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Cecere, G., Loew, D., Dingli, F., Cornes, E., Quarato, P., Didier, C., Li, B., Singh, M.
[
International Worm Meeting,
2019]
Caenorhabditis elegans has the presence of a large family of worm-specific Argonaute proteins (WAGOs). Among these WAGOs, only one Argonaute protein - CSR-1, is known to be essential and possess a catalytic activity in vitro. CSR-1 loads small RNAs antisense to most of the germline-expressed mRNAs. Recent studies indicate that the interaction between CSR-1 and its germline mRNA targets can result in 1) direct cleavage of the CSR-1 targets, and/or 2) protection from piRNA-mediated silencing. However, there is still a lack of understanding of the mechanism of CSR-1-mediated gene regulation and how these two activities of CSR-1 are compatible. Here, we combined a worm-sorting strategy with proteomic, biochemical, and high-throughput genomic approaches to dissect the role of CSR-1 catalytic activity-dependent and independent regulation of target genes at precise developmental stages. We observed that CSR-1 cleavage activity occurs on cytoplasmic mRNAs and results in the upregulation of a small subset of its targets characterized by a high density of small RNAs loaded onto CSR-1. These targets include CSR-1-interacting RNAi and p-granule factors identified by mass-spectrometry, indicating that this post-transcriptional slicing is a mode of feedback regulation. Mutation in the catalytic site does not affect the ability of CSR-1 to load small RNAs but impacts the biogenesis of small RNAs derived from the coding regions of its target mRNAs. Further, we show that upon complete removal of CSR-1 protein, piRNA-dependent downstream Argonaute proteins load small RNAs from a subset of CSR-1 targets and downregulate them at the transcriptional level. Overall, our study demonstrates mechanistic details for the two functions (protection and cleavage) attributed to CSR-1 which can, in fact, co-exist but regulate different sets of germline transcripts.
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[
J Helminthol,
1980]
The indirect fluorescent antibody technique has been applied to detect antibody levels in jirds (Meriones unguiculatus) infected with Brugia malayi. Sonicated antigens were prepared from microfilariae and adult worms. Sonicated microfilariae were found to be satisfactory for this purpose. Cyanogen bromide-activated sepharose coated with soluble antigens prepared from microfilariae and adult worms was also used to detect antibodies to Brugia infections. The present observations show that these techniques can be usefully applied for detention of filarial infections. Antibody titres in infected jirds generally ranged from 1:16-1:256 and were not affected by treatment with diethylcarbamazine.
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[
Parasitology,
2013]
DEAD Box RNA helicases are essential enzymes that are involved in RNA metabolic processes such as transcription, pre-mRNA splicing, translation initiation and RNA decay. We have previously over-expressed and biochemically characterized an immunodominant cDNA clone encoding DEAD box RNA helicase (BmL3-Helicase) isolated by immunoscreening of the larval stage cDNA library of Brugia malayi. In the current study, the 3D structure was determined and the immunoprophylactic efficacy of BmL3-Helicase was investigated by immunizing Mastomys coucha with the recombinant protein and subsequently challenging with B. malayi infective larvae. The immunization had an adverse outcome on the establishment of challenged larvae resulting in a 67.4% reduction in adult parasite recovery, a 86.7% decrease in the microfilarial density and profound sterility of the recovered female worms. The immune response thus generated was investigated by measuring the levels of specific antibodies including IgG subclasses, reactive oxygen species and cytokines.
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[
Nat Commun,
2021]
In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with translating ribosomes, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.
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[
Parasitol Res,
2009]
DEAD box proteins are putative RNA unwinding proteins found in organisms ranging from mammals to bacteria. We have identified a novel immunodominant cDNA clone, BmL3-helicase, encoding DEAD box RNA helicase by immunoscreening of a larval stage cDNA library of Brugia malayi. The cDNA sequence exhibited strong sequence homology to Caenorhabditis elegans and C. briggsae RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. The clone also showed similarity with RNA helicase of Wolbachia, an endosymbiotic bacterium of filarial parasite. It was overexpressed as approximately 50 kDa His-tag fusion protein, and ATP hydrolysis assay of recombinant enzyme showed that either ATP or dATP was required for the unwinding activity, indicating BmL3-helicase as an ATP/dATP-dependent RNA helicase. The recombinant protein also demonstrated cross-seroreactivity with human bancroftian sera. The presence of BmL3-helicase in various life stages of B. malayi was confirmed by immunoblotting of parasite-life-cycle extracts with polyclonal sera against the BmL3-helicase, which showed high levels of expression in microfilaria, L(3,) and adult (both male and female) stages. In the absence of an effective macrofilaricidal agent and validated anti-filarial drug targets, RNA helicases could be utilized as a rational drug target for developing agents against the human filarial parasite.
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[
Am J Trop Med Hyg,
1980]
The indirect hemagglutination (IHA) test done with turkey red cells was applied to 173 serum samples obtained from patients and persons exposed to Wuchereria bancrofti and Brugia malayi in endemic areas of Peninsular Malaysia. A crude extract of adult worms of the rat filaria, Breinlia booliati, was used as the antigen. When a titer of 1:16 was taken as negative, positive IHA test rates in sera from microfilaria-negative persons in endemic areas, microfilaremic cases, and patients with clinical filariasis were 13%, 75%, and 80%, respectively. Results of the IHA test correlated well with results obtained with the indirect fluorescent technique.
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[
J Biotechnol,
2012]
The DExD/H box families of RNA helicases are a multifunctional group of proteins involved in unwinding of inter- and intra-molecular base-paired regions. Successful knockdown of DEAD box RNA helicase gene (BmL3-Helicase) of human lymphatic filarial parasite Brugia malayi was done with specifically designed and chemically synthesized siRNA of <20bp to observe the role of enzyme in parasite biology and its worth as an antifilarial drug target. We made efforts to deliver siRNA into parasite by both electroporation and soaking that resulted into diminished helicase gene expression associated with decreased parasite motility, viability (97%) and release of microfilariae (81.0% reduction) from adult females in vitro. The specific gene knockdown also resulted into death of adult male worms in addition to phenotypic deformities in female worm intrauterine stages. RT-PCR of siRNA treated worms revealed a complete knockdown of BmL3-Helicase transcription within 16h. The present findings thus illustrate that targeting helicase gene of B. malayi would not only interfere with embryogenesis and microfilarial production but also result into decreased motility and viability of microfilariae and adult parasites. The B. malayi helicase enzyme thus represents a possible antifilarial drug target.
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[
Worm Breeder's Gazette,
1994]
More degenerins in the worm? Harbinder Singh Dhillon and Monica Driscoll. Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, 679 Hoes lane, Piscataway, N.J. 08855
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[
Dev Cell,
2014]
In this issue of Developmental Cell, Singh and Pohl (2014) report that myosin II cortical flow and the midbody remnant participate in the specification of the C.elegans embryo dorsal-ventral axis.
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[
Dev Cell,
2019]
Bacterial avoidance and innate immune response are two ways by which C.elegans respond to pathogenic bacteria. In this issue of Developmental Cell, Kumar etal. (2019) and Singh and Aballay (2019) demonstrate that bacterial colonization is essential to induce both responses, which may be associated with somatic and reproductive longevity.