In vivo PIWI slicing in mouse testes deviates from rules established in vitro
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE219200 | GEO | 2023/10/23
REPOSITORIES: GEO
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Project description:PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposable elements in animal gonads. Here we demonstrate that in the mouse embryonic male germline, endonucleolytic cleavage (slicing) of a transcript by cytosolic MILI acts as a trigger to initiate its further 5??3? processing into non-overlapping fragments. These fragments accumulate as new piRNAs within the nuclear PIWI protein MIWI2. We identify Exonuclease domain-containing 1 (EXD1) as a partner of the established MIWI2 piRNA biogenesis factor TDRD12. Although EXD1 homodimers are inactive as a nuclease, it functions as an RNA adapter within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 impacts biogenesis of MIWI2 piRNAs and displays a reduction in sequences generated by MILI slicing. This results in selective depletion of repeat piRNAs that target active retrotransposons like LINE1, which are de-repressed in the mutant. We propose that PIWI slicing and EXD1 promote coordination of nucleo-cytoplasmic silencing via piRNA biogenesis.
2015-12-04 | GSE74423 | GEO
Project description:PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposable elements in animal gonads. Here we demonstrate that in the mouse embryonic male germline, endonucleolytic cleavage (slicing) of a transcript by cytosolic MILI acts as a trigger to initiate its further 5??3? processing into non-overlapping fragments. These fragments accumulate as new piRNAs within the nuclear PIWI protein MIWI2. We identify Exonuclease domain-containing 1 (EXD1) as a partner of the established MIWI2 piRNA biogenesis factor TDRD12. Although EXD1 homodimers are inactive as a nuclease, it functions as an RNA adapter within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 impacts biogenesis of MIWI2 piRNAs and displays a reduction in sequences generated by MILI slicing. This results in selective depletion of repeat piRNAs that target active retrotransposons like LINE1, which are de-repressed in the mutant. We propose that PIWI slicing and EXD1 promote coordination of nucleo-cytoplasmic silencing via piRNA biogenesis. Immunoprecipitated or total small RNAs were purified and sequenced from P0 mouse testis of Exd1+/- and Exd1 -/- mice. Testes of three males were pooled together and MILI and MIWI2 immunoprecipitation was performed or total small RNAs were purified. Two replicas from different pools were prepared. For Rosa26-pi reporter mouse P0 testes of three males were pooled together and MILI and MIWI2 immunoprecipitation was performed.
2015-12-04 | E-GEOD-74423 | biostudies-arrayexpress
Project description:PIWI-clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ~23-30nt piRNAs that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endo-nuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the inter-dependencies between these piRNA biogenesis pathways in the developing Drosophila ovary. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and that defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear, transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways.
2015-08-31 | GSE71775 | GEO
Project description:The piRNA machinery is known for its role in mediating epigenetic silencing of transposons. Recent studies suggest that this function also involves piRNA-guided cleavage of transposon-derived transcripts. As many piRNAs also appear to have the capacity to target diverse mRNAs, this raises the intriguing possibility that piRNAs may act extensively as siRNAs to degrade specific mRNAs. To directly test this hypothesis, we compared mouse PIWI (MIWI)-associated piRNAs with experimentally identified cleaved mRNA fragments from mouse testes, and observed cleavage sites that predominantly occur at position 10 from the 5' end of putative targeting piRNAs. We also noted strong biases for U and A residues at nucleotide positions 1 and 10, respectively, in both piRNAs and mRNA fragments, features that resemble the pattern of piRNA amplification by the 'ping-pong' cycle. Through mapping of MIWI-RNA interactions by CLIP-seq and gene expression profiling, we found that many potential piRNA-targeted mRNAs directly interact with MIWI and show elevated expression levels in the testes of Miwi catalytic mutant mice. Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression. Importantly, we demonstrated that proper turnover of certain key piRNA targets is essential for sperm formation. Together, these findings reveal the siRNA-like function of the piRNA machinery in mouse testes and its central requirement for male germ cell development and maturation. CLIP-Seq (Crosslinking Immunoprecipitation coupled with high-throughput sequencing) experiments targeting Miwi in isolated round spermatids from mouse testis.
