PIWI proteins belong to an evolutionary conserved protein family as the sister sub-family of ARGONAUTE (AGO) proteins. While AGO proteins are functionally well-characterized and shown to mediate small RNA-guided gene regulation, the function of PIWI proteins remain elusive. Here we pursued functional characterization of PIWI proteins by studying MILI and MIWI, two PIWI proteins in the mouse.

We first show that both MIWI and MILI co-immunoprecipitate with a novel class of non-coding small RNAs from the post-natal mouse testis extract, which are named Piwi-interacting RNAs (piRNAs). Our cloning efforts identified thousands of different piRNA sequences, mostly derived from intergenic regions. Interestingly, both MILI and MIWI piRNAs correspond to the same regions on the genome and differ primarily in length. We propose piRNAs in the adult testis are produced by the processing of long, single stranded RNA precursors, based on the observation that they originate in clusters from a number of sites on the genome in a head-to-tail homology. In support, we bioinformatically predicted their putative promoters, and yeast-one-hybrid analysis on two such regions found out that they interact with Krueppel C2H2 type zinc finger transcription factors. We did not observe the features of the "ping-pong" mechanism in their biogenesis: Both MILI and MIWI piRNAs are biased for 5' Uracil without an Adenine bias on the 10^th nucleotide position, and do not significantly consist of sequences complementary to each other along their first 10nt. Moreover, MILI piRNAs are not down-regulated in Miwi-/- testis. These results indicate that the adult testicular piRNAs are produced independent of the "ping-pong" mechanism.

Although piRNAs are highly complex, PAGE and in situ analyses showed that they are germ cell-specific with a predominant expression in spermatocytes and round spermatids, suggestive of a meiotic function. Correspondingly, we found that Miwi-/-; Mili-/- mice undergo only male infertility with terminal spermatogenic arrest during meiosis. piRNAs show a nucleo-cytoplasmic distribution, with enrichment in the "chromatoid" and "dense" bodies, two male germ cell-specific structures. The "dense body" has been implicated in synapsis and in the heterochromatinization of the sex chromosomes during male meiosis, a process known as meiotic sex chromosome inactivation (MSCI). Our histological analysis on Miwi-/-; Mili-/- testes showed that, while the overall synapsis is not affected, the sex chromosomes retain the euchromatin marker acetyl-H4K16 and lacks the heterochromatin marker H3K9-dimethyl. These observations indicate that murine PIWI proteins are necessary for MSCI. Moreover, we identified piRNA production from the X chromosome before MSCI, and propose PIWI proteins utilize piRNAs to target and silence unpaired chromosomal regions during meiosis.