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Molecular characterization of RNA interference components in Chlamydomonas: A novel nucleotidyltransferase (MUT68) and a Vasa intronic gene homolog (MUT70)
RNA interference (RNAi) is an evolutionary conserved and highly specific genesilencing mechanism. The RNAi machinery plays an essential role in regulation of gene expression, developmental pathways and defense responses against genomic parasites in eukaryotes. In this study, we have employed the unicellular alga Chlamydomonas reinhardtii as a model system to gain insight into the mechanistic basis of RNAi by using a genetic screen to identify RNAi defective mutants; we have isolated two mutants with deletions in genes that we have named MUT68 and MUT70. MUT68 encodes a non-canonical poly(A) polymerase that adds untemplated adenines to the RNA induced silencing complex (RISC)-cleaved transcripts, and stimulates their exosome-dependent degradation. We also found that deletion of MUT68 and depletion of RRP6 exosome resulted in elevated small RNA levels. We showed that MUT68 plays a role in the untemplated uridylation of the 3’ ends of small RNAs in vivo and stimulates their degradation by the RRP6 exosome subunit in vitro. Thus, MUT68 in association with RRP6 participates in the degradation of RISC-associated miRNAs and siRNAs, most likely as a quality control mechanism to eliminate dysfunctional small RNA molecules. MUT70 encodes the Chlamydomonas homolog of the Drosophila Vasa intronic gene, previously identified as interacting with Argonaute2 (AGO2). Loss of MUT70 abolished small-RNA-guided target transcript cleavage, without a defect in the biogenesis of small RNAs or the steady-state levels of the AGO3 protein. Affinity purification using epitope tagged-MUT70 identified several interacting partners including AGO3/2, Dicer3/2 and ribosomal proteins. This complex is also associated with small RNAs and showed sequence-specific cleavage of a homologous RNA substrate. Polysome profile analyses demonstrated that depletion of MUT70 disrupted the association of AGO3 with polyribosomes. Therefore, MUT70 is an essential component of the Chlamydomonas RISC and is required for AGO3 association with polyribosomes. MUT70 may facilitate, through association with ribosomes, the recognition of target transcripts by RISC.^
Ibrahim, Fadia F, "Molecular characterization of RNA interference components in Chlamydomonas: A novel nucleotidyltransferase (MUT68) and a Vasa intronic gene homolog (MUT70)" (2009). ETD collection for University of Nebraska - Lincoln. AAI3368688.