In the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) and Argonaute proteins cooperatively play important roles. Primary siRNAs bearing monophosphorylated 5'' ends are produced from trigger dsRNAs by an RNaseIII-related enzyme called Dicer. In C. elegans, secondary siRNAs bearing triphosphorylated 5'' ends are also produced by RNA-dependent RNA polymerases, which act on target mRNAs. As a result of in vitro analyses using a cell-free assay system prepared from C. elegans, we previously showed that secondary-type siRNAs induce a prominent Slicer activity to cleave target mRNAs far more effectively than primary-type siRNAs. An Argonaute protein, CSR-1, is responsible for the Slicer activity induced by secondary-type siRNAs. Monophosphorylated siRNAs also induce a weak mRNA cleavage activity in the cell-free system, but the factor responsible for the weak Slicer activity was not clear. CSR-1 in cell lysates migrates to high-molecular-weight fractions by gel filtration. Somewhat similarly, recent fluorescent immunostaining experiments have shown that P-granules in germ cells and certain granules in somatic cells are intensely stained with anti-CSR-1 antibodies; non-granular regions of cytoplasm are also moderately stained. Using the same antibodies, we are now trying to examine interactions between CSR-1 and endogenous siRNAs. On the other hand, we biochemically tested various Argonaute mutants, and found that monophosphorylated siRNAs fail to induce a proper mRNA-cleavage activity in cell lysates prepared from a mutant strain, which corresponds to a 100-kDa Argonaute protein (
p100). This Argonaute-mutant strain is homozygous-viable and known to show a weak RNAi-deficient phenotype. We made a recombinant protein of
p100 and further examined its activity. The recombinant
p100 was able to show an mRNA cleavage activity in conjunction not only with monophosphorylated siRNAs but also with triphosphorylated siRNAs. Several experiments are in progress to investigate the position of
p100 in the RNAi pathway.