Biological Sciences, School of
Characterization of RNAi-defective mutants -- Mut13-2 and Mut20 -- in Chlamydomonas reinhardtii
Date of this Version
RNA interference (RNAi) is a highly conserved and sequence-specific process for regulation of gene expression. At the heart of the RNAi machinery lie 21-24 nt small non-coding RNA molecules which are able to downregulate the expression of cognate sequences, by causing degradation or translational repression of mRNAs. These noncoding RNAs act via the effector RNA-induced silencing complex (RISC), which includes core proteins such as the Argonautes (AGOs). Two Chlamydomonas reinhardtii RNAi-defective mutants, Mut13-2 and Mut20, show deletions of various domains of the Tudor Staphylococcal Nuclease 1 (TSN1) gene. TSN1 has already been recognized as a component of the RNA-induced silencing complex in metazoans. However, the specific function of this protein in the RNAi pathway remains undefined. In this study we report that TSN1 is essential for RNAi in Chlamydomonas reinhardtii. Interestingly, the two examined mutants show a widely different profile of endogenous miRNAs. The small RNAs are almost completely depleted in the case of Mut20 but are only slightly reduced in level in Mut13-2. Successful complementation with an epitope-tagged TSN1 and recovery of RNAi-induced phenotypes was achieved in Mut13-2 but not in Mut20. Through immunofluorescence microscopy, the epitope-tagged TSN1 protein was found to be predominantly cytoplasmic, although some degree of nuclear localization was also observed, which supports its association with the RISC as reported in metazoans. Yet, coimmunoprecipitation assays with the epitope-tagged TSN1 did not show association with AGO3, a core component of the RISC in Chlamydomonas. It is tempting to hypothesize that TSN1 may play an accessory role in the assembly of the RISC, perhaps in the loading of small RNAs onto Argonautes. This interpretation would be consistent with the reduced level of small RNAs in the mutant backgrounds and the lack of high affinity interactions with AGO proteins. However, elucidating the exact role of TSN1 will require further investigation.
Advisor: Heriberto Cerutti
A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Biological Sciences, Under the Supervision of Professor Heriberto Cerutti. Lincoln, Nebraska: July 2011
Copyright 2011 Arit Ghosh