Oey et al. BMC Genomics 2011, 12:315 http://www.biomedcentral.com/1471-2164/12/315 Discussion In this report we characterise novel members of a recently discovered class of 19nt long RNAs that origi- nate from piRNA-directed RNA cleavage. In a previous paper Berninger et al. [12] elegantly show that these small RNAs can be distinguished by their spatial rela- tionship to p-piRNAs and s-piRNAs. As expected, we find that this class of RNA is readily distinguished from other RNAs by the composition of the downstream flanking sequence. By making use of sequence composi- tion rather than overlap with piRNAs a greater number of pr19RNAs can be identified. Their precise length sug- gests they are formed from two separate enzymatic clea- vage events with the 3’ end likely to be linked to the formation of s-piRNA 5’ ends and the 5’ end likely to be representing a novel event. The biogenesis of s-piRNAs has been hypothesised to require only a single enzymatic cleavage event at the 5’ end, with the 3’ end defined by a less specific mechanism resulting in their variable lengths [9]. Curiously, the IAP and LINE elements, as well as the reverse strand of many piRNA clusters, were found to have a greater number of associated pr19RNAs than s- piRNAs. This difference is puzzling as their biogenesis appears linked and the latter is expected to accumulate more readily due to their association with Miwi and Mili. It may suggest that subsequent steps in the forma- tion of these RNAs are not synchronised. Alternatively, pr19RNAs may themselves be protected from degrada- tion via a mechanism independent of Miwi or Mili as neither appear to associate strongly with pr19RNAs. The abundance of pr19RNAs mappable to the LINE consensus sequence is particularly interesting as they also outnumber p-piRNAs. It has been shown that miR- NAs are incorporated into multiple-turnover enzymes such that a single microRNA can guide several consecu- tive cleavage events [30]. If p-piRNAs function in a simi- lar manner, then the number of pr19RNAs would depend on the abundance of substrate (e.g. LINE tran- scripts) rather than the abundance of p-piRNAs. By locating regions enriched for 19mers with an A 10nt downstream we find several genes with a reciprocal pattern in the distribution of 19mers and p-piRNAs. The transcripts from some of these genes appear to be the the target of piRNA-guided cleavage resulting in release of a 19mer. A similar mechanism where p-piR- NAs target the transcript of a protein-coding gene was recently proposed for piRNAs in Drosophila [31]. This process appears analogous to the endogenous short interfering RNAs that operate in some mammalian tis- sues [28,32,33]. However, most pr19RNAs co-localising with genes were found to occur in the opposite sense. These 19mers likely arise when genic piRNAs, which Page 9 of 10 are abundant in testis [7], direct the cleavage of overlap- ping antisense transcripts. The purpose of such events is not immediately clear. Conclusion Based on the sequence composition of the pr19RNA flanking regions and the positional relationship of pr19RNAs to p-piRNAs and s-piRNAs, we conclude that pr19RNAs are produced by enzymatic cleavage events guided mainly by p-piRNAs in adult mouse sper- matogenic tubules. This was also the conclusion reached by Berninger et al. [12] in their analyses of overlapping piRNAs and pr19RNAs. Like many newly described classes of small RNAs, a function for pr19RNAs has not yet been ascertained. However, the 5’ cleavage site, pre- cise length and abundance of these RNAs suggest the underlying mechanism serves an important role in piRNA-directed post-transcriptional gene regulation. The characterisation of this novel class of small RNA offers new insight into the biogenesis and function of piRNAs in the germline and is likely to facilitate new methods for elucidating the targets of these enigmatic small RNAs. Additional material Additional file 1: Supplementary Figures. Additional file 2: Proportion of non-piRNA reads in IP libraries. Acknowledgements We wish to thank Martin Asser Hansen for helpful bioinformatics advice and comments on the manuscript, and Lutz Krause and Lucia Clemens-Daxinger for providing helpful comments. This work was supported by NHMRC Australia. Authors’ contributions HMO carried out bioinformatics analyses and NAY carried out the experimental work. HMO and EW designed the study. HMO, NAY and EW wrote the manuscript. All authors read and approved the final manuscript. Received: 25 February 2011 Accepted: 15 June 2011 Published: 15 June 2011 References 1. 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