Mechanisms of small RNA-mediated translation repression in Chlamydomonas

该项目将产生广泛的科学影响，为微藻的遗传改良提供必要的基础知识，微藻正在成为生产生物材料、营养药品和生物燃料的合适资源。这项工作的具体重点将是了解小RNA分子如何调节蛋白质合成过程，并以单细胞绿藻为模型。这项研究将涉及本科生、研究生和博士后水平的研究人员，从而有助于培养“知识工作者”，他们的专业技能将使他们能够在STEM行业和职业中高效地工作。结果和产品将通过在科学期刊上发表、外展活动和向藻类资源中心捐款等方式广泛传播。该项目还将通过招募和教育科学领域代表性不足群体的成员和第一代学生来扩大参与。小RNA在多个过程中发挥着重要的调控作用，RNA干扰（RNA interference, RNAi）已成为逆向遗传研究和医学、农业实际应用的重要工具。尽管小RNA功能的许多方面已经被详细研究，但小RNA介导的翻译抑制机制仍然知之甚少。该项目将通过研究小rna如何在起始后抑制蛋白质合成来解决这一理解上的空白。实验将在单细胞绿藻衣藻中进行。在初步实验中，对小rna介导的翻译抑制受损突变体的基因筛选发现了涉及TOR（雷帕霉素靶蛋白）信号传导和多肽前置酰化途径的基因，从而暗示这些过程是小rna介导功能的关键步骤。为了跟进这些观察结果，将进行以下实验：(i)主要通过蛋白质组学方法表征RAPTOR（雷帕霉素靶蛋白的调控相关蛋白）和带有RNAi效应复合物的TOR激酶之间的负相互作用；（ii）通过鉴定VIG相互作用物及其对核糖体功能的影响，鉴定Vasa内含子基因（VIG）产物同源物缺失的突变体；（iii）通过插入突变筛选，确定srna介导的翻译抑制所需的新因子。这些发现有望为小rna在翻译抑制中的作用提供新的见解。

The project will have broad scientific impact by generating basic knowledge needed for the genetic improvement of microalgae, which are emerging as a suitable resource for the production of biomaterials, nutraceuticals, and biofuels. The specific focus of the work will be on understanding how small RNA molecules regulate the process of protein synthesis, using a unicellular green alga as the model. The research will involve researchers at undergraduate, graduate and postdoctoral levels, thus contributing to the training of 'knowledge workers' whose specialized skills will enable them to work productively within STEM industries and occupations. Results and products will be disseminated broadly through publication in scientific journals, outreach activities, and contributions to an algal resource center. The project will also broaden participation through the recruitment and education of members of underrepresented groups in the sciences and first generation students. Small RNAs play important regulatory roles in multiple processes, and RNA interference (RNAi) has become a valuable tool for reverse genetic studies and practical applications in medicine and agriculture. Whereas many aspects of small RNA function have been studied in detail, the mechanisms of small RNA-mediated translational repression remain poorly understood. This project will address this gap in understanding by studying how small RNAs function to inhibit protein synthesis at post-initiation steps. The experiments will be carried out in a unicellular green alga, Chlamydomonas. In preliminary experiments, a genetic screen for mutants impaired in small RNA-mediated translational repression identified genes involved in TOR (Target of Rapamycin) signaling and polypeptide prenylation pathways, thus implicating these processes as key steps in small RNA-mediated function. To follow up on these observations, experiments will be conducted to: (i) characterize the negative interplay between RAPTOR (Regulatory Associated Protein of Target Of Rapamycin) and the TOR kinase with RNAi effector complexes, mostly through proteomic approaches; (ii) characterize a mutant deleted for a homolog of the Vasa Intronic Gene (VIG) product, via the identification of VIG interactors and their effect(s) on ribosome function; and (iii) identify new factors required for sRNA-mediated translation repression, by means of an insertional mutagenesis screen. The findings are expected to provide new insights into how small RNAs function in translational repression.