Defects in mutator-class genes have active transposons, loss of endogenous siRNAs, and do not respond to exogenous dsRNA. We have characterized several factors in this pathway by cytological analysis and found that the mutator proteins localize to foci at the periphery of germline nuclei, near to, but not overlapping with P granules and nuclear pores. MUT-16, a Q/N-rich protein, is required for the integrity of the Mutator complex by cytology and immunoprecipitation. Currently, we are focusing on the role of another mutator-class gene,
mut-14, and its paralog Y38A10A.6/smut-1 (Synthetic MUTator-1), both of which are DEAD box helicases. The only available allele of
mut-14,
pk738, is a point mutation in the DEAD motif. Using this strain, we demonstrate that
mut-14 is required for the production of germline endogenous siRNAs (WAGO-class 22G and ERGO-class 26G) and is germline RNAi defective (Rde). However, we generated a deletion allele,
mut-14(
mg464), and unlike
pk738,
mg464 has only mild effects on endogenous siRNA levels and no Rde phenotype. In a subsequent RNAi screen for defects in endogenous siRNA formation and activity, we identified eight DEAD box helicases, including the
mut-14 paralog
smut-1. Like the
mut-14 deletion, a
smut-1 deletion has only very mild effects on endogenous siRNA levels and no Rde phenotype, but a
mut-14 smut-1 double mutant resembles the
pk738/DEAD mutant, suggesting that
pk738 may act dominantly, inhibiting
smut-1 function. Neither single mutant, nor the double mutant is required for somatic RNAi, suggesting a role for these DEAD box helicases specifically in the germline. Interestingly,
mut-14(
pk738) causes hyper-trimming and tailing of siRNAs, although both wild type MUT-14 and MUT-14
(pk738) localize to Mutator foci and trimming and tailing is thought to occur in P granules. We are currently investigating a model where MUT-14 and SMUT-1 transport RNAs from P granules to Mutator foci to initiation siRNA amplification.
CMP and TAM are funded by the Damon Runyon Cancer Research Foundation and the MGH ECOR Tosteson Award.