Small RNA-mediated Translation Repression in Chlamydomonas

智力优势：真核生物的生长和发育是由它们的基因组成和环境之间复杂的相互作用决定的。在多细胞生物中，不同类型的细胞、组织和器官最终是由不同的基因表达决定的。小rna（长度约为20-30个核苷酸）在基因调控以及对寄生基因组元件（如转座子和病毒）的防御反应中发挥重要作用。近年来，这些非编码RNA的生物发生和作用方式引起了人们的极大关注，但对小RNA （sRNA）功能的许多方面仍知之甚少。一个核心问题是我们对这些过程中涉及的因素的理解不完全。有趣的是，在绿藻莱茵衣藻（Chlamydomonas reinhardtii）中，与目标转录物完全互补的sRNAs可以抑制蛋白质合成，而不破坏或仅破坏最小的mRNA稳定性。这种机制在翻译起始后的阶段起作用，似乎改变了翻译核糖体的功能/结构构象，从而降低了它们对某些抗生素（如环己亚胺）抑制的敏感性。srna抑制转录物与多核糖体相关的证据也存在于其他真核生物中，这是支持起始后翻译抑制的有力论据。该项目将侧重于这一相对未被探索的过程的机制特征。它将涉及srna引导的效应复合物的分子成分鉴定，涉及起始后翻译抑制，以及在此过程中有缺陷的藻类突变体的分离和分析。该研究还将在翻译水平上描述被sRNAs优先抑制的内源性转录物，并开始解决小rna在藻类生物学中的作用。衣藻为分离和表征参与RNA介导的翻译抑制的新因子提供了优势，因为RNA干扰（RNAi）机制尽管相当复杂，但不是生物体生存所必需的。此外，通过这些研究可以获得新的机制见解，因为藻类和陆地植物似乎缺乏与后生动物中srna介导的转录后调节有关的几种蛋白质的同源物。在进化上不同的真核生物中对RNAi的表征有望有助于我们理解srna介导的沉默的共同机制和谱系特异性机制。更广泛的影响该项目将通过直接培养本科生、研究生和一名博士后，对人力资源发展产生影响。学生将通过亲身体验学习研究方法，目的是培养他们对科学发现的兴趣。在两门植物生物学课程中，实验室的研究结果将用于刺激学习过程，并促进对藻类/植物科学研究的好处的讨论。培养博士后从事研究与教育相结合的职业也是该项目的核心组成部分。此外，学生们还将参加一些专门针对少数民族个人的指导项目，以及与一家非营利组织一起开展的推广活动，该组织致力于向公众宣传科学的本质和重要性。该项目的结果也可能对用于生物燃料生产的藻类菌株的遗传改良具有实际意义。藻类原料商业开发的一个关键制约因素是对控制基因表达和代谢的调节机制的了解非常有限。预期的发现将为理解微藻中小rna的功能和开发调节基因表达的工具提供有用的信息。

Intellectual Merit: The growth and development of eukaryotes is determined by complex interactions between their genetic make-up and the environment. In multicellular organisms, the different types of cells, tissues, and organs are ultimately defined by differential gene expression. Small RNAs (~20-30 nucleotides in length) play important roles in gene regulation as well as in defense responses against parasitic genomic elements such as transposons and viruses. The biogenesis and mode of action of these noncoding RNAs have attracted great attention in recent years, but many aspects of small RNA (sRNA) function are still poorly characterized. One central problem is our incomplete understanding of the factors involved in these processes. Interestingly, in the green alga Chlamydomonas reinhardtii, sRNAs perfectly complementary to a target transcript can inhibit protein synthesis without or with only minimal mRNA destabilization. This mechanism operates at a post-initiation step of translation and appears to alter the function/structural conformation of translating ribosomes so that they display reduced sensitivity to inhibition by some antibiotics such as cycloheximide. Evidence for sRNA-repressed transcripts remaining associated with polyribosomes, a strong argument in support of post-initiation translation inhibition, also exists in several other eukaryotes. The project will focus on the mechanistic characterization of this relatively unexplored process. It will involve the identification of molecular components of sRNA-guided effector complexes, involved in post-initiation translation repression, and the isolation and analysis of algal mutants defective in this process. The research will also characterize endogenous transcripts preferentially repressed by sRNAs at the translational level and begin addressing the role(s) of small RNAs in algal biology. Chlamydomonas offers an advantage for the isolation and characterization of novel factors involved in sRNA-mediated translation repression since the RNA interference (RNAi) machinery, despite being fairly complex, is not required for organismal viability. Moreover, novel mechanistic insights may be obtained through these studies because algae and land plants appear to lack orthologs of several of the proteins implicated in sRNA-mediated post-transcriptional regulation in metazoans. Characterization of RNAi in evolutionarily divergent eukaryotes is expected to contribute to our understanding of both common as well as lineage-specific mechanisms of sRNA-mediated silencing.Broader ImpactsThe project will have an impact on human resource development through the direct training of undergraduate and graduate students and one postdoctoral fellow. Students will learn research methodology via hands on experience, with the goal of fostering their interest in scientific discovery. The laboratory findings will be used to stimulate the learning process and to promote discussions on the benefits of algal/plant science research in two plant biology courses. The training of a postdoctoral fellow for a career which combines research and education is also a central component of the project. Additionally, students will participate in a number of programs devoted to the instruction of minority individuals and in outreach activities with a non-profit organization working to educate the public on the nature and importance of science. The results of the project may also have practical implications for the genetic improvement of algal strains for biofuel production. One key constraint for the commercial development of algal feedstocks is the very limited knowledge on the regulatory mechanisms that control gene expression and metabolism. The anticipated findings will contribute useful information for understanding the function of small RNAs in microalgae and for the development of tools to modulate gene expression.