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

智力上的功绩。虽然经过几十年的研究，真核生物纤毛和鞭毛的蛋白质和脂肪成分已经形成了大量的目录，但以前还没有将RNA描述为鞭毛成分。然而，在衣藻的鞭毛中，可以在高度纯化的鞭毛部分中发现大量的小RNA(称为鞭毛RNA或fRNAs)，大小从30到200个核苷酸不等。关于这些fRNAs作用的一个吸引人的假说是调节鞭毛蛋白基因的表达。大量研究表明，鞭毛衣藻的组装状态与鞭毛蛋白质的合成直接相关。鉴于许多不同的小RNA在真核细胞中调节基因表达的多方面作用，似乎特定的小RNA在鞭毛室中的隔离可能在将鞭毛的组装状态传递到细胞中的基因表达机制中发挥作用。该项目寻求对这些RNA进行表征，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来鉴定单个fRNAs并确定它们是否与已知的小RNA相对应，如snoRNAs、miRNAs、piRNAs或其他小RNA；2)开发特定的杂交探针以定位细胞体和鞭毛中的fRNAs；3)对突变体和野生型鞭毛进行分离以确定fRNAs是否与特定的鞭毛下隔间，如膜或中央微管有关；以及4)研究鞭毛组装过程中fRNAs的积累动力学以及化学诱导的野生型细胞和具有组装和小RNA代谢缺陷的突变体的吸收。在50多年来对真核生物鞭毛的细胞生物学、生化和遗传学的研究中，没有任何迹象表明RNA在鞭毛的组装和功能中发挥作用。这一研究成果将改变我们对纤毛的认识，开辟一个以真核纤毛的组装和功能为中心的研究新领域。更广泛的影响。构成这项提议的基础的初步数据是由一名本科生产生的。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.