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

点击翻译按钮获取中文摘要

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.