piRNA biogenesis during adult spermatogenesis in mice is independent of the ping-pong mechanism

Abstract

piRNAs, a class of small non-coding RNAs associated with PIWI proteins, have broad functions in germline development, transposon silencing, and epigenetic regulation. In diverse organisms, a subset of piRNAs derived from repeat sequences are produced via the interplay between two PIWI proteins. This mechanism, termed "ping-pong" cycle, operates among the PIWI proteins of the primordial mouse testis; however, its involvement in postnatal testes remains elusive. Here we show that adult testicular piRNAs are produced independent of the ping-pong mechanism. We identified and characterized large populations of piRNAs in the adult and postnatal developing testes associated with MILI and MIWI, the only PIWI proteins detectable in these testes. No interaction between MILI and MIWI or sequence feature for the ping-pong mechanism among their piRNAs was detected in the adult testis. The majority of MILI-and MIWI-associated piRNAs originate from the same DNA strands within the same loci. Both populations of piRNAs are biased for 5′ Uracil but not for Adenine on the 10th nucleotide position, and display no complementarity. Furthermore, in Miwi mutants, MILI-associated piRNAs are not downregulated, but instead upregulated. These results indicate that the adult testicular piRNAs are predominantly, if not exclusively, produced by a primary processing mechanism instead of the ping-pong mechanism. In this primary pathway, biogenesis of MILI-and MIWIassociated piRNAs may compete for the same precursors; the types of piRNAs produced tend to be non-selectively dictated by the available precursors in the cell; and precursors with introns tend to be spliced before processed into piRNAs.

A Novel Class of Somatic Small RNAs Similar to Germ Cell Pachytene PIWI-interacting Small RNAs

“…This notion is supported by the following findings: 1) the major size of pilRNAs is 30 nt, which is closer to that of pachytene piRNAs; 2) pilRNAs largely fall within known pachytene piRNA instead of prepachytene piRNA clusters; and 3) similar to pachytene piRNAs, pilRNAs are mainly derived from intergenic regions (6). In contrast to pachytene piRNAs, which only have ϳ2% ping-pong activity, pilRNAs have a definite ping-pong signature with ϳ30% of stomach pilRNAs having a 10-nt overlap at their 5Ј ends (12). Therefore, we propose that pilRNAs are similar to germ cell pachytene piRNAs but are different in the fact that they have ping-pong activity like prepachytene piRNAs.…”

“…However, lone piRNAs derived from unique, unclustered genomic locations have been found in large quantities as well (12,14). To properly classify the pilRNAs identified, we collected genomic coordinates containing murine prepachytene and pachytene piRNA clusters, and pilRNAs were aligned and categorized within these clusters (9,12).…”

“…On the other hand, these transposonderived piRNAs induce transposon silencing through piRNAdependent DNA methylation (9). In contrast, pachytene piRNAs associate with MIWI and MILI, creating two subcategories of piRNAs: MILI-associated and MIWI-associated piRNAs (12). These piRNAs do not show ping-pong characteristics such as secondary transcripts that are partially complementary to primary transcripts.…”

“…These piRNAs do not show ping-pong characteristics such as secondary transcripts that are partially complementary to primary transcripts. Instead, pachytene piRNAs represent largely, if not exclusively, primary transcripts (12).…”

Conditional inactivation of Miwi2 reveals that MIWI2 is only essential for prospermatogonial development in mice

“…[4][5][6][7] piRNAs represent a distinct small noncoding RNA (sncRNA) species with an average length of 25-31nt, and piRNA biogenesis is independent of the RNase III enzymes DROSHA and DICER, which are essential for the production of miRNAs and siRNAs. [8][9][10] piRNAs can generally be divided into two groups based on their genomic origin: repeat-associated and non-repeat-associated. 4,7,11 Repeatassociated piRNAs are mostly derived from retrotransposon loci and appear to be synthesized through two successive steps: primary piRNA processing and secondary piRNA cleavage achieved by the so-called 'ping-pong' amplification cycle.…”