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.

Hiromi Moriguchi et al. (2004) East Asia Worm Meeting "A genetic screen to identify new components of the RNAi pathway in C. elegans"

Kazuma Aoki, Hiroaki Tabara, Hiromi Moriguchi

[East Asia Worm Meeting, 2004]

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 have revealed a rough mechanism of the RNAi response, it is far from a complete understanding. Toward identification of new components required for RNAi, we recently designed a new genetic screen for RNAi deficient ( rde ) mutants. In original screens, rde-1 and sid-1 mutations on LG V were recovered very frequently, and therefore it was not so easy to collect new rde mutations reflecting new loci. We recently performed a screen with avoiding this problem, utilizing nT1 which is a balancer chromosome of LG V. We mutagenized nT1 worms with a combination of TMP and UV, and next transferred the F2 population onto bacteria expressing cdk-1 dsRNA which induces a defect in cell cycle. Finally, viable worms in the F3 or F4 population were recovered as rde mutants. Instead of using TMP, similar screens with EMS mutagenesis have also been carried out. So far, we have successfully recovered total 19 mutants from several sets of screens. Among these mutations, 4 mutations were mapped on LG I, and the position of a mutation ( fj3 ) have been narrowed down into 1 mu interval. We will report the progress in mapping of fj3 and other mutations.

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.

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.

Masako ASAHINA et al. (2003) International Worm Meeting "A highly conserved yet non-essential coactivator MBF1 may play a role in oxidative stress in C. elegans"

Masako ASAHINA, Marek JINDRA, Susumu Hirose

[International Worm Meeting, 2003]

A small helix-turn-helix protein MBF1 has been maintained throughout the evolution of eukaryotes. Our previous genetic evidence from yeast and Drosophila implicates MBF1 in transcriptional activation mediated by bZIP transcription factors [1,2]. To understand the role of a C. elegans homolog of mbf1, we began to analyze its expression and function. The expression was visualized using an C. elegans mbf1(Cembf1)::GFP reporter and a polyclonal antibody against the Drosophila protein, revealing constitutive and ubiquitous presence of the gene product. Deletions of the transgenic GFP fusion construct showed that a relatively short (223 bp) upstream region was sufficient for Cembf1 expression. Functional conservation of mbf1 was tested in yeast (S. cerevisiae) mbf1 disruptant that is viable but sensitive to histidine starvation [1]. The growth of the mbf1 mutant yeast was partially restored by transformation with the C. elegans mbf1 homolog, indicating that the mechanism of MBF1 action could be conserved from unicellular to multicellular organisms. RNA interference silencing of mbf1 in C. elegans showed fairly wild type phenotype although the Cembf1 dsRNA effectively suppressed GFP in the Cembf1::GFP transgenic worms. This suggests that, as in yeast and flies, mbf1 is not an essential gene in C. elegans. The question then arises of what makes mbf1 important enough to be conserved during evolution. Our preliminary data show that the mbf1 RNAi treated worms are more sensitive to hydrogen peroxide, implying that MBF1 is required to withstand oxidative stress. A microarray analysis is underway to find what genes might be affected in mbf1 RNAi background during the hydrogen peroxide challenge. [1] Takemaru K, Harashima S, Ueda H and Hirose S. (1998) Mol. Cell Biol. 18, 4971-76 [2] Liu QX, Jindra M, Ueda H, Hiromi Y, Hirose S. (2003) Development 130, 719-28