The discovery of sperm-borne RNAs (mRNAs and small non-coding RNAs) has opened the possibility of additional paternal contributions aside from providing the DNA [1]. It has been reported that the incoming sperm can provide information for its host egg cytoplasm, which functionally influences the order of cell division [2], possibly via delivering RNAs. Indeed, the sperm-borne miRNA and mRNA have been demonstrated as active players in early embryo development [3] and transgenerational epigenetic inheritance [4]. However, given the diversity of small RNA classes (miRNA, endosiRNA, piRNA, etc.) generated during spermatogenesis, the contents and profiles of the small RNA population carried by mature sperm remain undefined. In the present study, we isolated mature sperm from the cauda epididymis of adult male mice (Supplementary information, Data S1). The purity of sperm was> 99% as evaluated by microscopy and was confirmed by RT-PCR analyses of different biomarkers (Supplementary information, Figure S1A and S1B). The RNA extracted from mature sperm, adult testis, and uterus were processed for small RNA (< 40 nt) deep sequencing (Supplementary information, Figure S1C, S1D, S1E and Data S1). The total small RNA reads and genome-mapping statistic data (Supplementary information, Table S1) showed an abundance of small RNAs carried by mature sperm. The overall length distribution of small RNAs (Figure 1A) revealed that the dominant reads from mature sperm were at 29-34 nt, slightly different from adult testis (26-32 nt), and distinct from uterus (21-23 nt). The majority of the 26-32 nt small RNAs in mouse testis are piwi-interacting RNAs (piRNAs), which are actively involved in retrotransposon silencing that protects the integrity of the genome [5]. As it was initially suggested that piRNAs are absent in the cauda epididymis [6] and that mammalian PIWI proteins (MILI, MIWI, MIWI2) are not expressed in mature sperm [5], the abundant existence of

29-34 nt small RNAs in the mature sperm is somewhat surprising to us and suggests that they might be different from the well-known piRNA population from testis. Further analysis has revealed a distinct signature for these mature-sperm-enriched small RNAs, which represent a novel class of abundantly expressed small RNAs that can be grouped into distinct families. The small RNAs within each family showed identical 5′ sequences and only differed at their distal 3′ ends (Figure 1B and Supplementary information, Figure S2), suggesting that they are derived from the same precursor sequence. Particularly, two of these small RNA families were extremely enriched, which comprised 38.19%(family-1) and 19.14%(family-2) of all small RNA reads, respectively (Figure 1B), and together they accounted for the majority of the 30-34 nt small RNA population (Figure 1C). To further characterize these mature-sperm-enriched small RNAs, we performed BLAT searches for the top two abundant families against the mouse genomic databases (mm9). As shown in Figure 1D, these small RNAs are located at multiple sites on the genome, with several clusters on chromosomes 1, 8 and 13. Most strikingly, each of these genomic locations corresponds to a tRNA locus (Figure 1D). By further comparing with the genomic tRNA database, we found that each of these small RNA families unanimously matches to the 5′ half of a specific tRNA, with cleavage sites located preferentially at the anticodon loop (30-34 nt from the 5′ end), as illustrated for families 1-2 (Figure 1E and 1F) and for families 3-7 (Supplementary information, Figure S2). Their ultra-high enrichment (Figure 1G) and the preferential …