6596–6607 Nucleic Acids Research, 2011, Vol. 39, No. 15 doi:10.1093/nar/gkr298 Published online 5 May 2011 Widespread expression of piRNA-like molecules in somatic tissues Zheng Yan 1 , Hai Yang Hu 1 , Xi Jiang 1 , Vera Maierhofer1, Elena Neb 1 , Liu He1,2, Yuhui Hu 3 , Hao Hu 4 , Na Li3, Wei Chen3,4,* and Philipp Khaitovich1,5,* 1Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Chinese Academy of Sciences, 320 Yueyang Road, 200031, Shanghai, 2Graduate School of Chinese Academy of Sciences, 19 Yuquan Road, 100039, Beijing, China, 3Max-Delbru¨ ck-Centrum fu¨ r Molekulare Medizin, Robert-Ro¨ ssle-str 10, D-13092, 4Max Planck Institute for Molecular Genetics, Ihnestrasse 63-73, D-14195 Berlin and 5Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany Received January 5, 2011; Revised and Accepted April 14, 2011 ABSTRACT Piwi-interacting RNA (piRNA) are small RNA abundant in the germline across animal species. In fruit flies and mice, piRNA have been implicated in maintenance of genomic integrity by transposable elements silencing. Outside of the germline, piRNA have only been found in fruit fly ovarian follicle cells. Previous studies have further reported presence of multiple piRNA-like small RNA (pilRNA) in fly heads and a small number of pilRNA have been reported in mouse tissues and in human NK cells. Here, we analyze high-throughput small RNA sequencing data in more than 130 fruit fly, mouse and rhesus macaque samples. The results show widespread presence of pilRNA, displaying all known character- istics of piRNA in multiple somatic tissues of these three species. In mouse pancreas and macaque epididymis, pilRNA abundance was compatible with piRNA abundance in the germline. Using in situ hybridizations, we further demonstrate pilRNA co-localization with mRNA expression of Piwi-family genes in all macaque tissues. Further, using western blot, we have shown the expression of Miwi protein in mouse pancreas. These findings indicate that piRNA-like molecules might play important roles outside of the germline. INTRODUCTION Typical piRNA are 24–32 nt in length (1,2–9). Primary piRNA have been suggested to originate via fragmenta- tion of longer transcripts and, therefore, can be traced to discrete genomic locations, or piRNA clusters, which show strong strand speciﬁcity (1,7,10,11). Another known property of primary piRNA is marked 50 uridine bias (1,7,10,11). Secondary piRNA are generated through an ampliﬁcation mechanism, known as the ping-pong mech- anism, which requires base pairing between the ﬁrst 10 nt of the primary piRNA and target transcripts (1,7,8,10). Thus, presence of the ping-pong mechanism can be deduced through an excess of small RNA with 10 nt long complementary regions (1,7,8–10). In Drosophila, piRNA have been described to be present in embryos and the germline (1,8,12), as well as in a speciﬁc type of somatic tissue: ovarian follicle cells (13,14). Fly germline piRNA include both primary and secondary piRNA, require the presence of three Piwi-family proteins; Piwi, Aub and Ago3, and are mainly derived from transposable elements (1,8). Presence of these piRNA has been shown to be essential for transposable element silencing (1). piRNA found in the ﬂy somatic ovarian follicle cells and a derived cell line, ovarian somatic sheet (OSS) cells, predominantly include primary piRNA that originate from discrete genomic loci (11,13,14–16). These piRNA are associated with one Piwi-family protein (Piwi) and are over- represented in both transposable elements and certain 30-UTR regions (11,13,14–16). Functionally, these piRNA play important roles in suppressing transposons in the ovarian somatic cells and are involved in suppres- sion of transposable element transfer into the female germline (13,14,17,18). In mammals, piRNA are particularly abundant in the male germline. Unlike Drosophila piRNA, piRNA present in adult mammalian testes tend to originate from unannotated genomic regions devoid of transposable elements and other repeats (4,5). The functional *To whom correspondence should be addressed. Tel: +49 30 9406 2995; Fax: +49 30 9406 3068; Email: wei.chen@mdc-berlin.de Correspondence may also be addressed to Philipp Khaitovich. Tel: +86 21 5492 0454; Fax: +86 21 5492 0451; Email: khaitovich@eva.mpg.de The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as First Author. The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.