2015-04-09 | E-GEOD-67683 | biostudies-arrayexpress
Project description:The piRNA machinery is known for its role in mediating epigenetic silencing of transposons. Recent studies suggest that this function also involves piRNA-guided cleavage of transposon-derived transcripts. As many piRNAs also appear to have the capacity to target diverse mRNAs, this raises the intriguing possibility that piRNAs may act extensively as siRNAs to degrade specific mRNAs. To directly test this hypothesis, we compared mouse PIWI (MIWI)-associated piRNAs with experimentally identified cleaved mRNA fragments from mouse testes, and observed cleavage sites that predominantly occur at position 10 from the 5' end of putative targeting piRNAs. We also noted strong biases for U and A residues at nucleotide positions 1 and 10, respectively, in both piRNAs and mRNA fragments, features that resemble the pattern of piRNA amplification by the 'ping-pong' cycle. Through mapping of MIWI-RNA interactions by CLIP-seq and gene expression profiling, we found that many potential piRNA-targeted mRNAs directly interact with MIWI and show elevated expression levels in the testes of Miwi catalytic mutant mice. Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression. Importantly, we demonstrated that proper turnover of certain key piRNA targets is essential for sperm formation. Together, these findings reveal the siRNA-like function of the piRNA machinery in mouse testes and its central requirement for male germ cell development and maturation.
2015-04-09 | GSE67683 | GEO
Project description:PIWI-clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ~23-30nt piRNAs that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endo-nuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the inter-dependencies between these piRNA biogenesis pathways in the developing Drosophila ovary. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and that defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear, transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. To understand the hierarchical order of primary versus secondary piRNA biogenesis in Drosophila ovaries, we sequenced piRNAs bound to total-Piwi, germline-Piwi, Aubergine and Argonaute3 from ovaries of germline specific knockdowns of control, piwi, aub, ago3 single knockdowns and aub/ago3 double knockdowns. To determine changes in Transposable Element (TE) transcription or TE RNA steady state in perturbed piRNA pathway conditions, we performed Pol2-ChIP-sequencing and polyA bound RNA-sequencing from ovaries of multiple germline knockdown genotypes. We also sequenced genomic DNA from ovaries of control knockdowns to experimentally estimate the TE copy number in our genetic background. Finally, we used CAP-seq from germline specific Piwi depletions to identify the Transcriptional Start Sites (TSS) in TEs in a deregulated background. Replicates are labeled with R1, R2, R3, R4 where indicated.
2015-08-31 | E-GEOD-71775 | biostudies-arrayexpress
Project description:During embryonic germ cell development in mice, transposon-enriched, piwi-interacting RNAs (piRNAs) guide MILI and MIWI2 to direct silencing of potentially active mobile element families. In contrast, we know much less about the function of the highly abundant and extremely diverse class of piRNAs, which partner with MIWI and MILI during meiosis. Both MIWI and its catalytic activity are required for successful spermatogenesis, strongly indicating that piRNA-guided cleavage is critical for germ cell development. To gain an understanding of meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing an entire human meiotic piRNA cluster. This triggered a spermatogenesis defect, presumably by inappropriately targeting the piRNA machinery to mouse RNAs essential for germ cell development. Through an analysis of such de novo targets, we derived a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein coding genes as targets of native piRNAs. Cleavage of genic targets begins at the pachytene stage when meiotic piRNAs first appear. As such, target mRNA levels attenuate starting from the pachytene stage and are further repressed throughout meiosis. Target mRNA-piRNA pairs also show evidence of an ongoing cleavage-dependent amplification cycle, which is not normally a strong feature of meiotic piRNAs. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells. 48 samples
2015-05-22 | E-GEOD-64138 | biostudies-arrayexpress
Project description:During embryonic germ cell development in mice, transposon-enriched, piwi-interacting RNAs (piRNAs) guide MILI and MIWI2 to direct silencing of potentially active mobile element families. In contrast, we know much less about the function of the highly abundant and extremely diverse class of piRNAs, which partner with MIWI and MILI during meiosis. Both MIWI and its catalytic activity are required for successful spermatogenesis, strongly indicating that piRNA-guided cleavage is critical for germ cell development. To gain an understanding of meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing an entire human meiotic piRNA cluster. This triggered a spermatogenesis defect, presumably by inappropriately targeting the piRNA machinery to mouse RNAs essential for germ cell development. Through an analysis of such de novo targets, we derived a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein coding genes as targets of native piRNAs. Cleavage of genic targets begins at the pachytene stage when meiotic piRNAs first appear. As such, target mRNA levels attenuate starting from the pachytene stage and are further repressed throughout meiosis. Target mRNA-piRNA pairs also show evidence of an ongoing cleavage-dependent amplification cycle, which is not normally a strong feature of meiotic piRNAs. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.
