EAGER: The role of small RNAs (fRNAs) in Chlamydomonas flagella

知识价值。尽管几十年的研究已经开发了真核纤毛和鞭毛的蛋白质和脂质成分的大目录，RNA以前没有被描述为鞭毛成分。然而，在鞭毛衣单胞菌中，在高度纯化的鞭毛片段中可以发现大量的小rna（称为鞭毛rna或fRNAs），其大小从30到200个核苷酸不等。对于这些fRNAs的作用，一个有吸引力的假设是调节鞭毛蛋白基因的表达。大量研究表明，鞭毛衣单胞菌的组装状态与鞭毛蛋白的合成直接相关。考虑到许多不同的小rna在真核细胞中调节基因表达的多方面作用，鞭毛室中特定小rna的隔离似乎可能在将鞭毛的组装状态传递给细胞中的基因表达机制方面发挥作用。该项目旨在描述这些rna的特征，以生成支持完整研究拨款提案的初步数据。本研究的目标是：1)使用高通量测序程序鉴定单个fRNAs，并确定它们是否对应于已知的小rna，如snoRNAs、miRNAs、piRNAs或其他；2)开发特异性杂交探针，定位细胞体和鞭毛中的frna；3)对突变型和野生型鞭毛进行分离，以确定frna是否与特定的鞭毛下隔室（如膜或中央对微管）相关；4)在野生型细胞和具有组装和小RNA代谢缺陷的突变体中，研究鞭毛组装和化学诱导吸收过程中fRNAs的积累动力学。在五十多年来对真核鞭毛细胞生物学、生物化学和遗传学的研究中，没有发现RNA在鞭毛组装和功能中的作用。本研究结果将改变我们对真核纤毛的认识，为真核纤毛的组装和功能研究开辟一个新的领域。更广泛的影响。形成该提案基础的初步数据是由一名本科生生成的。本科生将继续在这项研究中发挥作用，该项目还将为博士后研究员提供培训机会。

Intellectual Merit. Although decades of research have developed a large catalogue of protein and lipid components of eukaryotic cilia and flagella, RNA has not been previously described as a flagellar component. In Chlamydomonas flagella, however, abundant, small RNAs (called flagellar RNAs or fRNAs) ranging in size from 30 to 200 nucleotides can be found in highly purified flagellar fractions. One attractive hypothesis for the role of these fRNAs is to regulate the expression of the genes for flagellar proteins. There is a large body of research showing that the assembled state of Chlamydomonas flagella is directly correlated with the synthesis of flagellar proteins. Given the multi-faceted roles of many different small RNAs in regulating gene expression in eukaryotic cells, it seems likely that the sequestration of specific small RNAs in the flagellar compartment might play a role in communicating the assembled state of the flagella to the gene expression machinery in the cell. This project seeks to characterize these RNAs to generate preliminary data to support a full research grant proposal. The goals of the research are to: 1) use high throughput sequencing procedures to identify the individual fRNAs and determine whether they correspond to known small RNAs such as snoRNAs, miRNAs, piRNAs or others; 2) to develop specific hybridization probes to localize the fRNAs in cell bodies and flagella; 3) to use fractionation procedures on mutant and wild-type flagella to determine whether the fRNAs are associated with specific sub-flagellar compartments, such as the membrane or the central pair microtubules; and 4) to examine the kinetics of accumulation of fRNAs during flagellar assembly and chemically induced resorption in wild-type cells and mutants with defects in assembly and small RNA metabolism. In more than five decades of research focused on the cell biology, biochemistry and genetics of eukaryotic flagella, no hint of a role for RNA in flagellar assembly and function has emerged. The outcome of this research could transform our understanding and open a new field of research centered on the assembly and function of eukaryotic cilia. Broader Impacts. The preliminary data that formed the basis for this proposal were generated by an undergraduate student. Undergraduates will continue to play a role in this research, and the project will also provide training opportunities for a postdoctoral researcher.