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Piwi protein Miwi is shown to be a small RNA-guided RNase in mice; disrupting the catalytic activity of Miwi results in increased accumulation of LINE1 retrotransposon transcripts and male infertility. Transposon silencing by Piwi proteins The combination of Piwi proteins and their associated Piwi-interacting RNAs (piRNAs) mediates epigenetic transposon silencing in animal germlines. Piwi proteins are predicted to be endonucleases, but the significance of this activity had not been demonstrated in vivo . The laboratories of Dónal O'Carroll and Ramesh Pillai have now made mouse models in which residues expected to be critical for nuclease activity in the three mouse Piwi homologues, Mili, Miwi and Miwi2, are mutated. The mutant mice show phenotypic differences. The Mili and Miwi mutants are defective in piRNA production, transposon silencing and fertility, whereas the Miwi2 mutant has normal piRNA levels, seems to undergo piRNA amplification and silences transposons. These studies highlight distinctions between the murine enzymes responsible for piRNA biogenesis. Repetitive-element-derived Piwi-interacting RNAs (piRNAs) 1 , 2 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 3 , 4 . 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 5 , 6 , 7 . 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.

MicroRNAs (miRNAs) are [approximately]21-nucleotide-long RNA molecules regulating gene expression in multicellular eukaryotes. In metazoa, miRNAs act by imperfectly base-pairing with the 3' untranslated region of target messenger RNAs (mRNAs) and repressing protein accumulation by an unknown mechanism. We demonstrate that endogenous let-7 microribonucleoproteins (miRNPs) or the tethering of Argonaute (Ago) proteins to reporter mRNAs in human cells inhibit translation initiation. M⁷G-cap-independent translation is not subject to repression, suggesting that miRNPs interfere with recognition of the cap. Repressed mRNAs, Ago proteins, and miRNAs were all found to accumulate in processing bodies. We propose that localization of mRNAs to these structures is a consequence of translational repression.

Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5'→3' directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5' ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5'→3' helicase activity, and mutations that abolish this activity also reduce piRNA processing in vivo. Another somatic piRNA pathway factor Yb, an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery.

piRNAs have been implicated in transposon silencing. Tudor domain–containing protein-1 (Tdrd1) is now shown to interact with the mouse Piwi ortholog Mili and be part of a complex that contains Mili-associated piRNAs. Interaction occurs through the N terminus of Mili, which is dimethylated, a modification that promotes Tdrd1 interaction. The Tdrd1 mutant shares phenotypes with the Mili mutant but shows a strong effect on the nature of the small RNA pool associated with Mili. 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.

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell-free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5' cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.

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.

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.

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 retro-transposons 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.

Small RNAs guide nuclear Argonaute proteins to silence genomic target loci via recruitment of factors that lead to formation of repressive heterochromatin. Animal gonads use this pathway to repress transposable elements with PIWI-clade Argonaute proteins and their associated small RNAs called PIWI-interacting RNAs (piRNAs). Four research groups now identify a protein complex that acts as a molecular bridge between the piRNA pathway and the epigenetic silencing machinery.

  In mammals, in contrast, germ granules become discernible at later stages of germ cell differentiation, i.e., in spermatogenesis and oogenesis, and are not asymmetrically segregated. [...]their possible function seems different from those in early embryos of other species. In mice, the MAEL localization is dependent on the Mili function, but not vice versa, similarly to the requirement of Mili for MIWI2 and TDRD9 localizations [10],[18], and then Mael regulates the assembly of MIWI2 and TDRD9 onto piP-bodies. [...]MAEL acts downstream of MILI and upstream of MIWI2-TDRD9 with respect to the subcellular compartmentalization in fetal prospermatogonia in mice.