Oey et al. BMC Genomics 2011, 12:315 http://www.biomedcentral.com/1471-2164/12/315 R E S E A R C H A R T I C L E Open Access The characterisation of piRNA-related 19mers in the mouse Harald M Oey, Neil A Youngson and Emma Whitelaw* Abstract Background: Piwi interacting RNA, or piRNA, is a class of small RNA almost exclusively expressed in the germline where they serve essential roles in retrotransposon silencing. There are two types, primary and secondary piRNA, and the latter is a product of enzymatic cleavage of retrotransposons’ transcripts directed by the former. Recently, a new class of 19nt long RNA was discovered that is specific to testis and appears to be linked to secondary piRNA biogenesis. Results: We locate clusters of the testis-specific 19mers, which we call piRNA-related 19mers (pr19RNA), and characterise the transcripts from which they are derived. Most pr19RNA clusters were associated with retrotransposons and unannotated antisense transcripts overlapping piRNA clusters. At these loci the abundance of 19mers was found to be greater than that of secondary piRNAs. Conclusion: We find that pr19RNAs are distinguished from other RNA populations by their length and flanking sequence, allowing their identification without requiring overlapping piRNAs. Using such sequence features allows identification of the source transcripts, and we suggest that these likely represent the substrates of primary piRNA- guided RNA cleavage events. While pr19RNAs appear not to bind directly to Miwi or Mili, their abundance relative to secondary piRNAs, in combination with their precise length, suggests they may be more than by-products of secondary piRNA biogenesis. Background Piwi-interacting small RNAs (piRNAs) are almost exclu- sively expressed in the germline where they serve essen- tial roles in retrotransposon silencing [1-5]. There are two types, the primary piRNAs (p-piRNAs) and second- ary piRNAs (s-piRNAs), and they associate with three developmentally regulated Ago-related proteins, Miwi, Mili and Miwi2, in mice [6]. The p-piRNAs are identi- fied by their length, spanning ~24-30 ribonucleotides (nt) and the presence of a 5’ U. They are mostly derived from long non-coding RNAs transcribed from distinct genomic loci known as piRNA clusters [5,7]. Some p-piRNAs target the transcripts of active retro- transposons and retroviruses, such as the long inter- spersed nuclear elements (LINEs) and the intracisternal A particles (IAPs). The retrotransposon transcripts are then cleaved enzymatically resulting in the release of s- piRNAs. The initial cleavage site is located within the * Correspondence: emma.whitelaw@qimr.edu.au Queensland Institute of Medical Research, Epigenetics, 300 Herston Road, Herston, Brisbane 4006, Australia base-paired region 10 nt downstream of the 5’ terminal U of the p-piRNA and the resulting s-piRNAs are there- fore distinguished by an A at position 10 [8-10]. Some s-piRNAs are expected to be reverse complementary to the original p-piRNA precursor transcript and may themselves be able to direct cleavage of these to recreate the original p-piRNA. The formation of s-piRNAs direc- ted by p-piRNAs and p-piRNAs directed by s-piRNAs has been been called the ping pong amplification cycle [9]. The ping-pong amplification, identified by the pre- sence of secondary piRNAs, is particularly active at the pre-pachytene stage. At the pachytene stage the popula- tion of piRNAs shifts towards p-piRNAs [10,11]. The precise nature of these events in mice is still being investigated and appears to vary according to the com- plement of Piwi-related proteins that are expressed [10]. In this report we study a class of 19 nt long piRNA- related RNAs (pr19RNA) in mice testes that appears to result from piRNA directed cleavage of transcripts. While this manuscript was in preparation these RNAs were also reported by Berninger et al. [12]. However, © 2011 Oey et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.