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 MIWI-associated 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.

Figure 1

Postnatal testicular piRNAs are largely derived from non-transposon, intergenic regions. (A) MIWI and MILI associate with piRNAs in the testis. piRNAs isolated from the immunoprecipitates of MILI and MIWI were 5′ end-labeled and fractionated by 15% Urea-PAGE. Notice the size difference between MILI- and MIWI-associated piRNAs. M_D: 10 nt DNA marker, which migrates 10% faster than the corresponding size of RNA. Adult testicular MILI immunoprecipitate (IP) data was used in a publication before. (B) A diagram showing the expression of three mouse PIWI proteins during mouse testicular development. The bolts indicate the arrest points for the mutants. (C) Pie charts showing the number of the genomic hits of sequenced piRNAs from the MILI and MIWI complexes. MILI-associated piRNAs predominantly match to single (unique-mapping) sites on the genome throughout spermatogenesis as is the case for adult testicular MIWI-associated piRNAs. Repeat-associated piRNAs constitute a bigger fraction until 13 dpp relative to the adult testis. (D) Pie charts showing the genomic annotations of sequenced piRNAs from the MILI and MIWI complexes. MILI-associated piRNAs are largely derived from intergenic regions and poorly from transposon regions throughout spermatogenesis, as adult testicular MIWI-associated piRNAs. piRNAs from cellular RNAs and coding genes form a bigger fraction until 13 dpp relative to the adult stage. 13 dpp pachytene stage testis is rich in piRNAs that correspond to transposons relative to the other stages examined.

Figure 2

MILI- and MIWI-associated piRNAs do not contain “ping-pong signature” or complementary sequences required for the ping-pong mechanism. (A) Base composition of each piRNA population charted with the X-axis representing the nucleotide position relative to the 5′ (left panels) or 3′ (right panels) ends of the piRNAs (e.g., −1 at the 5′ end is the position 1-nt upstream of the piRNAs). The Y-axis represents the entropy score for the base bias. All populations of MILI-associated piRNAs are strongly biased for U on the 5′ end (position 1), and for G on the 2nd nucleotide position. In addition, they are biased for C on the 3′ end. MIWI-associated piRNAs show a bias for 5′ U only. None of the piRNA populations shows any A-bias at the 10th nucleotide position. (B) Pairwise comparison of the piRNA libraries shows that testicular piRNAs are largely devoid of matching piRNAs that are 10-nt complementary along their 5′ ends (left panel). The same has been observed for the transposon-derived sub-population of piRNAs (center panel), and for those derived from the coding genes (right panel). The values are the percentage of a given pair of piRNAs with complementary sequences, and belong to the libraries positioned on the rows. As a negative control, piRNA sequences from each library were randomized to generate a corresponding simulated library. Notice the similar percentage of complementary sequences in the libraries relative to their corresponding simulation.

Figure 3

MILI-associated piRNAs are not downregulated in the absence of MIWI. (A) MILI and MIWI interact with independent populations of piRNAs. MILI and subsequently MIWI complexes were immunoprecipitated from the same adult testicular extract. Associated piRNAs were isolated and analyzed as in Figure 1. (B) Northern blotting for representative piRNAs. 20 μg of total testicular RNA samples from 24 dpp Miwi^+/− and Miwi^−/− were resolved with 15% Urea-PAGE and northern blots were probed for individual piRNAs. U6snRNA (U6) was used as an internal loading control. Ethidium bromide staining was also used to assess the global level of piRNAs. Five piRNA probes are used: Tp2, transposon 2; A-exo, anti-sense exonic; Rapi1, repeat-associated 1; S-Intro, sense intronic; T4, piRNA-T4. The annotations indicate the genomic regions from which piRNAs are derived (e.g., “anti-sense exonic” is a piRNA that corresponds to the anti-sense strand of an exon). Each probe hybridizes to both MIWI-associated (long arrows) and MILI-associated (short arrow) piRNAs. MILI-associated piRNAs are not downregulated in the absence of MIWI. (C) Immunoprecipitation of MILI from Miwi^−/− testes, which was loaded with ∼1/3 of total RNAs as compared to that of Miwi^+/− testes. Even though the non-piRNA controls (due to non-sepcific binding, empty arrow) are expectedly ∼1/3 lower in Miwi^+/− testes, the total MILI-associated piRNAs in Miwi^−/− testes (solid arrow) are equal to slightly more than that in Miwi^+/− testes, indicating that the total MILI-associated piRNAs in increased by at least three times in Miwi^−/− testes. IP, immunoprecipitates.

Figure 4

MILI- and MIWI-associated piRNAs in the adult testis are mostly processed from the same single-stranded precursor transcripts by the primary pathway instead of the ping-pong mechanism. (A) Mapping of the MILI- and MIWI-associated piRNAs reveals that they are both derived from the same sets of piRNA cluster loci. Cluster locations are indicated with triangles, whose height is proportional to the number of piRNAs in the cluster. The triangles on the left side of a chromosome denote piRNAs derived from the plus strand, while those on the right are from the minus strand. Blue and red triangles indicate MILI- and MIWI-associated piRNA clusters, respectively. Only clusters with more than 300 piRNAs are displayed. (B) piRNAs in clusters are derived from one or the other genomic strand. While piRNAs in a few clusters are derived from the same genomic strand along the entire cluster (bottom panel), in most clusters, they abruptly switch to the opposing strand at the center of the cluster resulting in a bidirectional profile (upper panel). Only MILI-associated piRNAs in the adult testis are shown. Each vertical bar represents one piRNA with the height of the bar reflecting the size of the corresponding piRNA. piRNAs from the plus strand are colored green, those from the minus strand are yellow. Arrows indicate the transcription direction of the piRNAs. (C) Higher resolution of the bidirectional cluster displayed in B shows that MILI- and MIWI-associated piRNAs are derived from the same genomic strands and switch origin of synthesis to the other strand at a same piRNA-free region. Only piRNAs cloned from the adult testicular immunoprecipitates are shown. EST and mRNA information from GenBank is displayed at the bottom. (D) Alignment of clustered piRNAs shows that they have overlapping sequences. Shown is part of the genomic sequence (top line) of the Chromosome 17 cluster and the piRNAs that correspond to this sequence.

Figure 5

Transposon-derived piRNAs are processed as part of the precursors. The density distribution of piRNAs at the largest piRNA cluster on Chromosome 17 shows no correlation with the location of transposons.