Mitochondrial membrane-based initial separation of MIWI and MILI functions during pachytene piRNA biogenesis.
ABSTRACT: PIWI-interacting RNAs (piRNAs) engage PIWI proteins to silence transposons and promote germ cell development in animals. In diverse species, piRNA biogenesis occurs near the mitochondrial surface, and involves mitochondrial membrane-anchored factors. In mice, two cytoplasmic PIWI proteins, MIWI and MILI, receive processed pachytene piRNAs at intermitochodrial cement (IMC). However, how MIWI and MILI are initially recruited to the IMC to engage multiple steps of piRNA processing is unclear. Here, we show that mitochondria-anchored TDRKH controls multiple steps of pachytene piRNA biogenesis in mice. TDRKH specifically recruits MIWI, but not MILI, to engage the piRNA pathway. It is required for the production of the entire MIWI-bound piRNA population and enables trimming of MILI-bound piRNAs. The failure to recruit MIWI to the IMC with TDRKH deficiency results in loss of MIWI in the chromatoid body, leading to spermiogenic arrest and piRNA-independent retrotransposon LINE1 de-repression in round spermatids. Our findings identify a mitochondrial surface-based scaffolding mechanism separating the entry and actions of two critical PIWI proteins in the same piRNA pathway to drive piRNA biogenesis and germ cell development.
Project description:Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposition, regulate translation, and guide epigenetic programming in the germline. Here, we show that an evolutionarily conserved Tudor and KH domain-containing protein, Tdrkh (a.k.a. Tdrd2), is required for spermatogenesis and involved in piRNA biogenesis. Tdrkh partners with Miwi and Miwi2 via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is a mitochondrial protein often juxtaposed to pi-bodies and piP-bodies and is required for Tdrd1 cytoplasmic localization and Miwi2 nuclear localization. Tdrkh mutants display meiotic arrest at the zygotene stage, attenuate methylation of Line1 DNA, and upregulate Line1 RNA and protein, without inducing apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs but accumulate a distinct population of 1'U-containing, 2'O-methylated 31-37?nt RNAs that largely complement the missing mature piRNAs. Our results demonstrate that the primary piRNA biogenesis pathway involves 3'?5' processing of 31-37?nt intermediates and that Tdrkh promotes this final step of piRNA biogenesis but not the ping-pong cycle. These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.
Project description:Germ cells implement elaborate mechanisms to protect their genetic material and to regulate gene expression during differentiation. Piwi proteins bind Piwi-interacting RNAs (piRNAs), small germline RNAs whose biogenesis and functions are still largely elusive. We used high-throughput sequencing after cross-linking and immunoprecipitation (HITS-CLIP) coupled with RNA-sequencing (RNA-seq) to characterize the genome-wide target RNA repertoire of Mili (Piwil2) and Miwi (Piwil1), two Piwi proteins expressed in mouse postnatal testis. We report the in vivo pathway of primary piRNA biogenesis and implicate distinct nucleolytic activities that process Piwi-bound precursor transcripts. Our studies indicate that pachytene piRNAs are the end products of RNA processing. HITS-CLIP demonstrated that Miwi binds spermiogenic mRNAs directly, without using piRNAs as guides, and independent biochemical analyses of testis mRNA ribonucleoproteins (mRNPs) established that Miwi functions in the formation of mRNP complexes that stabilize mRNAs essential for spermiogenesis.
Project description:Pachytene piRNAs are MIWI-/MILI-bound small RNAs abundantly expressed in pachytene spermatocytes and round spermatids in adult mouse testes. Miwi knockout (KO) male mice are sterile due to spermiogenic arrest. In Caenorhabditis elegans, sperm-borne piRNAs appear to have an epigenetic role during fertilization and development because progeny of individuals with piRNA-deficient gametes display a progressive loss of fertility after several generations. In mice, it remains unknown whether pachytene piRNA-deficient round spermatids can produce offspring, and whether the progeny of Miwi mutants also exhibits transgenerational, progressive fertility loss. Here, we report that Miwi KO round spermatids could fertilize both wild-type (WT) and Miwi KO oocytes through round spermatid injection, and could produce healthy and fertile offspring despite the global downregulation of both MIWI-/MILI-bound pachytene piRNAs. Progeny of ROSI-derived heterozygotes, both male and female, displayed normal fertility for at least three generations when bred with either WT or Miwi KO females. Our data indicate that aberrant MIWI-/MILI-bound pachytene piRNA profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting that pachytene piRNAs might not be required for paternal transgenerational epigenetic inheritance in mice.
