Proteomics screens identified T23G7.5/PIR-1 as a major component of Dicer complexes recovered from both embryonic and adult populations (Duchaine et al., 2006). PIR-1 exhibits strong conservation in metazoans and is closely related to the phosphatase domain of the essential mRNA capping enzyme, which removes the <font face=symbol>g</font>-phosphate from nascent mRNAs prior to capping. The human counterpart, PIR1 (phosphatase that interacts with RNA/RNP complex 1; Yuan et al., 1998), has been shown to catalize the sequential removal of the <font face=symbol>g</font>- and <font face=symbol>b</font>-phosphates at the 5-terminus of triphosphorylated RNA molecules in vitro, leaving a 5-monophosphate end (Deshpande et al., 1999). The biological role of this enzyme, however, remains unknown. In C. elegans,
pir-1 mutants arrest as L4 larvae. Remarkably, many of these arrested animals remain active and viable and lead a normal life span without reaching sexual maturity. While microRNA processing and function are apparently unaffected, these arrested animals are deficient in RNAi as judged by the lack of twitching and paralysis when fed dsRNA targeting
unc-22. Consistent with the idea that the phosphatase activity of PIR-1 is essential for its role in promoting RNAi, expression of wild-type PIR-1, but not of a catalytically dead form of the protein can rescue
unc-22 RNAi in
pir-1 mutants. We initially reported that exposure of
pir-1 mutant animals to dsRNA for 12 hours did not lead to detectable secondary siRNA accumulation (Duchaine et al., 2006). However, weve recently discovered that exposure to dsRNA for 24 hours results in a level of siRNA production at least as high as in wild-type. This stands in contrast to most other RNAi-defective mutants in which resistance to RNAi correlates with a total absence of secondary siRNAs. We are currently determining the RNAi resistance of the
pir-1 mutants by directly looking at changes in cognate mRNA levels, not only of
unc-22 but additional targets. If, as preliminary results seem to suggest, the targeted gene expression is not downregulated in
pir-1 mutants, we will explore the phosphorylation status of these siRNAs and examine their interactions with various Argonautes implicated in silencing. Finally, we are investigating PIR-1s involvement in the production and/or activity of endogenous small RNAs. Additionally, we are employing whole-genome microarray analysis to compare staged
pir-1 larvae to their wild-type counterparts. We hope that changes in endogenous small RNA and mRNA expression profiles will provide insight into the remarkable L4 developmental arrest of the
pir-1 homozygotes.