Changes in small RNAs upon Viral infection of C.elegans
ABSTRACT: Attempt to identify small non-coding RNAs that change in levels as a result of viral infection of C.elegans Small non-coding RNA (18-30nt) was extracted from animals either infected with Orsay virus or uninfected as indicated.
Project description:Attempt to identify progressive changes in small non-coding RNA levels in C.elegans lacking RSD-2 or RSD-6. Small non-coding RNA (18-30nt) was extracted from animals the indicated number of generations after homozygosity was established
Project description:An attempt to identify small non-coding RNAs that change with increasing generations after becoming homozygous for the loss of PRG-1 Small non-coding RNA (18-30nt) was extracted from animals the indicated number of generations after homozygosity was established
Project description:Dissection of the small RNA pathway required for generation of an antiviral small RNA response Small non-coding RNA (~15-30nt) was extracted from animals infected with the Orsay virus
Project description:piRNAs are required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. We find that target silencing is independent of Piwi endonuclease activity or “slicing”. Instead, we show that piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene, pseudogene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes but not pseudogenes or transposons. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans. 7 small RNA libraries were sequenced as part of 25 flow cell lanes on the Illumina GA II platform. Samples were treated with tobacco acid pyrophosphatase to allow cloning of small RNAs with a 5'-triphosphate. Samples were labelled for multiplexing using 4-bp 5'-barcodes or barcodes included in Illumina TruSeq adapters. In most cases a single flow cell lane included several multiplexed libraries.
Project description:C. elegans has served as a laboratory model organism due to its ease of manipulation and the availability of both forward and reverse genetics. In recent years, efforts to study host-pathogen interactions in C. elegans have increased. For example, analysis of infections by bacteria such as Pseudomonas, Salmonella or Serratia has revealed the existence of innate immune pathways in C. elegans that are also conserved in vertebrates. To date, there has been no natural virus infection reported in C. elegans or C. briggsae. Here we describe evidence of natural virus infection in wild isolates of both C. elegans and C. briggsae. Two highly divergent but related RNA viruses in the family Nodaviridae, tentatively named Orsay nodavirus and Santeuil nodavirus, were detected and their genomes partially sequenced. Infected worm lysates passed through 0.2 um filters could be used to infect uninfected worms, which could be further passaged for many generations. Furthermore, the viruses were subject to processing by the RNAi machinery as evidenced by the detection of virally derived small RNAs. Infection of mutant worms defective in small RNA pathways yielded more robust levels of viral RNA as compared to infection of isogenic N2 reference worms. These data demonstrate that nodaviruses are natural parasites of nematodes in the wild. Further study of the interactions between these viruses and nematodes is likely to provide insight into the natural ecology of nematodes and may reveal novel innate immune mechanisms that respond to viral infection. Two small RNA libraries (18-30 nt) from nodavirus-infected and cured C. elegans wild isolate JU1580 were sequenced on the Illumina Genome Analyzer II platform. Samples were treated with tobacco acid pyrophosphatase to allow cloning of small RNA molecules with 5'-triphosphates. Each sample was labelled with a unique four base pair barcode and libraries were multiplexed together with a third library (not included in this submission). The multiplexed libraries were sequenced in triplicate.
Project description:This SuperSeries is composed of the following subset Series: GSE28617: Function, targets and evolution of Caenorhabditis elegans piRNAs (small RNA-Seq) GSE37432: Function, targets and evolution of Caenorhabditis elegans piRNAs (mRNA) Refer to individual Series
Project description:Analysis of the transcriptional response to viral infection in C.elegans. Comparison of infected and uninfected animals for three strains: N2 (resistant), JU1580 (sensitive), RDE-1 (sensitive)
Project description:Identification of defects in piRNAs and piRNA precursors Sequencing of small RNAs (18-30nt). Sample treatments: Secondary siRNAs in C. elegans carry a 5' triphosphate and this has to be removed prior to cloning, hence the use of 5' polyphosphatase treatment to reveal this population. TAP (tobacco acid pyrophosphatase) is another enzyme that removes 5' triphosphate but it also removes 5' CAP structures- we used this to look for piRNA precursor sequences in C. elegans that have a 5' cap.