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MIWI catalysis is required for piRNA amplification-independent LINE1 transposon silencing [microarray]

ABSTRACT: Here we show that MIWI is a small RNA-guided ribonuclease (Slicer) that requires extensive complementarity for target cleavage in vitro. Disruption of its catalytic activity in mice by a single point mutation results in male infertility and displays increased accumulation of LINE1 transposon transcripts. Round spermatids were purified from adult mouse testes of indicated Miwi genotypes. Total RNA was extracted and subjected to microarray analysis for gene expression profiling.

ORGANISM(S): Mus musculus

SUBMITTER: Ramesh Pillai

PROVIDER: E-GEOD-32180 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing.

Reuter Michael M Berninger Philipp P Chuma Shinichiro S Shah Hardik H Hosokawa Mihoko M Funaya Charlotta C Antony Claude C Sachidanandam Ravi R Pillai Ramesh S RS

Nature 20111127 7376

Repetitive-element-derived Piwi-interacting RNAs (piRNAs) act together with Piwi proteins Mili (also known as Piwil2) and Miwi2 (also known as Piwil4) in a genome defence mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility. A third Piwi family member, Miwi (also known as Piwil1), is expressed in ...[more]

PMID: 22121019

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MIWI catalysis is required for piRNA amplification-independent LINE1 transposon silencing

Project description:This SuperSeries is composed of the following subset Series: GSE32180: MIWI catalysis is required for piRNA amplification-independent LINE1 transposon silencing [microarray] GSE32184: MIWI catalysis is required for piRNA amplification-independent LINE1 transposon silencing [deep sequencing] Refer to individual Series

2011-11-30 | E-GEOD-32183 | biostudies-arrayexpress

MIWI catalysis is required for piRNA amplification-independent LINE1 transposon silencing [deep sequencing]

Project description:Here we show that MIWI is a small RNA-guided ribonuclease (Slicer) that requires extensive complementarity for target cleavage in vitro. Disruption of its catalytic activity in mice by a single point mutation results in male infertility and displays increased accumulation of LINE1 transposon transcripts. MIWI-associated piRNAs from different genotypes were sequenced. Total RNA from purified round spermatids were subjected to Ribozero purification and strand-specific RNAseq lib prepared. Global 5' RACE library was prepare from indicated genotypes.

2011-11-30 | E-GEOD-32184 | biostudies-arrayexpress

MIWI catalysis is required for piRNA amplification-independent LINE1 transposon silencing [microarray]

2011-11-30 | GSE32180 | GEO

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Transcriptome analysis in mouse late spermatocyte deficient in pachytene piRNAs (MIWI-immunoprecipitated RNAs)

Project description:The eukaryotic genome has vast intergenic regions containing transposons, pseudogenes and other repetitive sequences. They produce numerous long non-coding RNAs (lncRNAs) and PIWI-interacting RNAs (piRNAs), yet the functions of the vast intergenic regions remain largely unknown. Mammalian piRNAs are abundantly expressed in late spermatocytes and round spermatids. Their expression coincides with the widespread expression of lncRNAs from the genome of these cells. Here, we show that piRNAs mediate the degradation of a large number of mRNAs and lncRNAs in mouse late spermatocytes. In particular, they have a large impact on the lncRNA transcriptome, as a quarter of lncRNAs expressed in late spermatocytes are upregulated in mice deficient in piRNA pathway. Furthermore, our genomic and in vivo functional analyses reveal that retrotransposon sequences in the 3´UTR of mRNAs are potentially targeted by piRNAs for degradation. Similarly, a large number of spermatogenic cell-specific lncRNAs are degraded by piRNAs via retrotransposon sequences. Moreover, we show that pseudogenes regulate mRNA stability via the piRNA pathway. The degradation of mRNAs and lncRNAs by piRNAs requires MIWI and, at least in part, depends on its slicer activity. Together, these findings reveal the presence of the highly complex and global RNA regulatory network mediated by piRNAs with retrotransposons and pseudogenes as regulatory sequences. RNA-seq of MIWI-immunoprecipitated RNAs from Late spermatocytes in Miw+/-, MiwiADH/-, Miwi-/-

2014-11-20 | E-GEOD-62416 | biostudies-arrayexpress

TDRD6 instructs correct assembly of mRNPs in the chromatoid body to potentiate UPF1 dependent mRNA decay.

Project description:RNA sequencing of heterozygote or Tudor domain contian protein 6 (TDRD6) knockout round spermatid cells. Chromatoid bodies (CBs) are germ cell-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family such as TDRD6. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay machinery such as UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 interaction, and for assembly of UPFs and other RNA binding proteins into super-complexes. In absence of TDRD6, the association of some mRNAs with UPF1 is impaired, and the long 3â UTR-stimulated but not the exon junction complex-stimulated pathway of NMD is distorted. Reduced association of mRNAs with UPF1 correlated with increased stability and presence in polysome fractions, i.e. enhanced translational activity. Thus, we define CBs as sites of UPF1-dependent mRNA degradation and provide evidence for the requirement for NMD in spermiogenesis. This function of CBs depends on TDRD6-promoted assembly of mRNA decay enzymes within mRNPs. RNA was extracted from quadruplicate samples and libraries generated for sequencing using the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) at the Deep Sequencing Group SFB 655, Biotechnology Center of Technische UniversitÃ¤t Dresden. After enrichment and XP bead (Agencourt AMPure Kit; Beckman Coulter, Inc.) purification, quality control was done using Fragment AnalyzerTM (Advanced Analytical). The bar-coded libraries were equimolarly pooled and subjected to 76 bp single-end sequencing on Illumina HiSeq 2000, resulting in an average of 33 million reads per sample.

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Mili-IP, Miwi-IP, and total RNA from mouse adult testis

Project description:We deep-sequenced small RNAs after immunoprecipitation of Mili or Miwi, as well as total small RNA from adult mouse testis. The goal of this experiment is to more deeply characterize the piRNA pool from adult mouse testes, using the Illumina platform. Comparison of 2 IP libraries with a non-IP library

2010-01-04 | E-GEOD-19172 | biostudies-arrayexpress

MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes

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.

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2015-04-09 | E-GEOD-67683 | biostudies-arrayexpress