Download Identification of RNAi-Related Genes in Archaea

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.