Target RNA repertoire of mouse Mili and Miwi proteins reveals piRNA biogenesis and Miwi function in spermiogenesis [HITS-CLIP]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE27622 | GEO | 2012/07/20
SECONDARY ACCESSION(S): PRJNA141769
REPOSITORIES: GEO
Similar Datasets
Project description:Germ cells employ elaborate mechanisms to maintain and protect their genetic material, and also to regulate gene expression during the complex differentiation process of gametogenesis. Piwi proteins, a subclade of the Argonaute family, are expressed mainly in the germline and bind piRNAs, a novel and diverse class of small RNAs whose biogenesis and putative functions are still largely elusive. We employed High Throughput Sequencing after Crosslinking and Immunoprecipitation (HITS-CLIP) coupled with RNA-Seq to characterize the genome-wide target RNA repertoire of Mili and Miwi, two mouse Piwi proteins. Our analysis outlines a model for primary piRNA biogenesis in postnatal mouse and indicates that piRNAs do not mediate target RNA recognition, but rather are the end products of RNA processing. Moreover, we identify a set of mRNAs essential for spermiogenesis that are bound and regulated by Miwi, directly implicating Piwi proteins in the control of gene expression at key time points of spermiogenesis. HITS-CLIP (High Throughput Sequencing after Crosslinking and Immunoprecipitation) experiments targeting two mouse Piwi proteins Mili and Miwi.
2012-07-19 | E-GEOD-27622 | biostudies-arrayexpress
Project description:Germ cells employ elaborate mechanisms to maintain and protect their genetic material, and also to regulate gene expression during the complex differentiation process of gametogenesis. Piwi proteins, a subclade of the Argonaute family, are expressed mainly in the germline and bind piRNAs, a novel and diverse class of small RNAs whose biogenesis and putative functions are still largely elusive. We employed High Throughput Sequencing after Crosslinking and Immunoprecipitation (HITS-CLIP) coupled with RNA-Seq to characterize the genome-wide target RNA repertoire of Mili and Miwi, two mouse Piwi proteins. Our analysis outlines a model for primary piRNA biogenesis in postnatal mouse and indicates that piRNAs do not mediate target RNA recognition, but rather are the end products of RNA processing. Moreover, we identify a set of mRNAs essential for spermiogenesis that are bound and regulated by Miwi, directly implicating Piwi proteins in the control of gene expression at key time points of spermiogenesis.
2012-07-20 | GSE27609 | 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
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:piRNAs are a novel class of small noncoding RNAs that were recently identified in animal germ cells. This class of small noncoding RNAs is specifically associated with an evolutionarily conserved PIWI family proteins, which belong to the germline-specific members of the Argonaute protein family and are indispensable to germline development in animals. The PIWI/piRNA pathway has been deemed as an innate immune system that prevents mobile genetic elements from destabilizing DNA and that protects genome integrity in animal germ cells. By ribosome profiling using Miwi-null testes, we identified a group of ~600 mRNAs as likely direct piRNA targets for translational regulation in mouse spermatids. These results suggest that the mouse PIWI (MIWI)/piRNA is responsible for activating translation of a subset of spermiogenic mRNAs to coordinate with morphological transformation into elongated spermatids.
2019-12-15 | GSE139399 | GEO
Project description:Nearly half of the mammalian genome is composed of repeated sequences. In Drosophila, PIWI proteins exert control over transposons. However, mammalian PIWI proteins, Miwi and Mili, partner with piRNAs that are depleted of repeat sequences, raising questions about a role for mammalian PIWIs in transposon control. A search for murine small RNAs that might program PIWI proteins for transposon suppression revealed developmentally regulated piRNA loci, some of which resemble transposon master control loci of Drosophila. We also find evidence of an adaptive amplification loop in which PIWI catalyzes formation of piRNA 5’ ends. Mili mutants de-repress L1 and IAP and lose DNA methylation of L1 elements, demonstrating an evolutionarily conserved role for PIWI proteins in transposon suppression. Keywords: small RNA profile, piRNA
2007-04-20 | GSE7414 | GEO
Project description:Piwi proteins and their associated piRNAs are essential in the germline where they repress transposition, regulate translation, and guide epigenetic programming. Little is known, however, about the molecular mechanisms through which Piwi proteins and piRNAs mediate these processes. Here, we show that an evolutionarily conserved Tudor and KH-domain containing protein, Tdrkh (a.k.a. Tdrd2), partners with Miwi and Miwi2 in mice via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is localized to pi-bodies and piP-bodies and is required for nuclear localization of Miwi2. Genetic deletion of Tdrkh arrests meiosis at the zygotene stage, demethylates Line1 DNA, and up-regulates Line1 transposition, but does not promote apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs. Specifically, in Tdrkh mutants, piRNAs accumulate as a distinct population of 5’U-containing 31-37nt RNA that largely complements the missing mature piRNAs. These results demonstrate that the primary piRNA biogenesis pathway involves 3à5’ processing of the 31-37nt intermediates and that Tdrkh is required for this final step of piRNA biogenesis. However, Tdrkh is not required for the secondary piRNA biogenesis pathway (i.e., the ping pong cycle). These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent. Tdrkh-floxed mice were generated by the University of Connecticut Gene Targeting and Transgenic Facility. The targeting vector utilized C57Bl6 Tdrkh genome sequences from BAC clone RP23-263K17 (Chori BACPAC) and floxed exons 2-4 (the start codon is in exon 2) and was electroporated into D2 ES cells, a male hybrid C57Bl6/129SEV line. Clones that survived positive selection with G418 and negative selection with gancyclovir were expanded and screened by PCR with primers recognizing endogenous and exogenous (vector-derived) sequences. Positive clones were fused to CD-1 embryos, and germline transmission from resulting pups was confirmed by test crosses to CD-1 mice (which also confirmed that all gametes were derived from the targeted clones). Positive animals were bred to FLP mice to delete the neomycin-targeting cassette, resulting in Tdrkh cKO mice on a C57Bl6/129 background. cKO mice were bred with EIIa-Cre transgenic mice to excise the floxed exons 2-4 and generate Tdrkh +/- animals. Heterozygous animas were intercrossed to remove the EIIA transgene. Two independent knockout lines were generated from independent cKO founder lines and showed identical phenotypes in all experiments performed.
2013-09-01 | E-GEOD-47151 | biostudies-arrayexpress
Project description: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.
| S-EPMC3414646 | biostudies-literature
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 proteins and their associated piRNAs are essential in the germline where they repress transposition, regulate translation, and guide epigenetic programming. Little is known, however, about the molecular mechanisms through which Piwi proteins and piRNAs mediate these processes. Here, we show that an evolutionarily conserved Tudor and KH-domain containing protein, Tdrkh (a.k.a. Tdrd2), partners with Miwi and Miwi2 in mice via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is localized to pi-bodies and piP-bodies and is required for nuclear localization of Miwi2. Genetic deletion of Tdrkh arrests meiosis at the zygotene stage, demethylates Line1 DNA, and up-regulates Line1 transposition, but does not promote apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs. Specifically, in Tdrkh mutants, piRNAs accumulate as a distinct population of 5’U-containing 31-37nt RNA that largely complements the missing mature piRNAs. These results demonstrate that the primary piRNA biogenesis pathway involves 3à5’ processing of the 31-37nt intermediates and that Tdrkh is required for this final step of piRNA biogenesis. However, Tdrkh is not required for the secondary piRNA biogenesis pathway (i.e., the ping pong cycle). These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.
2013-09-01 | GSE47151 | GEO