Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A Genome-wide RNAi screen identifies factors required for distinct stages of C. elegans piRNA biogenesis

ABSTRACT: In animals, piRNAs, and their associated Piwi proteins, guard germ cell genomes against mobile genetic elements via an RNAi-like mechanism. In C. elegans, 21U-RNAs comprise the piRNA class and these collaborate with 22G RNAs, via unclear mechanisms, to discriminate self from non-self and selectively and heritably silence the latter. Recent work indicates that 21U-RNAs are post-transcriptional processing products of individual transcription units that produce ~26 nucleotide capped precursors. Yet, nothing is known of how the expression of precursors is controlled or of how primary transcripts give rise to mature small RNAs. We conducted a genome-wide RNAi screen to identify components of the 21U biogenesis machinery. Screening by direct, qPCR-based measurements of mature 21U-RNA levels, we identified 22 genes important for 21U-RNA production, termed TOFUs (Twenty-One-u Fouled Up). We also identified 7 genes that normally repress 21U production. By measuring mature 21U-RNA and precursor levels for the 7 strongest hits from the screen, we have assigned factors to discrete stages of 21U-RNA production. Our work has identified factors separately required for the transcription of 21U precursors, and the processing of these precursors into mature 21U-RNAs, thereby providing an essential resource for studying the biogenesis of this important small RNA class. Small RNA and capped small RNA sequencing from total RNA of C. elegans subjected to different RNAi and different C. elegans mutants

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Gregory Hannon

PROVIDER: E-GEOD-53970 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In plants, MicroRNAs (miRNAs) are a new class of endogenous small RNAs that play essential regulatory roles in plant growth, development and stress response. Extensive studies of miRNAs have been performed in model plants such as rice, Arabidopsis thaliana and other plants. However, the number of miRNAs discovered in maize is relatively low and little is known about miRNAs involved in the four stages during maize ear development. Here, we use deep-sequencing, miRNA microarray assays and computational methods to identify, proM-oM-,M-^Ale, and describe conserved and non-conserved miRNAs at four developmental stages. A total of 27 conserved miRNA families were identiM-oM-,M-^Aed in all four stages, In addition to known miRNAs, we also found 23 new maize-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. We have found that many known and new miRNAs showed temporally expression. Finally, a total of 251 transcripts (140 genes) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.This study led to the confirmation of the authenticity of 27 conserved miRNA families and the discovery of 23 novel miRNAs in maize. In addition, we have identified 130 targets of known and new miRNAs and ta-siRNA using recently developed tools for the global identification of miRNA targets. Identification and characterization of this important class of regulatory genes in maize may improve our understanding of molecular mechanism controlling flower development. The seeds of maize inbred line B73 were first sterilized and germinated in an incubator, then grown in a controlled environment with 28M-BM-0C/21M-BM-0C (day/night) under a 16-h day/8-h night photoperiod with a relative humidity of 70%. Ear development can be divided into four stages: the growth point elongation phase, spikelet differentiation phase, the floret primordium differentiation phase and floret organ differentiation phase. Plant materials (ears) were collected as described previously. In brief, ears were manually collected at the four developmental stages according to the plant features (number of visible leaves, leaf age index, and number of unfolded and folded leaves) combined with microscopic observation.

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A Genome-wide RNAi screen identifies factors required for distinct stages of C. elegans piRNA biogenesis

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