Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis

Abstract

Germ cells implement elaborate mechanisms to protect their genetic material and to regulate gene expression during differentiation. Piwi proteins bind piRNAs, a class of small germline RNAs whose biogenesis and functions are still largely elusive. We employed high throughput sequencing after crosslinking and immunoprecipitation (HITS-CLIP) coupled with RNA-Seq to characterize the genome-wide target RNA repertoire of Mili (Piwil2) and Miwi (Piwil1), two Piwi proteins expressed in mouse postnatal testis. We report the in vivo pathway of primary piRNA biogenesis and implicate distinct nucleolytic activities that process Piwi-bound precursor transcripts. Our studies indicate that pachytene piRNAs are the end products of RNA processing. HITS-CLIP demonstrates that Miwi binds spermiogenic mRNAs directly, without utilizing piRNAs as guides, and independent biochemical analyses of testis mRNA-ribonucleoproteins (mRNPs) establishes that Miwi functions in the formation of mRNP complexes that stabilize mRNAs essential for spermiogenesis.