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 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.

piRNAs: from biogenesis to function

“…There has been some debate regarding the overall length of the precursors: Cecere et al have detected a >60 nt capped transcript using 5′ RACE, whereas Gu et al detected shorter 26 nt putative precursor species by small RNA sequencing (Cecere et al, 2012;Gu et al, 2012b). We and others have recently refined this finding showing that it is indeed short species of 28 or 29 nt that are made from most piRNA loci (Goh et al, 2014;Weick et al, 2014).…”

“…PRDE-1 is either involved in generating these precursors by direct or indirect interaction with the motif or may stabilise the precursors once they have been transcribed from the motif (Weick et al, 2014). Furthermore, using an RNAi based genome-wide screen, the Hannon lab has identified several other factors required for C. elegans piRNA biogenesis, which they collectively refer to as TOFUs for 'twenty-one U fouled up' (Goh et al, 2014). Although further work will be required to investigate the localisation, interactions and mechanisms of these proteins, analysis of the precursor and mature piRNA populations following perturbation of these TOFUs has provided valuable insights into the hierarchy of the biogenesis mechanism.…”

piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis

“…Size-selected small RNAs were then cloned as described (Goh et al 2014). Libraries were sequenced on an Illumina HiSeq SE36 or SE76 platform.…”

Diversity of the piRNA pathway for nonself silencing: worm-specific piRNA biogenesis factors

“…Transcription of piRNA precursors requires specific transcription factors, which vary according to species and the precise type of piRNAs. For example, mouse pachytene piRNAs are transcribed by A-Myb, a master regulator of male meiosis (Li et al 2013), while transcription from the fly flamenco locus, a major unistrand piRNA cluster, requires the transcription factor Cubitus interruotus (Goriaux et al 2014). On the other hand, piRNA production from dual-strand clusters in flies depends on Rhino (a variant of heterochromatin protein 1…”

“…In fact, most of piRNA biogenesis factors identified in other animals, including the endonuclease Zucchini/MitoPLD, are not conserved in worms. In this issue of Genes & Development, three groups (de Albuquerue et al 2014;Goh et al 2014;Weick et al 2014) report on their discoveries of novel factors that are involved in the production of 21U-RNAs in C. elegans. Goh et al (2014) performed an RNAi screen covering >80% of annotated C. elegans protein-coding genes and identified 22 genes that affect mature 21U-RNA levels (named TOFUs).…”