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PIWI slicing and EXD1 drive biogenesis of nuclear piRNAs from cytosolic targets of the mouse piRNA pathway

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Ramesh Pillai

PROVIDER: E-GEOD-74423 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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PIWI slicing and EXD1 drive biogenesis of nuclear piRNAs from cytosolic targets of the mouse piRNA pathway

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

Exonuclease domain-containing 1 enhances MIWI2 piRNA biogenesis via its interaction with TDRD12

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.

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2008-10-01 | E-GEOD-12757 | biostudies-arrayexpress

Loss of the Mili-interacting Tdrd1 activates transposons and alters Mili-associated small RNA profile

Project description:Piwi proteins and their associated Piwi-interacting RNAs (piRNAs) are implicated in transposon silencing in the murine germline. There is currently little information on additional proteins in the murine Piwi complex and how they might regulate the entry of transcripts that accumulate as piRNAs in the Piwi ribonucleoprotein (piRNP). We isolated Mili-containing complexes from adult mouse testes and identified Tudor domain-containing protein 1 (Tdrd1) as a factor specifically associated with the Mili piRNP throughout spermatogenesis. Complex formation is promoted by the recognition of symmetrically dimethylated arginines at the N-terminus of Mili by the Tudor domains of Tdrd1. Similar to a Mili mutant, mice lacking Tdrd1 show derepression of L1 transposons accompanied by a loss of DNA methylation of their regulatory elements, and delocalization of Miwi2 from the nucleus to the cytoplasm. Finally, we show that Mili piRNPs devoid of Tdrd1 accept the entry of abundant cellular transcripts into the piRNA pathway and accumulate piRNAs with a profile that is drastically different from the wild-type. Our data suggests that Mili recruits Tdrd1 to ensure only the entry of transcripts that contribute to its normal piRNA pool. Immunoprecipitation of Tdrd1 or Mili from mouse testes isolated from wild-type or tdrd1 ko animals. Associated RNAs were sequenced in this study.

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A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice

2008-10-01 | GSE12757 | GEO

A MILI-independent piRNA biogenesis pathway empowers partial germline reprogramming.

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

Analysis of small RNAs in piRNA pathway mutant mouse testes

Project description:Piwi-interacting RNAs are 25-32 nt small RNAs bound to Piwi proteins. To know the steps where factors involved in the piRNA biogenesis (MILI, MIWI2, ZUC (MitoPLD), MVH) work, we sequenced small RNAs from mutant mouse testes and analyzed piRNAs.

2010-05-27 | GSE20327 | GEO

Mouse MOV10L1 associates with Piwi proteins and is an essential component of the piRNA pathway

Project description:Piwi-interacting RNAs (piRNAs) are essential for silencing of transposable elements in the germline but their biogenesis is poorly understood. Here we demonstrate that MOV10L1, a germ cell-specific putative RNA helicase, is associated with Piwi proteins. Genetic disruption of the MOV10L1 RNA helicase domain in mice renders both MILI and MIWI2 devoid of piRNAs. Absence of a functional piRNA pathway in Mov10l1 mutant testes causes loss of DNA methylation and subsequent de-repression of retrotransposons in germ cells. The Mov10l1 mutant males are sterile due to complete meiotic arrest. This is the first mouse mutant that expresses Piwi proteins but lacks piRNAs, suggesting that MOV10L1 is required for piRNA biogenesis and/or loading to Piwi proteins. Examination of piRNAs in Mov10l IP and differences in total small RNA profiles in RNA helicase Mov10l+/- and Mov10l-/- mutants.

2010-06-12 | E-GEOD-21763 | biostudies-arrayexpress

Molecular role of RNA helicases MVH and TDRD9 in PIWI slicing-triggered mammalian piRNA biogenesis and function

Project description:PIWI-interacting RNAs (piRNAs) function in the nucleus and cytoplasm of animal germ cells to suppress mobile genetic elements. In the mouse male germline, biogenesis of MIWI2-bound nuclear piRNAs depends on endonuclease activity of cytosolic MILI, but the process is poorly understood. Here we use a mouse model expressing an artificial piRNA precursor to show that MILI slicing of the precursor generates a 16-nt by-product and a pre-piRNA intermediate that requires 3ʹ end processing to mature as a new piRNA. The ability to use the slicer products requires ATPase activity of the RNA helicase MVH, as the catalytic-dead Mvh mutant mice (Mvh-/KI) fail to convert the intermediate into piRNAs, abrogating biogenesis of MIWI2 piRNAs. This results in an early arrest in spermatogenesis and de-repression of transposons. Furthermore, the mutant MVH protein is dominant-negative (Mvh+/KI) as it causes a late-spermatogenic arrest by trapping complexes containing the mysterious pachytene piRNAs and slicer products, uncovering a role for the protein beyond the embryonic germline. In contrast, we find that the ATPase activity of TDRD9 is dispensable for piRNA biogenesis, but is essential for silencing by MIWI2. Our studies implicate distinct RNA helicases in specific steps along the mammalian nuclear piRNA pathway.

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