Exonuclease domain-containing 1 enhances MIWI2 piRNA biogenesis via its interaction with TDRD12
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PROVIDER: PRJNA489266 | ENA |
REPOSITORIES: ENA
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Project description:PIWI proteins and their associated small RNAs called PIWI-interacting RNAs (piRNAs) restrict transposon activity in animal gonads to ensure fertility. Distinct biogenesis pathways load piRNAs into the PIWI proteins MILI and MIWI2 in the mouse male embryonic germline. While most of MILI piRNAs derive via a slicer-independent pathway, a MILI slicer endonuclease-initiated pathway loads nuclear MIWI2 with a series of phased piRNAs. Tudor domain-containing 12 (TDRD12) and its interaction partner Exonuclease domain-containing 1 (EXD1) are required for loading MIWI2, but only Tdrd12 is essential for fertility, leaving us with no explanation for the physiological role of Exd1. Using an artificial piRNA precursor, we demonstrate that MILI-triggered piRNA biogenesis is greatly reduced in the Exd1 mutant. The situation deteriorates in the sensitized Exd1 mutant (Exd1-/-; Tdrd12+/-), where diminished MIWI2 piRNA levels de-repress LINE1 retrotransposons, causing infertility. Thus, EXD1 enhances slicing-triggered MIWI2 piRNA biogenesis via a functional interaction with TDRD12.
2018-09-27 | GSE119447 | GEO
Project description:In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use piRNA guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2-deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 suggesting that the proteins have independent roles in establishing DNA methylation patterns.
2015-07-11 | GSE70731 | GEO
Project description:In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use piRNA guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2-deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 suggesting that the proteins have independent roles in establishing DNA methylation patterns. Small RNA profiles (19-30 nt range) of embryonic (E16.5) and post-natal (P10) testis of Miwi2 mutant mice and matched heterozygote controls. mRNA profiles of embryonic testis (E16.5) of heterozygote control mice and of postnatal testis (P10) of Miwi2 and Mili mutants and heterozygote controls. CpG methylation BS-seq profile of postnatal (P10) spermatocytes of Miwi2 mutant mice and matched heterozygote controls.
2015-07-11 | E-GEOD-70731 | biostudies-arrayexpress
Project description:Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3′–5′ domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.
2023-12-21 | GSE194043 | GEO
Project description:Piwi-interacting RNAs (piRNAs) are gonad-specific small RNAs that provide defence against transposable genetic elements called transposons. Our knowledge of piRNA biogenesis is sketchy, partly due to an incomplete inventory of the factors involved. Here, we identify Tudor domain-containing 12 (TDRD12; also known as ECAT8) as a novel piRNA biogenesis factor in mice. TDRD12 is detected in complexes containing MILI (PIWIL2), its associated primary piRNAs, and TDRD1, all of which are already implicated in secondary piRNA biogenesis. Male mice carrying either a nonsense point mutation (repro23 mice) or a targeted deletion in the Tdrd12 locus are infertile, and de-repress retrotransposons. We find that TDRD12 is dispensable for primary piRNA biogenesis but essential for production of secondary piRNAs that enter MIWI2 (PIWIL4). Cell culture studies with the insect orthologue of TDRD12 suggest a role for the multi-domain protein in mediating complex formation with other participants during secondary piRNA biogenesis. Total small RNA and immunoprecipitated small RNA were purified from mouse testis extract and Bmn4 cells for preparation of high-throughput sequencing libraries.
2013-11-04 | E-GEOD-50544 | biostudies-arrayexpress
Project description:Piwi-interacting RNAs (piRNAs) are gonad-specific small RNAs that provide defence against transposable genetic elements called transposons. Our knowledge of piRNA biogenesis is sketchy, partly due to an incomplete inventory of the factors involved. Here, we identify Tudor domain-containing 12 (TDRD12; also known as ECAT8) as a novel piRNA biogenesis factor in mice. TDRD12 is detected in complexes containing MILI (PIWIL2), its associated primary piRNAs, and TDRD1, all of which are already implicated in secondary piRNA biogenesis. Male mice carrying either a nonsense point mutation (repro23 mice) or a targeted deletion in the Tdrd12 locus are infertile, and de-repress retrotransposons. We find that TDRD12 is dispensable for primary piRNA biogenesis but essential for production of secondary piRNAs that enter MIWI2 (PIWIL4). Cell culture studies with the insect orthologue of TDRD12 suggest a role for the multi-domain protein in mediating complex formation with other participants during secondary piRNA biogenesis.
2013-11-04 | GSE50544 | 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: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:In mice, the PIWI-piRNA pathway is essential to re-establish transposon silencing during male germline reprogramming. The cytoplasmic PIWI protein MILI mediates piRNA-guided transposon RNA cleavage as well as piRNA amplification. MIWI2-bound piRNAs and its nuclear localization are proposed to be dependent upon MILI function. Here, we demonstrate the existence of a piRNA biogenesis pathway that in the absence of MILI that sustains partial MIWI2 function and reprogramming activity.
2017-04-25 | E-MTAB-5561 | biostudies-arrayexpress