Mechanisms of small RNA-mediated translation repression in Chlamydomonas

衣藻中小 RNA 介导的翻译抑制机制

• 批准号: 1616863
• 负责人: Heriberto Cerutti
• 金额: $ 56万
• 依托单位: University of Nebraska-Lincoln
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616863&HistoricalAwards=false
• 关键词: Mechanisms; small; RNA; mediated; translation

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.

该项目将通过产生微藻遗传改良所需的基础知识而产生广泛的科学影响，微藻正在成为生产生物材料、营养保健品和生物燃料的合适资源。这项工作的具体重点是了解小 RNA 分子如何调节蛋白质合成过程，以单细胞绿藻为模型。该研究将涉及本科生、研究生和博士后水平的研究人员，从而有助于培训“知识工人”，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、外展活动以及对藻类资源中心的贡献来广泛传播。该项目还将通过招募和教育科学领域代表性不足的群体成员和第一代学生来扩大参与范围。 小RNA在多个过程中发挥着重要的调节作用，RNA干扰（RNAi）已成为反向遗传研究和医学和农业实际应用的宝贵工具。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 如何在启动后步骤抑制蛋白质合成来解决这一理解空白。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的突变体进行遗传筛选，鉴定出参与TOR（雷帕霉素靶标）信号传导和多肽异戊二烯化途径的基因，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，将进行实验：(i) 主要通过蛋白质组学方法，表征 RAPTOR（雷帕霉素靶点调节相关蛋白）和 TOR 激酶与 RNAi 效应复合物之间的负相互作用； (ii) 通过鉴定 VIG 相互作用因子及其对核糖体功能的影响，表征 Vasa Intronic Gene (VIG) 产品同源物缺失的突变体； (iii) 通过插入诱变筛选确定 sRNA 介导的翻译抑制所需的新因子。这些发现有望为小RNA如何在翻译抑制中发挥作用提供新的见解。