Ramesh S. Pillai

The TDRD9-MIWI2 Complex Is Essential for piRNA-Mediated Retrotransposon Silencing in the Mouse Male Germline

Host-defense mechanisms against transposable elements are critical to protect the genome information. Here we show that tudor-domain containing 9 (Tdrd9) is essential for silencing Line-1 retrotransposon in the mouse male germline. Tdrd9 encodes an ATPase/DExH-type helicase, and its mutation causes male sterility showing meiotic failure. In Tdrd9 mutants, Line-1 was highly activated and piwi-interacting small RNAs (piRNAs) corresponding to Line-1 were increased, suggesting that feedforward amplification operates in the mutant. In fetal testes, Tdrd9 mutation causes Line-1 desilencing and an aberrant piRNA profile in prospermatogonia, followed by cognate DNA demethylation. TDRD9 complexes with MIWI2 with distinct compartmentalization in processing bodies, and this TDRD9-MIWI2 localization is regulated by MILI and TDRD1 residing at intermitochondrial cement. Our results identify TDRD9 as a functional partner of MIWI2 and indicate that the tudor-piwi association is a conserved feature, while two separate axes, TDRD9-MIWI2 and TDRD1-MILI, cooperate nonredundantly in the piwi-small RNA pathway in the mouse male germline.

Miwi catalysis is required for piRNA amplification- independent LINE1 transposon silencing

Repetitive-element-derived Piwi-interacting RNAs (piRNAs) act together with Piwi proteins Mili (also known as Piwil2) and Miwi2 (also known as Piwil4) in a genome defence mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility. A third Piwi family member, Miwi (also known as Piwil1), is expressed in specific postnatal germ cells and associates with a unique set of piRNAs of unknown function. Here we show that Miwi is a small RNA-guided RNase (slicer) that requires extensive complementarity for target cleavage in vitro. Disruption of its catalytic activity in mice by a single point mutation causes male infertility, and mutant germ cells show increased accumulation of LINE1 retrotransposon transcripts. We provide evidence for Miwi slicer activity directly cleaving transposon messenger RNAs, offering an explanation for the continued maintenance of repeat-derived piRNAs long after transposon silencing is established in germline stem cells. Furthermore, our study supports a slicer-dependent silencing mechanism that functions without piRNA amplification. Thus, Piwi proteins seem to act in a two-pronged mammalian transposon silencing strategy: one promotes transcriptional repression in the embryo, the other reinforces silencing at the post-transcriptional level after birth.

Loss of the Mili-interacting Tudor domain-containing protein-1 activates transposons and alters the Mili-associated small RNA profile

Piwi proteins and their associated Piwi-interacting RNAs (piRNAs) are implicated in transposon silencing in the mouse germ line. 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 at 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 that of the wild type. Our data suggest that Tdrd1 ensures the entry of correct transcripts into the normal piRNA pool.

Mouse MOV10L1 associates with Piwi proteins and is an essential component of the Piwi-interacting RNA (piRNA) pathway

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 derepression of retrotransposons in germ cells. The Mov10l1 mutant males are sterile owing to complete meiotic arrest. This mouse mutant expresses Piwi proteins but lacks piRNAs, suggesting that MOV10L1 is required for piRNA biogenesis and/or loading to Piwi proteins.

piRNAs and their involvement in male germline development in mice

Piwi‐interacting RNAs (piRNAs) are a class of small non‐coding RNAs expressed in the animal gonads. They are implicated in silencing the genome instability threat posed by mobile genetic elements called transposons. Unlike other small RNAs, which use double‐stranded precursors, piRNAs seem to arise from long single‐stranded precursor transcripts expressed from discrete genomic regions. In mice, the Piwi pathway is essential for male fertility, and its loss‐of‐function mutations affect several distinct stages of spermatogenesis. While this small RNA pathway primarily operates post‐transcriptionally, it also impacts DNA methylation of target retrotransposon loci, representing an intriguing model of RNA‐directed epigenetic control in mammals. Remarkably the Piwi pathway components are specifically localized at germinal granule/nuage, an evolutionarily conserved but still enigmatic ribonucleoprotein compartment in the germline. The inaccessibility of the germline for easy experimental manipulation has meant that this class of RNAs has remained enigmatic. However, recent advances in the use of cell culture models and cell‐free systems have greatly advanced our understanding. In this review, we briefly summarize our current understanding of the Piwi pathway, focusing on its developmental regulation, piRNA biogenesis and key function in male germline development from fetal spermatogonial stem cell stage to postnatal haploid spermiogenesis in mice.

