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沉默是一种基因调节机制 蛋白质联合因子调节外源和内源性mRNA的表达。小rnas起关键 通过完全或部分互补的mRNA和靶向来维持适当基因表达的作用 它们进行转录或转录后调节。 RNA沉默在基因调节中的作用 在大多数真核生物中，途径是保守的，因此对于发展性和 人类的细胞生物学。秀丽隐杆线虫是研究RNA沉默的绝佳系统，因为它的短 生成时间，其透明度非常适合显微镜以及可用于的强大遗传工具 基因组操纵。在秀丽隐杆线虫的生殖细胞中，参与RNA沉默的蛋白质被组织成亚 核周冷凝水的隔室，每个子室都起着独特而关键的作用。许多 已经确定了蛋白质因子本地化到这些结构，但我们对它们的状况一无所知 组织和组装。在本提案的第一部分中，我们将确定促进的因素和条件 将RNA沉默组件组装到核周冷凝水中。我们将进一步确定每个RNA 子分区，其最终目标是通过其每个靶向mRNA的轨迹进行剖析 阶段：从转录开始，继续通过这种核周的凝结物的组合，然后 最终通过RNA沉默途径的翻译抑制或退化。因为有〜27 C. 秀丽隐杆线虫蛋白质（其中许多共定位在P花岗岩中，我们接下来都会确定促成的因素 将小RNA分选为适当的Argonaute蛋白。这种排序至关重要，因为许多人 蛋白质彼此结合不同的小RNA，靶向不同的mRNA组，并且可以非常有 对这些目标mRNA的不同调节作用。最后，我们将重点介绍蛋白质修饰（例如 磷酸化和甲基化可以调节RNA沉默途径，包括影响定位， 相互作用的伴侣以及Argonaute蛋白质和其他关键因素的动态。一起，提议 实验将揭示RNA沉默途径如何组织的关键细节，如何特异性 定义了途径，哪些机制调节了这些途径。这项工作将导致更好 了解小RNA如何调节健康细胞中的基因表达，最终可以应用于 辨别这些途径的变化如何导致人类基因的不正调并过渡到疾病 状态。