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. 2012 Aug;19(8):773-81.
doi: 10.1038/nsmb.2347. Epub 2012 Jul 29.

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

Anastassios Vourekas  1 , Qi Zheng, Panagiotis Alexiou, Manolis Maragkakis, Yohei Kirino, Brian D Gregory, Zissimos Mourelatos
Affiliations

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

Anastassios Vourekas et al. Nat Struct Mol Biol. 2012 Aug.

Abstract

Germ cells implement elaborate mechanisms to protect their genetic material and to regulate gene expression during differentiation. Piwi proteins bind Piwi-interacting RNAs (piRNAs), small germline RNAs whose biogenesis and functions are still largely elusive. We used high-throughput sequencing after cross-linking and immunoprecipitation (HITS-CLIP) coupled with RNA-sequencing (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 demonstrated that Miwi binds spermiogenic mRNAs directly, without using piRNAs as guides, and independent biochemical analyses of testis mRNA ribonucleoproteins (mRNPs) established that Miwi functions in the formation of mRNP complexes that stabilize mRNAs essential for spermiogenesis.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Mili and Miwi HITS-CLIP
(a, b) CLIPs were performed using highly stringent conditions with buffers containing 2% Empigen, a potent zwitterionic detergent. Autoradiograms and Western blots of immunoprecipitated, UV-crosslinked, RNA-protein complexes ligated to radiolabeled 3′ adapter are shown for Mili (a) and Miwi (b). Negative control CLIPs were performed using non-immune mouse (NMS) or rabbit serum (NRS) with crosslinked testis, and anti-Mili or anti-Miwi antibodies with crosslinked kidney. Cells were lysed after crosslinking and the lysates were incubated in the absence or presence of RNase T1 prior to immunoprecipitation, as indicated. RNA (CLIP tags) was extracted from the membranes after cutting at indicated areas: a blue line marks the major radioactive signal containing mainly piRNA-protein complexes; a red line marks larger RNA-protein complexes which appear as a smear extending to higher molecular weights. (c, d) cDNA libraries from Mili (c) and Miwi (d) CLIP tags were prepared by RT-PCR, gel purified and sequenced with Illumina. Blue and red brackets denote piRNA and large tag enriched cDNA samples, respectively.
Figure 2
Figure 2. Size distribution, nucleotide preference and genomic classification of mapped CLIP reads
(a, b) Size distribution of mapped HITS-CLIP reads for Mili (a) and Miwi (b) (c, d) Nucleotide preference of the first ten positions for piRNAs (c) and lgClips (d). (e, f) Genomic classification of piRNAs (e) and lgClips (f). RMSK: Repeat Masker, TR: terminal repeats, TE: transposable elements.
Figure 3
Figure 3. Processing of intergenic piRNA precursors
(a) Genome browser snapshot showing mapped CLIP-tags on an intergenic piRNA hotspot (IPH). Magenta = forward, blue = reverse tags. Numbers on the side of tracks denote number of stacked tags. Genomic coordinates (mm9) are marked below the tracks. (b) Zoom-in view of (a), revealing piRNA processing. Note the concurrence of 5′ ends between piRNAs and lgClips (intermediate piRNA precursors), and progressive shortening of lgClip 3′ ends. (c) Density plot of piRNA-lgClip distances (piLg-dist) for CLIP tags that map within IPHs. The distance is defined as the genomic position of lgClip end (5′ or 3′), minus the genomic position of the respective piRNA end (5′ or 3′) for the forward strand, and vice versa for the reverse strand. On the Y-axis, the percent of piLg-dist over distances for all pairwise combinations between lgClips and piRNAs is plotted. Only piRNAs that lie within a distance of 30 nucleotides upstream or downstream of any given lgClip were considered. Distance measuring requires unique genomic coordinates; therefore only uniquely mapped tags were used in this analysis. (d) Nucleotide preference at the 5′ end position of piRNAs and lgClips originating from IPHs, and total lgClips, for the indicated proteins. IPH lgClips are enriched for 5′ U in comparison with total lgClips.
Figure 4
Figure 4. Non-repeat piRNAs lack complementary RNA targets
(a) BLASTN was used to examine complementarity potential for all piRNAs versus all lgClips for each protein. Complementary hits between piRNAs and lgClips were reported as identity ≥85% and a match-ratio of ≥60% for the piRNA sequence. (b) Left panel: Percent of piRNAs complementary to one or more lgClips. Less than 10% of unique RefSeq exon or intergenic piRNAs match to a complementary lgClip. Right panel: Percent of lgClips with one or more complementary piRNAs. Less than 10% of unique RefSeq exon or intergenic lgClips can be targeted by a complementary piRNA. (c) The mean percentage of pairing events per nucleotide position, divided by the number of events for each piRNA is plotted, for Mili (left panel), and Miwi (right panel). All piRNA classes show a random pattern of complementarity, with no preference for pairing the 5′ or 3′ ends, almost identical to the control (shuffled piRNA sequences, yellow line). (d) Conservation of piRNA complementary sites within RefSeq exon derived lgClips (black line). As a control, the complementary sites of shuffled piRNA sequences are analyzed (red line); no differences are observed.
