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The Function of Piwi is Independent of Its Slicer Activity

ABSTRACT: In diverse organisms, the highly-conserved Piwi proteins bind to a complex class of small non-coding RNAs called piRNAs, and are essential for germline stem cell maintenance, transposon silencing, fertility, and offspring viability1. By homology with other Argonautes, Piwi proteins have been proposed and later reported to possess endonuclease activity in vitro, effected by its conserved aspartate catalytic triad2,3. Indeed, mutation of these residues in one of the three mouse PIWI proteins, MILI, affects the production of Line 1 piRNAs and spermatogenesis4. In Drosophila, the conserved slicer function has been proposed to be crucial for all Piwi proteins in piRNA amplification and transposon silencing5. Here, we report in vivo evidence that, in contrast to common belief, the catalytic triad of Piwi is not required for the function of PIwi in the germline for stem cell maintenance, fertility, transposon silencing, or piRNA biogenesis. In addition, it is not required for piRNA biogenesis in the soma where Piwi is the sole Piwi-subfamily protein involved in the process. These observations, together with the recent findings on MILI and MIWI2 triad mutant, indicate that even though the slicer function may be required for a subset of piRNA-mediated process, it is overall inconsequential for the function of Piwi proteins

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE34032 | GEO | 2013/02/04

SECONDARY ACCESSION(S): PRJNA148075

REPOSITORIES: GEO

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The Function of Piwi is Independent of Its Slicer Activity

Project description:In diverse organisms, the highly-conserved Piwi proteins bind to a complex class of small non-coding RNAs called piRNAs, and are essential for germline stem cell maintenance, transposon silencing, fertility, and offspring viability1. By homology with other Argonautes, Piwi proteins have been proposed and later reported to possess endonuclease activity in vitro, effected by its conserved aspartate catalytic triad2,3. Indeed, mutation of these residues in one of the three mouse PIWI proteins, MILI, affects the production of Line 1 piRNAs and spermatogenesis4. In Drosophila, the conserved slicer function has been proposed to be crucial for all Piwi proteins in piRNA amplification and transposon silencing5. Here, we report in vivo evidence that, in contrast to common belief, the catalytic triad of Piwi is not required for the function of PIwi in the germline for stem cell maintenance, fertility, transposon silencing, or piRNA biogenesis. In addition, it is not required for piRNA biogenesis in the soma where Piwi is the sole Piwi-subfamily protein involved in the process. These observations, together with the recent findings on MILI and MIWI2 triad mutant, indicate that even though the slicer function may be required for a subset of piRNA-mediated process, it is overall inconsequential for the function of Piwi proteins Assess the role of the endonuclease activity of Piwi in all known biological processes that involve Piwi function in Drosophila ovary

2013-02-04 | E-GEOD-34032 | biostudies-arrayexpress

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.

2018-09-27 | GSE119447 | GEO

Profiling of Mili and Miwi2 associated piRNAs in WT, MiliDAH, Miwi2DAH and Miwi2KO from E16.5 gonadocytes.

Project description:Piwi proteins and piRNAs have conserved functions in transposonM- silencing. The murine Piwi proteins Mili and Miwi2 direct epigeneticM- LINE1 (L1) and intracisternal A particle (IAP) transposon silencingM- during genome reprogramming in the embryonic male germline. PiwiM- proteins are proposed to be piRNA-guided endonucleases that initiateM- secondary piRNA biogenesis . However the actual contribution of theirM- endonuclease activities to piRNA biogenesis and transposon silencingM- remain unknown. To investigate the role of Piwi-catalyzedM- endonucleolytic activity, we engineered point mutations in the mouseM- that substitute the second D to an A in the catalytic triad (DDH) ofM- Mili and Miwi2, generating the MiliDAH and Miwi2DAH alleles,M- M- respectively. Analysis of Mili-bound piRNAs from homozygous MiliDAHM- fetal gonadocytes revealed the failure of transposon piRNA amplification resulting in the stark reduction of piRNA bound withinM- Miwi2 ribonuclear particles (RNPs). We find that Mili-mediated piRNA amplification is selectively required for L1 but not IAP silencing.M- The defective piRNA pathway in MiliDAH mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2DAH mice areM- fertile, transposon silencing is established normally and no defectsM- in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. WeM- conclude that cycles of intra-Mili secondary piRNA biogenesis fuelM- piRNA amplification that is selectively required for L1 silencing.M-

2011-10-28 | E-MTAB-730 | biostudies-arrayexpress

Developmentally regulated piRNA clusters implicate MILI in transposon control

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

the analysis of piRNA shows the function of Slicer activity of AGO3 in piRNA biogenesis.

Project description:Piwi-interacting RNAs (piRNAs) silence transposons in animal germ cells. In Drosophila, the reciprocal “Ping-Pong” cycle of piRNA-directed RNA cleavage, catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (AGO3) through their Slicer activity, is believed to expand the population of antisense piRNAs in response to transposon expression. Whether and how the Slicer activity of AGO3/Aub promotes the process of the secondary piRNA amplification remain unclear. Here we generated transgenic flies that could express AGO3 Slicer mutant forms to ellucidate the Slicer activity of AGO3.

2014-06-30 | GSE49436 | GEO

the analysis of piRNA shows the function of Slicer activity of AGO3 in piRNA biogenesis.

2014-06-30 | E-GEOD-49436 | biostudies-arrayexpress

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

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

Project description:We examined the construction of the piRNA system in the restricted developmental window in which methylation patterns are set during mammalian embryogenesis. We find robust expression of two Piwi family proteins, MIWI2 and MILI. Their associated piRNA profiles reveal differences from Drosophila wherein large piRNA clusters act as master regulators of silencing. Instead, in mammals, dispersed transposon copies initiate the pathway, producing primary piRNAs, which predominantly join MILI in the cytoplasm. MIWI2, whose nuclear localization and association with piRNAs depend upon MILI, is enriched for secondary piRNAs antisense to the elements that it controls. The Piwi pathway lies upstream of known mediators of DNA methylation, since piRNAs are still produced in Dnmt3L mutants, which fail to methylate transposons. This implicates piRNAs as specificity determinants of DNA methylation in germ cells. Examination of total small RNA and MILI associated piRNAs in embryonic and post-birth mouse testes, MIWI2-associated piRNAs in embryonic testes.

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

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

2008-10-01 | GSE12757 | GEO

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.

2009-05-24 | E-GEOD-16023 | biostudies-arrayexpress

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