Regulation of gene expression and genome organization by small RNAs

小RNA调控基因表达和基因组组织

• 批准号: 10202138
• 负责人: Carolyn Marie Phillips
• 金额: $ 44.14万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10202138
• 关键词: Affect; Binding Proteins; Caenorhabditis elegans; Cells; Cellular biology; Congenital Abnormality; Cytoplasmic Granules; Developmental Biology; Disease; Eukaryota; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Transcription; Genome; Germ Cells; Goals; Human; Human Biology; Human Development; Infertility; Malignant Neoplasms; Messenger RNA; Methylation; Microscopy; Nucleotides; Pathway interactions; Phase; Phosphorylation; Phylogeny; Play; Post-Transcriptional Regulation; Post-Translational Protein Processing; Proteins; RNA; RNA Degradation; RNA Interference; Regulator Genes; Regulatory Pathway; Role; Small RNA; Sorting - Cell Movement; Specificity; Structure; System; Time; Transcriptional Regulation; Translational Repression; Work; experimental study; genome integrity; human disease; tool

RNA silencing is a gene regulatory mechanism by which small RNAs (18-30 nucleotides) and their Argonaute protein co-factors modulate the expression of both foreign and endogenous mRNAs. Small RNAs play a critical role in maintaining proper gene expression by identifying fully or partially complementary mRNAs and targeting them for either transcriptional or post-transcriptional regulation. The role of RNA silencing in gene regulatory pathways is conserved across most eukaryotes and is thus of fundamental importance to the developmental and cellular biology of humans. C. elegans is an excellent system to study RNA silencing because of its short generation time, its transparency which is ideal for microscopy, and the powerful genetic tools available for genome manipulation. In C. elegans germ cells, proteins involved in RNA silencing are organized into sub- compartments of perinuclear condensates, with each sub-compartment playing a unique and critical role. Many protein factors have been identified that localize to these structures, yet we know little about how they are organized and assembled. In the first part of this proposal, we will identify factors and conditions that promote assembly of RNA silencing components into perinuclear condensates. We will further identify the RNAs in each sub-compartment, with the ultimate goal of dissecting the trajectory of a targeted mRNA through each of its phases: beginning with transcription, continuing through this assemblage of perinuclear condensates, and ultimately with translational repression or degradation by the RNA silencing pathway. Because there are ~27 C. elegans Argonaute proteins, many of which colocalize at P granules, we will next identify the factors contributing to sorting of small RNA into the appropriate Argonaute proteins. This sorting is critical because many Argonaute proteins bind different small RNAs from one another, target distinct groups of mRNAs, and can have very different regulatory effects on these target mRNAs. Finally, we will focus on how protein modifications such as phosphorylation and methylation can regulate RNA silencing pathways, including affecting localization, interacting partners, and dynamics of Argonaute proteins and other key factors. Together, the proposed experiments will uncover the key details of how RNA silencing pathways are organized, how the specificity of the pathways is defined, and what mechanisms regulate these pathways. This work will lead to a better understanding of how small RNAs modulate gene expression in healthy cells, which can ultimately be applied to discerning how changes in these pathways cause misregulation of genes in humans and transition to a disease state.

RNA沉默是一种基因调控机制，小RNA（18-30个核苷酸）及其Argonaute通过该机制 蛋白质辅因子调节外源和内源 mRNA 的表达。小RNA发挥着关键作用 通过识别完全或部分互补的 mRNA 和靶向来维持正确的基因表达 它们用于转录或转录后调节。 RNA沉默在基因调控中的作用 该途径在大多数真核生物中都是保守的，因此对于发育和发育至关重要。 人类细胞生物学。线虫是研究 RNA 沉默的优秀系统，因为它的时间短 生成时间、其透明度（非常适合显微镜）以及强大的遗传工具 基因组操纵。在秀丽隐杆线虫生殖细胞中，参与 RNA 沉默的蛋白质被组织成亚 核周凝聚物的隔室，每个子隔室都发挥着独特而关键的作用。许多 蛋白质因子已被发现定位于这些结构，但我们对它们是如何运作的知之甚少 组织和集合。在本提案的第一部分中，我们将确定促进 将 RNA 沉默成分组装成核周凝聚物。我们将进一步鉴定每个中的RNA 子区室，最终目标是通过其每个子区剖析目标 mRNA 的轨迹 阶段：从转录开始，继续通过核周凝聚物的聚集，以及 最终通过RNA沉默途径进行翻译抑制或降解。因为有〜27 C。 线虫 Argonaute 蛋白，其中许多共定位于 P 颗粒，接下来我们将确定影响因素 将小 RNA 分选为适当的 Argonaute 蛋白。这种排序至关重要，因为许多阿尔戈英雄 蛋白质彼此结合不同的小 RNA，针对不同的 mRNA 组，并且可以具有非常 对这些目标 mRNA 具有不同的调节作用。最后，我们将重点关注蛋白质修饰的方式，例如 磷酸化和甲基化可以调节 RNA 沉默途径，包括影响定位、 相互作用的伙伴、Argonaute 蛋白的动态和其他关键因素。共同提出的 实验将揭示 RNA 沉默途径如何组织、RNA 沉默的特异性如何的关键细节。 定义了途径，以及调节这些途径的机制。这项工作将带来更好的结果 了解小 RNA 如何调节健康细胞中的基因表达，最终可应用于 辨别这些途径的变化如何导致人类基因失调并转变为疾病 状态。