Program of the 2004 East Coast Worm Meeting - Caenorhabditis ...

1. Candidate EGO-1 interactors that function in germline development.
Xiang Yu 1 , Valarie Vought 1 , Jamie Wasilenko 1 , Tom Ratliff 2 , Bill Kelly 2 , Eleanor Maine 1
1Department of Biology, Syracuse University, Syracuse, NY
2Biology Department, Emory University, Atlanta, GA
ego-1 was identified in a genetic screen for enhancers of glp-1 (Qiao et al.1995). Later studies
showed that ego-1 mutants have defects in germline proliferation, meiotic progression,
gametogenesis and RNA interference (RNAi) (Smardon et al. 2000). EGO-1 belongs to the
RNA-dependent RNA Polymerase (RdRP) family. RdRP family members have been shown to
function in RNA silencing in many organisms, although their specific role is unclear. Our working
model is that the ego-1 developmental defects result from defects in RNA metabolism, perhaps
specifically from the failure to produce or amplify small, non-coding RNA important for various
cellular processes. Two approaches that we have taken to understanding the role of EGO-1 in
germline development are to screen for interaction partners using the yeast two-hybrid system
and to investigate chromatin structure in ego-1 mutant germ lines.
EGO-1 is predicted to be a 1632 amino acid (aa) protein with a conserved RdRP domain (813
aa) located between N-terminal (491 aa) and C-terminal (328 aa) domains. We used both
N-terminal and C-terminal domains as "bait" in two-hybrid screens. We then tested candidate
interactors by RNAi to identify those that function in the germ line, and particularly those whose
germline function might be related to ego-1. We identified three genes that bind the EGO-1
N-terminal domain and have severe proliferation defects in RNAi injection experiments. We then
used tempered RNAi to produce milder proliferation defects, and checked for genetic interactions
with glp-1. We find that R08C7.12 RNAi causes a meiotic progression defect similar to ego-1
mutants, but does not enhance glp-1(ts). In contrast, ZC518.2 RNAi causes a masculinized germ
line (a Mog defect) in wildtype animals and enhances glp-1(ts). Finally, the Y54F10BM.2 RNAi
proliferation defect is more severe in a glp-1(ts) background than in a wildtype background,
suggesting that it is enhanced by glp-1(ts). We are now extending these RNAi studies to include
other relevant genes, and are collaborating with the Conradt lab (Dartmouth College) to recover
deletion alleles of all three genes. To complement these studies, we are using a directed yeast
two-hybrid approach to better define the physical interactions between EGO-1 and these three
proteins.
Studies in S. pombe have shown that chromatin silencing modifications depend, at least in part,
on components of the RNAi machinery, including the pombe RdRP, RdP1 (Volpe et al., 2002).
For example, accumulation of a silencing modification on centromeres, methylation of histone H3
on the lysine 9 residue (H3meK9), is defective in RdP1 mutants. In the C. elegans germ line,
H3meK9 silencing modifications accumulate on the X chromosome during male meiosis and on
other unpaired DNA (Bean et al., 2004). We are investigating whether EGO-1 function is
important for this aspect of germline chromatin assembly. Preliminary data suggest that silencing
modifications on the male X are dependent on EGO-1 function.
Bean et al. (2004) Nature Genetics 36, 100-105
Qiao et al. (1995) Genetics 141, 551-569
Smardon et al. (2000) Current Biology 10, 169-178
Volpe et al. (2002) Science 297, 1833-1837.