Project description:Tudor domains are protein modules that mediate protein-protein interactions, potentially by binding to methylated ligands. A group of germline specific single and multiTudor domain containing proteins (TDRDs) represented by drosophila Tudor and its mammalian orthologs Tdrd1, Tdrd4/RNF17, and Tdrd6 play evolutionarily conserved roles in germinal granule/nuage formation and germ cell specification and differentiation. However, their physiological ligands, and the biochemical and structural basis for ligand recognition, are largely unclear. Here, by immunoprecipitation of endogenous murine Piwi proteins (Miwi and Mili) and proteomic analysis of complexes related to the piRNA pathway, we show that the TDRD group of Tudor proteins are physiological binding partners of Piwi family proteins. In addition, mass spectrometry indicates that arginine residues in RG repeats at the N-termini of Miwi and Mili are methylated in vivo. Notably, we found that Tdrkh/Tdrd2, a novel single Tudor domain containing protein identified in the Miwi complex, is expressed in the cytoplasm of male germ cells and directly associates with Miwi. Mutagenesis studies mapped the Miwi-Tdrkh interaction to the very N-terminal RG/RA repeats of Miwi and showed that the Tdrkh Tudor domain is critical for binding. Furthermore, we have solved the crystal structure of the Tdrkh Tudor domain, which revealed an aromatic binding pocket and negatively charged binding surface appropriate for accommodating methylated arginine. Our findings identify a methylation-directed protein interaction mechanism in germ cells mediated by germline Tudor domains and methylated Piwi family proteins, and suggest a complex mode of regulating the organization and function of Piwi proteins in piRNA silencing pathways.
Project description:Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during postnatal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feedforward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals.
Project description:Pachytene piRNAs are a class of Piwi-interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from piRNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon-derived piRNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved piRNA pathway protein Maelstrom (MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor-domain proteins and processing intermediates of pachytene piRNA primary transcripts. We provide evidence of functional significance of these complexes in Mael129 knockout mice that exhibit spermiogenic arrest with acrosome and flagellum malformation. Mael129-null mutant testes possess low levels of piRNAs derived from MAEL-associated piRNA precursors and exhibit reduced translation of numerous spermiogenic mRNAs including those encoding acrosome and flagellum proteins. These translation defects in haploid round spermatids are likely indirect, as neither MAEL nor piRNA precursors associate with polyribosomes, and they may arise from an imbalance between pachytene piRNAs and MIWI.
Project description:PIWI-interacting RNAs (piRNAs) are germ cell-specific small RNAs essential for retrotransposon gene silencing and male germ cell development. In piRNA biogenesis, the endonuclease MitoPLD/Zucchini cleaves long, single-stranded RNAs to generate 5' termini of precursor piRNAs (pre-piRNAs) that are consecutively loaded into PIWI-family proteins. Subsequently, these pre-piRNAs are trimmed at their 3'-end by an exonuclease called Trimmer. Recently, poly(A)-specific ribonuclease-like domain-containing 1 (PNLDC1) was identified as the pre-piRNA Trimmer in silkworms. However, the function of PNLDC1 in other species remains unknown. Here, we generate Pnldc1 mutant mice and analyze small RNAs in their testes. Our results demonstrate that mouse PNLDC1 functions in the trimming of both embryonic and post-natal pre-piRNAs. In addition, piRNA trimming defects in embryonic and post-natal testes cause impaired DNA methylation and reduced MIWI expression, respectively. Phenotypically, both meiotic and post-meiotic arrests are evident in the same individual Pnldc1 mutant mouse. The former and latter phenotypes are similar to those of MILI and MIWI mutant mice, respectively. Thus, PNLDC1-mediated piRNA trimming is indispensable for the function of piRNAs throughout mouse spermatogenesis.
