Defining piRNA primary transcripts.

PIWI proteins and their associated small RNAs (PIWI-interacting RNAs, piRNAs) are essential for fertility in mammals. piRNAs (24–35 nt) are longer than miRNAs (21–23 nt) and have 2'-O-methyl-modified 3' termini. Like miRNAs, piRNAs bind members of the Argonaute family of proteins, but piRNAs are unique in that they guide PIWI proteins, a specialized subfamily of Argonaute proteins that are expressed mainly in germ cells. The sequences of piRNAs are more diverse than any other known class of cellular RNAs. For example, our 8.8 million piRNA reads in a deep sequencing library from adult mouse testis comprised 2.7 million different piRNAs; > 90% of piRNA species were sequenced just once. piRNAs map to large blocks of genomic sequence called clusters. The architecture of piRNA clusters suggests that mature piRNAs derive from precursor transcripts via multiple RNA processing steps. While > 80% of piRNA reads in flies map to transposons, ~93% of adult mouse piRNA reads map to a single site in the genome. That so many mouse piRNAs map uniquely to the genome facilitates the study of their precursor transcripts. Using total RNA sequencing, we detected long RNAs (> 100 nt) from piRNA clusters. Chromatin immunoprecipitation of RNA polymerase II and III suggests that these transcripts are transcribed by RNA polymerase II. Consistent with mouse piRNA precursors being RNA polymerase II transcripts, the long RNAs bear a standard cap structure (detected by cap analysis of gene expression; CAGE), and their 3' ends possess a poly(A) tail Defining piRNA primary transcripts