Regulation of gene expression and genome organization by small RNAs

RNA沉默是一种基因调控机制，通过这种机制，小RNA（18-30个核苷酸）及其Argonaute 蛋白质辅因子调节外源和内源mRNA的表达。小分子RNA在 通过鉴定完全或部分互补的mRNA和靶向 它们用于转录或转录后调节。RNA沉默在基因调控中的作用 在大多数真核生物中是保守的，因此对发育和 人类细胞生物学C.秀丽隐杆线虫由于其短而成为研究RNA沉默的优秀系统 世代时间，它的透明度，这是理想的显微镜，和强大的遗传工具，可用于 基因组操作In C.在线虫生殖细胞中，参与RNA沉默的蛋白质被组织成亚细胞， 核周冷凝物的隔间，每个子隔间发挥独特的和关键的作用。许多 蛋白质因子已被确定定位于这些结构，但我们对它们是如何定位的知之甚少。 组织和组装。在本提案的第一部分，我们将确定促进 RNA沉默组分组装成核周浓缩物。我们将进一步鉴定每个细胞中的RNA， 亚区室，最终目标是解剖靶向mRNA通过其每个亚区室的轨迹。 阶段：从转录开始，继续通过核周浓缩物的聚集， 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，其中许多共定位在P颗粒，我们将确定下一步的因素 将小RNA分选成合适的Argonaute蛋白。这种分类是至关重要的，因为许多Argonaute 蛋白质相互结合不同的小RNA，靶向不同的mRNA组， 对这些靶mRNA的不同调节作用。最后，我们将重点讨论蛋白质修饰，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蛋白和其他关键因素的动力学。在一起，拟议的 实验将揭示RNA沉默途径如何组织的关键细节， 这些途径是确定的，什么机制调节这些途径。这项工作将导致更好的 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 识别这些途径的变化如何导致人类基因的失调并转变为疾病 状态

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.