A genome-wide RNAi screen identifies factors required for distinct stages of C. elegans piRNA biogenesis
- Wee-Siong Sho Goh1,2,
- Jun Wen Eugene Seah1,2,
- Emily J. Harrison1,2,
- Caifu Chen3,
- Christopher M. Hammell1 and
- Gregory J. Hannon1,2,4
- 1Watson School of Biological Sciences,
- 2Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA;
- 3Genetic Applications R&D, Life Technologies Corporation, Foster City, California 94404, USA
Abstract
In animals, piRNAs and their associated Piwi proteins guard germ cell genomes against mobile genetic elements via an RNAi-like mechanism. In Caenorhabditis elegans, 21U-RNAs comprise the piRNA class, and these collaborate with 22G RNAs via unclear mechanisms to discriminate self from nonself 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. However, nothing is known of how the expression of precursors is controlled or 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, quantitative PCR (qPCR)-based measurements of mature 21U-RNA levels, we identified 22 genes important for 21U-RNA production, termed TOFUs (Twenty-One-u Fouled Ups). We also identified seven genes that normally repress 21U production. By measuring mature 21U-RNA and precursor levels for the seven strongest hits from the screen, we assigned factors to discrete stages of 21U-RNA production. Our work identifies for the first time factors separately required for the transcription of 21U precursors and the processing of these precursors into mature 21U-RNAs, thereby providing a resource for studying the biogenesis of this important small RNA class.
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Footnotes
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↵4 Corresponding author
E-mail hannon{at}cshl.edu
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Supplemental material is available for this article.
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.235622.113.
- Received November 27, 2013.
- Accepted February 20, 2014.
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