2015-05-22 | GSE64138 | GEO
Project description:Piwi-interacting small RNAs (piRNAs) of fetal prospermatogonia of mice have been strongly implicated in transposon control. In contrast, little is known about biogenesis and function of abundant piRNAs from adult testes expressed in late spermatocytes and round spermatids. These so-called "pachytene" piRNAs are processed from long non-coding piRNA precursors and have no defined RNA targets in the transcriptome even though their binding partner Piwi, MIWI, is essential for spermiogenesis and fertility. Here we report that 129SvJae mice lacking Maelstrom (MAEL), a conserved piRNA pathway protein, exhibit spermiogenic arrest with defects in acrosome and flagellum formation. Further analysis revealed MAEL association with RNPs containing MIWI, TDRD6, and processed intermediates of pachytene piRNA precursors of various length. Loss of MAEL causes a 10-fold drop in pachytene piRNA levels but an increase in piRNAs from abundantly expressed mRNAs. These results suggest a MAEL-dependent mechanism for the selective processing of pachytene piRNA precursor into piRNAs. Strikingly, ribosome profiling of Mael-null testes revealed that reduced piRNA production is accompanied by reduced translation of over 800 spermiogenic mRNAs including those encoding acrosome and flagellum proteins. In light of recent reports of piRNA-independent protection of translationally repressed mRNPs by MIWI and piRNA-dependent turnover of MIWI, we propose that pachytene piRNAs function by controlling the availably of MIWI for the translational repression of spermiogenic mRNAs.
2014-07-25 | GSE50983 | GEO
Project description:Piwi-interacting small RNAs (piRNAs) of fetal prospermatogonia of mice have been strongly implicated in transposon control. In contrast, little is known about biogenesis and function of abundant piRNAs from adult testes expressed in late spermatocytes and round spermatids. These so-called "pachytene" piRNAs are processed from long non-coding piRNA precursors and have no defined RNA targets in the transcriptome even though their binding partner Piwi, MIWI, is essential for spermiogenesis and fertility. Here we report that 129SvJae mice lacking Maelstrom (MAEL), a conserved piRNA pathway protein, exhibit spermiogenic arrest with defects in acrosome and flagellum formation. Further analysis revealed MAEL association with RNPs containing MIWI, TDRD6, and processed intermediates of pachytene piRNA precursors of various length. Loss of MAEL causes a 10-fold drop in pachytene piRNA levels but an increase in piRNAs from abundantly expressed mRNAs. These results suggest a MAEL-dependent mechanism for the selective processing of pachytene piRNA precursor into piRNAs. Strikingly, ribosome profiling of Mael-null testes revealed that reduced piRNA production is accompanied by reduced translation of over 800 spermiogenic mRNAs including those encoding acrosome and flagellum proteins. In light of recent reports of piRNA-independent protection of translationally repressed mRNPs by MIWI and piRNA-dependent turnover of MIWI, we propose that pachytene piRNAs function by controlling the availably of MIWI for the translational repression of spermiogenic mRNAs. piRNA sequencing, RNA immunoprecipitation, and expression measurements (RNA-Seq and ribosome profiling) in wild-type and Mael -/- testes
2014-07-25 | E-GEOD-50983 | biostudies-arrayexpress