Piwi proteins and their associated small RNAs (piRNAs) have been implicated in germline maintenance and genomic surveillance in metazoans. C. elegans encodes two piwi related genes,
prg-1 and
prg-2, whose gene products bind a class of 21nt RNAs (21U RNAs) and function in the germline piRNA pathway(1,2). PRG-1 and piRNAs are required for fertility at elevated temperatures and repression of the Mariner transposon Tc3, but the mechanisms of these outcomes have not yet been explicitly determined(1,2). Little is known about the biosynthesis, transcription, and possible processing of piRNAs in the C. elegans germline. Unlike in fly, where piRNAs are processed from long, single-stranded precursors transcribed from relatively few discrete genomic loci (3), the 21U RNAs are encoded on both strands in two broad regions of chromosome IV (1,2,4). Beyond the defining 5' uridine, there is no discernable sequence motif present in the 15,000+ unique 21U RNAs identified thus far(4), but most 21U RNA-encoding loci exhibit a signature upstream motif, suggesting that each 21U RNA comprises a modular transcriptional unit(4). We hypothesize that the conserved upstream motif represents a binding site for factors that drives expression of each 21U RNA in the developing germline. To test this hypothesis, we are conducting a yeast one-hybrid screen to identify proteins that bind the conserved upstream motif of 21U RNAs. Multiple bait sequences were designed using this motif and surrounding genomic sequences. These bait sequences were integrated into the yeast genome and used to screen fusion libraries of C. elegans proteins expressed at the L4 and young adult stages through selection for an antibiotic resistance factor encoded downstream of the bait sequence integration site. This resulted in the identification of 26 proteins thus far that allow yeast growth on antibiotic-containing media. To determine which of these interact specifically with the 21U RNA upstream motif, we are testing whether mutation of the bait abrogates the interaction. True positives are being investigated in vivo for a role in piRNA biogenesis through RNAi and/or mutant analysis by Taqman assay of 21U RNA expression levels. The effect on fertility, transposon activity, and germline development will also be characterized. This approach will provide insight into piRNA biogenesis and forge new paths for exploring the mechanisms of Piwi-piRNA function in the worm germline. 1. Das, P.P. et al., Cell 31: 79-90 (2008) 2. Batista, P.J. et al., Cell: 67-78 (2008) 3. Brennecke, j. et al., Cell 128: 1089-103 (2007) 4. Ruby, G. et al., Cell 127:1193-207(2008).