Kazuma Aoki et al. (2007) International Worm Meeting "Biochemical genetic analyses of RdRP and Slicer activities related to RNAi in C. elegans."

Kazuma Aoki, Hiroaki Tabara, Katsuya Okawa, Hiromi Moriguchi

[International Worm Meeting, 2007]

In the pathway of RNAi, Dicer activity processes long dsRNA into siRNAs, and subsequently the siRNAs induce Slicer activity resulting in mRNA cleavage or transcriptional silencing. RdRP activity is thought to participate in a sort of signal amplification step. In C. elegans, biochemical knowledge about RdRP and Slicer activities remains to be insufficient. Utilizing an in vitro assay system constructed from C. elegans, we analyzed RdRP and Slicer activities. As a result of incubating template RNAs and labeled ribonucleotides in partially fractionated cell lysates, we detected an RdRP activity responsible for the synthesis of 21-23 nt small RNAs. Long single-stranded RNAs (such as mRNA derivatives) were much more effective at inducing the RdRP activity than dsRNA. In vivo studies by several other groups have mentioned that the majority of siRNAs accumulated during RNAi in C. elegans are secondary siRNAs synthesized de novo on mRNA targets, because the major fraction of the accumulated siRNAs contains triphosphates at their 5'' end. This fact raises a new question if 3'' end formation of secondary siRNAs is coupled with Dicer activity. We found that the RdRP activity synthesizing small RNAs is severely impaired in mutants of an RdRP, rrf-1(fj18 etc.). In contrast, dcr-1 and rde-4 mutants retains the RdRP activity. We then analyzed the RRF-1 complex, and found DRH-3 as a component in the complex. The RRF-1 complex was able to synthesize small RNAs on long single-stranded RNAs with a primer-independent initiation. Further analyses revealed that drh-3(fj52) and rde-3 mutants also lack the RdRP activity. These data suggest that RRF-1, cooperatively with DRH-3 and RDE-3, produces secondary siRNAs with a Dicer-independent manner. Meanwhile, to seek for Slicer activities cleaving target mRNAs, we introduced duplex siRNAs, monophosphorylated or triphosphorylated single-stranded siRNAs into cell lysates. Slicer activities were induced weakly by the duplex siRNAs and moderately with the monophosphorylated single-stranded siRNAs. Interestingly, the triphosphorylated single-stranded siRNAs (mimicking secondary-type) induced a strongest Slicer activity. This observation suggests that secondary siRNAs may be a major driving force in the destruction of target mRNAs during RNAi in C. elegans. We examined various mutants, and found that the Slicer activity induced by the triphosphorylated single-stranded siRNAs is severely impaired in an Argonaute mutant, csr-1(fj54). A recombinant protein of CSR-1 showed a Slicer activity weakly with monophosphorylated siRNAs and strongly with triphosphorylated siRNAs. CSR-1 together with secondary siRNAs may act as a major RISC in C. elegans.

Kazuma Aoki et al. (2005) International Worm Meeting "Construction of an in vitro RNAi system using cell lysates from C. elegans"

Kazuma Aoki, Hiromi Moriguchi, Hiroaki Tabara, Katsuya Okawa

[International Worm Meeting, 2005]

RNAi is a form of sequence-specific gene silencing induced by the introduction of double-stranded RNA (dsRNA). In C. elegans, genetic analyses have revealed various RNAi-related genes as well as analogies among RNAi and several endogenous silencing events. However, biochemical analyses of the RNAi pathway have been insufficient. By contrast, studies of RNAi in Drosophila have progressed mainly with biochemical approaches and revealed two major enzymatic activities. Introduced long dsRNAs are processed into 21- to 22-nt fragments termed small interfering RNAs (siRNAs). A downstream complex receives the siRNAs and forms RNA-induced silencing complex (RISC) mediating the cleavage of target mRNA. Unfortunately, studies in C. elegans have not succeeded in detecting the RISC-like activities with in vitro experiments. To analyze RNAi-related enzymatic activities in C. elegans, we decided to construct an in vitro system reproducing RNAi, using cell lysates from C. elegans. At the start point, an apparent obstruction was the fact that non-specific nuclease activities are abundantly present in crude worm lysates. Therefore, we fractionated the lysates by ion-exchange chromatographies and took a wide fraction whose non-specific nuclease activities were minimized. This partially purified lysates have been used for constructing our in vitro RNAi system. We first checked whether our lysates contain the RNaseIII activity producing siRNAs. In vitro reactions with our lysates processed long dsRNAs into 23- to 24-nt siRNAs. We next introduced 23-nt synthetic siRNAs and a target mRNA into our lysates, and investigated whether the siRNAs can induce the sequence-specific cleavage of target mRNA (RISC activity). Finally, we succeeded in detecting RISC-like activities reconstituted in the worm lysates. This in vitro system requires ATP. Interestingly, single-strand siRNAs are more efficient than duplex siRNAs for inducing the RISC-like activities in our system. Possibly, this effectiveness of single-strand siRNAs may correlate with a phenomenon that antisense strands but not sense strands of siRNAs tend to accumulate in vivo by feeding RNAi. It was shown that major components of RISC in Drosophila and mammals are homologs of RDE-1 found in C. elegans. In order to dissect RISC in C. elegans, we are interested in RDE-1 and its homologs. To investigate proteins interacting with RDE-1, we analyzed the RDE-1 complex and found that RDE-1 interacts with DCR-1, DRH-1and RDE-4. Currently, we are investigating possible involvements of these RNAi factors in the RISC activity.