A Role for Fkbp6 and the Chaperone Machinery in piRNA Amplification and Transposon Silencing

Epigenetic silencing of transposons by Piwi-interacting RNAs (piRNAs) constitutes an RNA-based genome defense mechanism. Piwi endonuclease action amplifies the piRNA pool by generating new piRNAs from target transcripts by a poorly understood mechanism. Here, we identified mouse Fkbp6 as a factor in this biogenesis pathway delivering piRNAs to the Piwi protein Miwi2. Mice lacking Fkbp6 derepress LINE1 (L1) retrotransposon and display reduced DNA methylation due to deficient nuclear accumulation of Miwi2. Like other cochaperones, Fkbp6 associates with the molecular chaperone Hsp90 via its tetratricopeptide repeat (TPR) domain. Inhibition of the ATP-dependent Hsp90 activity in an insect cell culture model results in the accumulation of short antisense RNAs in Piwi complexes. We identify these to be byproducts of piRNA amplification that accumulate only in nuage-localized Piwi proteins. We propose that the chaperone machinery normally ejects these inhibitory RNAs, allowing turnover of Piwi complexes for their continued participation in piRNA amplification.

Tudor domain containing 7 (Tdrd7) is essential for dynamic ribonucleoprotein (RNP) remodeling of chromatoid bodies during spermatogenesis

In the male germline in mammals, chromatoid bodies, a specialized assembly of cytoplasmic ribonucleoprotein (RNP), are structurally evident during meiosis and haploidgenesis, but their developmental origin and regulation remain elusive. The tudor domain containing proteins constitute a conserved class of chromatoid body components. We show that tudor domain containing 7 (Tdrd7), the deficiency of which causes male sterility and age-related cataract (as well as glaucoma), is essential for haploid spermatid development and defines, in concert with Tdrd6, key biogenesis processes of chromatoid bodies. Single and double knockouts of Tdrd7 and Tdrd6 demonstrated that these spermiogenic tudor genes orchestrate developmental programs for ordered remodeling of chromatoid bodies, including the initial establishment, subsequent RNP fusion with ubiquitous processing bodies/GW bodies and later structural maintenance. Tdrd7 suppresses LINE1 retrotransposons independently of piwi-interacting RNA (piRNA) biogenesis wherein Tdrd1 and Tdrd9 operate, indicating that distinct Tdrd pathways act against retrotransposons in the male germline. Tdrd6, in contrast, does not affect retrotransposons but functions at a later stage of spermiogenesis when chromatoid bodies exhibit aggresome-like properties. Our results delineate that chromatoid bodies assemble as an integrated compartment incorporating both germline and ubiquitous features as spermatogenesis proceeds and that the conserved tudor family genes act as master regulators of this unique RNP remodeling, which is genetically linked to the male germline integrity in mammals.

Tudor domain containing 12 (TDRD12) is essential for secondary PIWI interacting RNA biogenesis in mice

Significance Large parts of eukaryotic genomes are composed of transposons. Mammalian genomes use DNA methylation to silence these genomic parasites. A class of small RNAs called Piwi-interacting RNAs (piRNAs) is used to specifically guide the DNA methylation machinery to the transposon DNA elements. How germ cells make piRNAs is not entirely understood. We identify a mouse protein and demonstrate its importance for transposon silencing. We find that the protein collaborates with other factors already implicated in piRNA production. Moreover, the protein is required for piRNA production and assembly of the nuclear silencing complex. Physiological importance of the protein is highlighted by the fact that male mice lacking the protein are infertile. This study will greatly benefit the field of germ-cell biology. Piwi-interacting RNAs (piRNAs) are gonad-specific small RNAs that provide defense 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 unique piRNA biogenesis factor in mice. TDRD12 is detected in complexes containing Piwi protein 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 (reproductive mutant 23 or repro23 mice) or a targeted deletion in the Tdrd12 locus are infertile and derepress retrotransposons. We find that TDRD12 is dispensable for primary piRNA biogenesis but essential for production of secondary piRNAs that enter Piwi protein MIWI2 (PIWIL4). Cell-culture studies with the insect ortholog of TDRD12 suggest a role for the multidomain protein in mediating complex formation with other participants during secondary piRNA biogenesis.

Crystal structure of the primary piRNA biogenesis factor Zucchini reveals similarity to the bacterial PLD endonuclease Nuc

Piwi-interacting RNAs (piRNAs) are a gonad-specific class of small RNAs that associate with the Piwi clade of Argonaute proteins and play a key role in transposon silencing in animals. Since biogenesis of piRNAs is independent of the double-stranded RNA-processing enzyme Dicer, an alternative nuclease that can process single-stranded RNA transcripts has been long sought. A Phospholipase D-like protein, Zucchini, that is essential for piRNA processing has been proposed to be a nuclease acting in piRNA biogenesis. Here we describe the crystal structure of Zucchini from Drosophila melanogaster and show that it is very similar to the bacterial endonuclease, Nuc. The structure also reveals that homodimerization induces major conformational changes assembling the active site. The active site is situated on the dimer interface at the bottom of a narrow groove that can likely accommodate single-stranded nucleic acid substrates. Furthermore, biophysical analysis identifies protein segments essential for dimerization and provides insights into regulation of Zucchinis activity.

PIWI Slicing and EXD1 Drive Biogenesis of Nuclear piRNAs from Cytosolic Targets of the Mouse piRNA Pathway

Summary PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse PIWI protein MILI acts as a trigger to initiate its further 5′→3′ processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by PIWI slicing, and promoted by EXD1, ensures that the same guides instruct PIWI proteins in the nucleus and cytoplasm.