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Target RNA repertoire of mouse Mili and Miwi proteins reveals piRNA biogenesis and Miwi function in spermiogenesis [RNA-Seq]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE27609 | GEO | 2012/07/20

SECONDARY ACCESSION(S): PRJNA141777

REPOSITORIES: GEO

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Target RNA repertoire of mouse Mili and Miwi proteins reveals piRNA biogenesis and Miwi function in spermiogenesis [HITS-CLIP]

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

Target RNA repertoire of mouse Mili and Miwi proteins reveals piRNA biogenesis and Miwi function in spermiogenesis [HITS-CLIP]

2012-07-20 | GSE27622 | GEO

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

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.

2009-05-24 | GSE16023 | 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

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

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

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.

2010-01-04 | GSE19172 | 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.

2018-09-27 | GSE119447 | GEO

MAEL-dependent selective processing of pachytene piRNA precursors and translation of spermiogenic mRNAs

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

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