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David M. Ng BME 230, Winter 2005 Project Abstract, Version 2 Identification of RNAi-Related Genes in Archaea Background RNA interference (RNAi) was discovered in 1998 in C. elegans. When a double-stranded RNA (dsRNA) is introduced into a eukaryotic cell, the gene that matches the sequence of the dsRNA is silenced. This method of gene regulation may have originated as a protection mechanism against viruses. Another mechanism for gene expression regulation uses endogeneous regulatory RNAs known as microRNAs; this mechanism uses the same types of proteins as RNAi. RNAi is of interest for several reasons. As a mechanism of regulating gene expression it is of scientific interest. However, RNAi also has applications as an experimental technique and it has possible medical applications. The Project RNA interference is a mechanism that is present in eukaryotes. The goal of my project is to identify the presence of RNAi-related genes in Archaeal genomes. One such gene, Argonaute, has already been identified in Pyrococcus furiosus; finding additional related genes would be suggestive of RNAi in Archaea. Archaea is of particular interest because it is a different domain than Eukarya, but it is phylogenetically the closest. Genes related to RNAi include Argonaute; genes that encode nucleases such as Dicer and Drosha; and genes that encode various helicases and RNA-dependent RNA polymerases1. This project is also my BME 296 Lab Rotation project, supervised by Kevin Karplus. Approach First, I will do a literature search to identify RNAi-related genes; this will include genes from eukaryotes, and also the Argonaute gene of P. furiosus. I will then search for homologs in Archaeal genomes using successively more sensitive gene and protein sequence searches: 1. Gene and protein sequence alignment (i.e., BLAST). 2. Iterative sequence search techniques (i.e., SAM-T2K and SAM-T02). Using the idea that protein structure is more conserved than the primary sequence of amino acids, I will next perform structure-based searches. For RNAi-related proteins with known structures, I will search for proteins of similar structure (and thus presumably of similar function) in Archaea. Not many archaeal protein structures are known, so I will focus my search in two ways. First, I will focus on P. furiosus and P. aerophilum because relatively more information is available for these species. Second, I will search for proteins corresponding to genes in the same operon as Argonaute in P. furiosus; the reason is that genes under common regulatory control are presumably for related functions (i.e., RNAi). For relevant proteins 1 Hammond, et al., “Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi”, Science, 10 August 2001. without known structures, I will use protein structure preduction tools to determine their (putative) structure. Note that the crystal structure was recently determined for an Argonaute protein from Pyrococcus furiosus2. 2 Song, et al., “Crystal Structure of Argonaute and Its Implications for RISC Slicer Activity”, Science, 3 September 2004.