Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes1,2,3,4. A subgroup of the Argonaute proteins—known as the ‘Piwi family’5—is required for germ- and stem-cell development in invertebrates6,7, and two Piwi members—MILI and MIWI—are essential for spermatogenesis in mouse8,9. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5′ uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26–31 nucleotides (nt) in length—clearly distinct from the 21–23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)—and we refer to them as ‘Piwi-interacting RNAs’ or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.
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We thank J. Brennecke, M. Landthaler, Y. Pei, K. Fejes Toth and A. Sewer for discussion and help with the manuscript. We also thank L. Mitchell and K. Hwang for expert assistance in germ-cell purification, M. Poy and M. Stoffel for providing mouse tissues, and D. Weir and R. Choksi for assistance with sequencing. This work was supported by a FRAXA Research Foundation postdoctoral fellowship to A.A., an NIH grant to P.M., NIH grants to T.T., and an SNF grant to M.Z. Author Contributions A.A., S.P. and M.L.-Q. prepared the mouse, and P.L. and N.I. the human, testes small RNA libraries. A.A. recognized the presence of piRNAs, performed the MILI IPs, prepared the MILI-interacting small RNA library, and performed, together with N.I., the northern blotting analysis. S.K.-M. and T.N. produced, characterized and purified the MILI antibody. T.T. developed the concept of cloning from Ago/Piwi IPs. P.M. isolated germline cells. M.C., J.J.R. and J.J. performed the large-scale sequencing. The bioinformatic analyses of piRNAs were designed and carried out by D.G. and M.Z. with input from A.A. and T.T. The database of RNAs with known function was compiled by M.Z., R.S., P.L. and S.P., the software used for small RNA annotation was developed by M.Z., R.S. and C.S., and the manuscript was written by A.A., M.Z. and T.T.
This file contains Supplementary Methods, Supplementary Tables 1–10 (excluding 3, 4, 6–9) and their legends, and Supplementary Figure Legends. (DOC 168 kb)
Summary of the mouse piRNA clusters. (XLS 28 kb)
List of mouse piRNA clones. (XLS 1039 kb)
Mouse piRNA minicluster alignments. (DOC 270 kb)
Human piRNA minicluster alignments. (DOC 30 kb)
Summary of the human piRNA clusters. (XLS 21 kb)
Supplementary Table 9 nature04916-s7.xls List of human piRNA clones. (XLS 194 kb)
This file contains Supplementary Figures 1–6. (PDF 5095 kb)
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Aravin, A., Gaidatzis, D., Pfeffer, S. et al. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, 203–207 (2006). https://doi.org/10.1038/nature04916
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Molecular Biology Reports (2022)
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