Insights into mechanisms of fertility through study of Caenorhabditis elegans piRNAs
piRNAs are a class of short interfering RNAs (siRNAs) characterized by 21 nt length, 5’ uridine, and no sequence similarity or conservation.1 piRNAs require Piwi protein PRG-1 for accumulation and are involved in germline maintenance and fertility.[2-4] piRNAs suppress harmful genetic elements, including transposable elements, by priming biogenesis of secondary siRNAs that target these elements for degradation.[5-8] piRNAs map primarily to two broad regions of chromosome IV, but their mechanism of biogenesis remains largely unknown. A recently characterized class of germline endogenous siRNAs, 26G RNAs, show distinct expression in either spermatogenic cells or oocytes and embryos.[9,10] We wondered whether piRNAs also show patterns of male or female germline specificity. Although some piRNAs have been detected in both male and female germlines,[2,3] what might distinguish germline-specific patterns of enrichment among all piRNAs is poorly understood. Here, we computationally analyze published sequencing datasets to assess germline specificity of piRNAs. More than 70% of piRNAs are >5-fold enriched in male or female germline. Initial piRNA studies identified a short 8 nt motif (CTGTTTCA) located 46 nt upstream of piRNA loci.[1,2] Strikingly, 80% of male piRNA upstream regions contain the 5 nt core motif GTTTC, compared to less than half for female piRNAs. Additionally, position 1 of the short motif is enriched for C upstream of male piRNAs, which is associated with more robust male piRNA expression; no such pattern is observed for female piRNAs. By generating transgenic C. elegans animals expressing a synthetic piRNA sequence, we show that placing a C-containing short motif upstream of a female piRNA alters its expression pattern to look more like a male piRNA. Furthermore, placing a non-C-containing motif upstream of a male piRNA alters its expression pattern to look more like a female piRNA. These data suggest that the upstream motif orchestrates germline expression patterns of piRNAs in C. elegans.