Figure 5
Figure 5. Absence of piRNA processing in Miwi-bound mRNAs essential for spermiogenesis
(a) Density plots of piRNAs (dashed lines) and lgClip (solid lines) relative positions on RefSeq mRNAs, for Mili (red) and Miwi (blue). In contrast to Miwi, Mili piRNA and lgClip densities are highly correlated. (b) MA plot of generalized-log-odds (glog-odds) for lgClips over piRNA abundance (M value), versus total CLIP-tag glog abundance (lgClip + piRNA) (A value) for all RefSeq exons. mRNAs that have more lgClips than piRNAs tend to appear on the top of the graph. RefSeq mRNAs that had a Miwi lgClip/piRNA ratio more than 8-fold (glog-odds ≥ 3) across all three Miwi replicates (p<0.05, t-test) were marked as covered (outlined circles). As a result, we identified 575 Miwi-covered mRNAs representing 460 unique RefSeq genes that have significantly more Miwi lgClips than piRNAs (see Supplementary Table 5). (c) Heatmap of log2 abundances of Miwi-covered mRNAs in five time points in testis development. Most of Miwi-covered mRNAs are highly enriched or are only expressed in the post meiotic stages of spermatogenesis (21 dpp and after).
Figure 6
Figure 6. Miwi, devoid of piRNAs, binds and stabilizes spermiogenic mRNAs in repressed mRNPs
(a) Isopyknic density gradients of post-nuclear testis lysate. (b) Total RNA content. (c) Heat map of mRNA levels determined by qRT-PCR, normalized to spiked luciferase RNA. Repressed spermiogenic mRNAs are predominantly in fractions 4 and 5 (bracketed). (d) Western blots. (e) Small RNA content analyzed by 5′-end labeling and UREA-PAGE. piRNAs are in fractions 6 and 7. (f) Silver stain of Oligo-dT pull downs and Western for Miwi. Proteins were identified by mass spectrometry from excised bands and from entire pulldown eluates (see Supplementary Table 7). (g) Miwi protein (top panel), piRNAs (middle) and mRNAs (bottom) from Miwi immunoprecipitates. Spermiogenic mRNAs and piRNAs are bound to Miwi in a non-overlapping manner. (h) Western blots from adult (wild type, WT) and 28 dpp (WT, Miwi HET and KO) testis lysates. Tnp2 (expressed in elongating spermatids ,), is not detected in 28 dpp testis, verifying the absence of elongating spermatids at that age. (i) qRT-PCR from 28dpp Miwi HET and KO. Gapdh was used as endogenous control (average Ct, HET=14.94; KO=14.65), and the Miwi HET 28dpp sample as reference. Upper panel: distribution of six mRNAs from adult and 28dpp WT mice across the polysomal (fractions 1 and 2) and repressed mRNPs (fractions 4 and 5; see Supplementary Fig. 7). Bottom panel: Relative levels of the same mRNAs from total RNA isolated from 28 dpp Miwi HET and KO testes. Spermiogenic mRNAs that are highly enriched in fractions 4 and 5 (Odf1, Smcp, Prm1, Tnp2), show a dramatic decrease in the absence of Miwi. Ldhc, which is equally distributed between polysomes and repressed mRNPs at 28dpp, shows a smaller reduction in Miwi KO.
Figure 7
Figure 7. Model for primary piRNA biogenesis, and functions of Mili and Miwi piRNPs during mouse male germ cell differentiation
Two distinct biological functions of Piwi proteins are outlined in this model: piRNA biogenesis and stabilization of spermiogenic mRNAs by Miwi. piRNA processing of Piwi bound transcripts occurs in distinct steps (circled numbers). Two nucleolytic activities, an endonuclease (scissors) that generates the 5′ ends, and a 3′-5′ exonuclease (pacman) that generates the 3′-ends are implicated; their identities are unknown. Mili and Miwi bind to long intergenic transcripts that appear during meiosis, before and/or after endonucleolytic cuts of the long precursors generate intermediate fragments. The function of these intergenic transcripts in meiosis is unknown but may include roles in meiotic chromatin remodeling. 3′-ends of piRNAs are methylated by Hen1 and protected from further trimming. The piRNA-generating nucleases, or associated cofactors, are not expressed, cease to be active or are sequestered in haploid spermatids. Pachytene Piwi/piRNA complexes are thus end products of RNA clearance but they may also have as yet undetermined non-RNA targeting functions (i.e. chromatoid body organization). Another pool of Miwi binds spermiogenic mRNAs throughout their lengths. This binding is an integral part of the cooperative formation of the mRNPs that maintain these messages prior to spermatid elongation. Red dot indicates the chromatoid body in spermatids that is enriched in Miwi/piRNAs, MVH and Tdrds and which is ultimately eliminated from the maturing spermatid as part of the residual body. NPC: Nuclear Pore Complex.
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References

    1. Cox DN, et al. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev. 1998;12:3715–27. - PMC - PubMed
    1. Deng W, Lin H. miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev Cell. 2002;2:819–30. - PubMed
    1. Kuramochi-Miyagawa S, et al. Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development. 2004;131:839–49. - PubMed
    1. Boswell RE, Mahowald AP. tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster. Cell. 1985;43:97–104. - PubMed
    1. Chen C, Nott TJ, Jin J, Pawson T. Deciphering arginine methylation: Tudor tells the tale. Nat Rev Mol Cell Biol. 2011 - PubMed

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