Hiromi Moriguchi et al. (2005) International Worm Meeting "A search for new components of the RNAi pathway in C. elegans"

Hiroaki Tabara, Kazuma Aoki, Hiromi Moriguchi, Katsuya Okawa

[International Worm Meeting, 2005]

RNAi is a form of sequence-specific gene silencing induced by the introduction of double-stranded RNA (dsRNA). The ability of dsRNA to induce silencing was first discovered in C. elegans, and similar responses have been observed in a variety of eukaryotes. Although genetic and biochemical studies in several model organisms have revealed a rough mechanism of the RNAi response, it is far from a complete understanding. With several approaches, we are searching for new components required for RNAi. In C. elegans, genetic factors required for RNAi have been revealed by screens for RNAi deficient (rde or other names) mutants. In original screens, rde-1 and sid-1 mutations on LG V were recovered very frequently, and therefore it was not easy to collect new rde mutations reflecting new loci. In order to avoid this problem, we performed a genetic screen utilizing nT1 which is a balancer chromosome of LG V (EAWM 2004, ab112). The presence of nT1 prevents from recovering recessive mutaions on the balanced chromosomal region. Among total 51 mutants isolated from this and similar screens, we are currently focusing and analyzing mutants whose RNAi responses are severely deficient. The mutant stocks include rde-3 (one allele), mut-16 (one allele), rrf-1 (two alleles), rde-4 (10 alleles) as well as several new mutants possibly reflecting new components in the RNAi pathway. In addition, we are trying to identify proteins interacting with known RNAi factors (such as RDE-1) to find candidates for new RNAi factors (see also Aoki et al., this meeting). For this purpose, we generated several transgenic worms expressing the known RNAi factors attached with an epitope tag. Utilizing the epitope tag, we next immuno-purified the RNAi factor complex and analyzed the protein components of the complex with mass spectrometry. From these experiments, a protein possessing DExD/H helcase domain was identified. We isolated a deletion mutant lacking the helicase gene (with the kind help of Nishiwaki and Kuroki) to investigate its function. This mutant (fj52) is sterile showing a defect in oogenesis. Currently, we are analyzing the detailed phenotype of fj52.

Kobayashi S et al. (2001) Biochem J "Membrane recruitment of DOCK180 by binding to PtdIns(3,4,5)P3."

Kobayashi S, Shirai T, Fukui Y, Mochizuki N, Matsuda M, Kiyokawa E

[Biochem J, 2001]

DOCK180 was originally identified as one of two major proteins bound to the Crk oncogene product and became an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. Further study has suggested that DOCK180 is involved in the activation of Rac by the CrkII-p130(Cas) complex. With the use of deletion mutants of DOCK180, we found that the C-terminal region containing a cluster of basic amino acids was required for binding to and activation of Rac. This region showed high amino-acid sequence similarity to the consensus sequence of the phosphoinositide-binding site; this led us to examine whether this basic region binds to phosphoinositides. For this purpose we used PtdIns(3,4,5)P(3)-APB beads, as reported previously [Shirai, Tanaka, Terada, Sawada, Shirai, Hashimoto, Nagata, Iwamatsu, Okawa, Li et al. (1998) Biochim. Biophys. Acta 1402, 292-302]. By using various competitors, we demonstrated the specific binding of DOCK180 to PtdIns(3,4,5)P(3). The expression of active phosphoinositide 3-kinase (PI-3K) did not enhance a DOCK180-induced increase in GTP-Rac; however, the expression of PI-3K translocated DOCK180 to the plasma membrane. Thus DOCK180 contained a phosphoinositide-binding domain, as did the other guanine nucleotide exchange factors with a Dbl homology domain, and was translocated to the plasma membrane on the activation of PI-3K.