RNA-mediated processes result in suppression of gene expression in eukaryotes and can produce a variety of outcomes such as mRNA degradation, heterochromatin formation, or DMA methylation. The RNA interference machinery has also been implicated in the processing and function of microRNAs, a class of small RNAs that regulate gene expression by translational repression or mRNA cleavage. The widespread occurrence of these phenomena in eukaryotes suggests that they entail ancestral, conserved mechanisms postulated to play essential roles in limiting the expression of parasitic elements, such as transposons and viruses, as well as in controlling developmental programs. Our long term goal is to elucidate the molecular basis of RNA-mediated silencing. By using the unicellular alga Chlamydomonas reinhardtii as a model system, we have isolated mutants in two classes of genes involved in RNA silencing. One group encodes factors that appear to be directly involved in RNAi: Mut68p, a putative DNA polymerase beta-like nucleotidyltransferase;MutTOp, a homolog of the vasa intronic gene product;and Mut91p, a novel but evolutionary conserved protein with a C2H2 zinc finger and a RNA binding motif. Another group of genes, typified by Mut6 (encoding a putative DEAH-box RNA helicase), seems to regulate the pre-mRNA processing and, thus, the mRNA levels of certain RNAi components. These genes may modulate RNAi activity in response to abiotic stresses. This proposal will focus on three main goals. (1) Molecular characterization of mutants defective in dsRNA-mediated silencing (Mut-68, Mut-70, and Mut-91). All these strains appear to be defective in processes downstream from the processing of long dsRNA to small RNAs. Our hypothesis is that these factors either play a role as components of RISC (the RNA-guided endonucleolytic complex) or may modulate its activity/assembly and coordinate the degradation of cleaved transcripts. We will attempt to define their molecular roles by isolation of proteins interacting with the cloned gene products;by testing the biochemical activity of purified complexes and recombinant polypeptides;by examining the subcellular localization of fusion proteins;and by complementation of mutant strains with wild type and mutated forms of the proteins followed by detailed phenotypic and molecular characterization. (2) Examination of the biological role(s) of RNA-silencing in the response to abiotic stresses. This goal will be achieved by testing the survival of mutant strains under different environmental conditions. Potential target genes of RNA-silencing will be identified in microarray experiments comparing wild-type and mutant strains. (3) Isolation of additional genes involved in dsRNA-mediated gene silencing by insertional mutagenesis screens. The overall findings are expected to improve our ability to exploit RNAi as an experimental and/or therapeutic tool, with likely impacts in both medicine and agriculture.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics C Study Section (MGC)
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Bender, Michael T
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University of Nebraska Lincoln
Schools of Arts and Sciences
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Kim, Eun-Jeong; Cerutti, Heriberto (2009) Targeted gene silencing by RNA interference in Chlamydomonas. Methods Cell Biol 93:99-110
Merchant, Sabeeha S; Prochnik, Simon E; Vallon, Olivier et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245-50
Cerutti, Heriberto; Casas-Mollano, J Armando (2006) On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50:81-99
Ibrahim, Fadia; Rohr, Jennifer; Jeong, Won-Joong et al. (2006) Untemplated oligoadenylation promotes degradation of RISC-cleaved transcripts. Science 314:1893
Sarkar, Nandita; Lemaire, Stephane; Wu-Scharf, Danxia et al. (2005) Functional specialization of Chlamydomonas reinhardtii cytosolic thioredoxin h1 in the response to alkylation-induced DNA damage. Eukaryot Cell 4:262-73
van Dijk, Karin; Marley, Katherine E; Jeong, Byeong-ryool et al. (2005) Monomethyl histone H3 lysine 4 as an epigenetic mark for silenced euchromatin in Chlamydomonas. Plant Cell 17:2439-53
Rohr, Jennifer; Sarkar, Nandita; Balenger, Susan et al. (2004) Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J 40:611-21
Cerutti, Heriberto (2003) RNA interference: traveling in the cell and gaining functions? Trends Genet 19:39-46
Zhang, Chaomei; Wu-Scharf, Dancia; Jeong, Byeong-ryool et al. (2002) A WD40-repeat containing protein, similar to a fungal co-repressor, is required for transcriptional gene silencing in Chlamydomonas. Plant J 31:25-36