RNA 的巨大多样性，翻译可以以密码子依赖的方式影响 mRNA 的稳定性。 真核生物中的结合蛋白和小RNA，以及选择性剪接和聚腺苷酸化， mRNA 降解的调控可能非常复杂，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，尽管发育调节合子 mRNA 的程度 为了探索这一点，我正在研究秀丽隐杆线虫的合子 mRNA 降解。 在目标 1 中，我将生成 mRNA 衰减率的全转录组图。 整个胚胎发生的空间和时间分辨率将由转录本半衰期确定。 单细胞 RNA 测序可测量经转录抑制剂处理的胚胎细胞中 mRNA 的丰度。 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 RNA 聚合酶 II 的降解作为两种正交方法来测量降解速率。 将探索不同的 mRNA 降解，即不同细胞类型中具有不同降解速率的基因 在目标 2 中，我将使用转基因方法确定主要 5' 至 3' 和 3' 至 5' mRNA 衰变的作用。 我将通过分阶段的 RNA 测序来识别这两种途径的 mRNA 靶标。 与对照相比，同源核糖核酸外切酶基因被显着上调的胚胎。 此外，我将确定这两种途径在细胞命运中的作用。 通过使用实时成像分析核糖核酸外切酶耗尽的胚胎中细胞命运标记的表达来规范。 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 差异 mRNA 降解的研究，我将开始揭示合子 mRNA 更新在胚胎细胞命运中的作用 这些发现将提供对所使用的监管策略的更全面的了解。 在胚胎发生过程中介导发育转变和模式形成。