Project description:The mouse PIWI-interacting RNA (piRNA) pathway produces a class of 26-30-nucleotide (nt) small RNAs and is essential for spermatogenesis and retrotransposon repression. In oocytes, however, its regulation and function are poorly understood. In the present study, we investigated the consequences of loss of piRNA-pathway components in growing oocytes. When MILI (or PIWIL2), a PIWI family member, was depleted by gene knockout, almost all piRNAs disappeared. This severe loss of piRNA was accompanied by an increase in transcripts derived from specific retrotransposons, especially IAPs. MIWI (or PIWIL1) depletion had a smaller effect. In oocytes lacking PLD6 (or ZUCCHINI or MITOPLD), a mitochondrial nuclease/phospholipase involved in piRNA biogenesis in male germ cells, the piRNA level was decreased to 50% compared to wild-type, a phenotype much milder than that in males. Since PLD6 is essential for the creation of the 5? ends of primary piRNAs in males, the presence of mature piRNA in PLD6-depleted oocytes suggests the presence of compensating enzymes. Furthermore, we identified novel 21-23-nt small RNAs, termed spiRNAs, possessing a 10-nt complementarity with piRNAs, which were produced dependent on MILI and independent of DICER. Our study revealed the differences in the biogenesis and function of the piRNA pathway between sexes.
Project description:piRNAs, a class of small non-coding RNAs associated with PIWI proteins, have broad functions in germline development, transposon silencing, and epigenetic regulation. In diverse organisms, a subset of piRNAs derived from repeat sequences are produced via the interplay between two PIWI proteins. This mechanism, termed "ping-pong" cycle, operates among the PIWI proteins of the primordial mouse testis; however, its involvement in postnatal testes remains elusive. Here we show that adult testicular piRNAs are produced independent of the ping-pong mechanism. We identified and characterized large populations of piRNAs in the adult and postnatal developing testes associated with MILI and MIWI, the only PIWI proteins detectable in these testes. No interaction between MILI and MIWI or sequence feature for the ping-pong mechanism among their piRNAs was detected in the adult testis. The majority of MILI- and MIWI-associated piRNAs originate from the same DNA strands within the same loci. Both populations of piRNAs are biased for 5' Uracil but not for Adenine on the 10th nucleotide position, and display no complementarity. Furthermore, in Miwi mutants, MILI-associated piRNAs are not downregulated, but instead upregulated. These results indicate that the adult testicular piRNAs are predominantly, if not exclusively, produced by a primary processing mechanism instead of the ping-pong mechanism. In this primary pathway, biogenesis of MILI- and MIWI-associated piRNAs may compete for the same precursors; the types of piRNAs produced tend to be non-selectively dictated by the available precursors in the cell; and precursors with introns tend to be spliced before processed into piRNAs.
Project description:PIWI-interacting RNAs (piRNAs) play a crucial role in transposon silencing in animal germ cells. In piRNA biogenesis, single-stranded piRNA intermediates are loaded into PIWI-clade proteins and cleaved by Zucchini/MitoPLD, yielding precursor piRNAs (pre-piRNAs). Pre-piRNAs that are longer than the mature piRNA length are then trimmed at their 3' ends. Although recent studies implicated the Tudor domain protein Papi/Tdrkh in pre-piRNA trimming, the identity of Trimmer and its relationship with Papi/Tdrkh remain unknown. Here, we identified PNLDC1, an uncharacterized 3'-5' exonuclease, as Trimmer in silkworms. Trimmer is enriched in the mitochondrial fraction and binds to Papi/Tdrkh. Depletion of Trimmer and Papi/Tdrkh additively inhibits trimming, causing accumulation of ?35-40-nt pre-piRNAs that are impaired for target cleavage and prone to degradation. Our results highlight the cooperative action of Trimmer and Papi/Tdrkh in piRNA maturation.