RNase κ promotes robust piRNA production by generating 2′,3′-cyclic phosphate-containing precursors

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2′,3′-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5′-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5′-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.

Maternally inherited piRNAs direct transient heterochromatin formation at active transposons during early Drosophila embryogenesis

The PIWI-interacting RNA (piRNA) pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. In Drosophila, piRNAs are intergenerationally inherited through the maternal lineage, and this has demonstrated importance in the specification of piRNA source loci and in silencing of I- and P-elements in the germ cells of daughters. Maternally inherited Piwi protein enters somatic nuclei in early embryos prior to zygotic genome activation and persists therein for roughly half of the time required to complete embryonic development. To investigate the role of the piRNA pathway in the embryonic soma, we created a conditionally unstable Piwi protein. This enabled maternally deposited Piwi to be cleared from newly laid embryos within 30 minutes and well ahead of the activation of zygotic transcription. Examination of RNA and protein profiles over time, and correlation with patterns of H3K9me3 deposition, suggests a role for maternally deposited Piwi in attenuating zygotic transposon expression in somatic cells of the developing embryo. In particular, robust deposition of piRNAs targeting roo, an element whose expression is mainly restricted to embryonic development, results in the deposition of transient heterochromatic marks at active roo insertions. We hypothesize that roo, an extremely successful mobile element, may have adopted a lifestyle of expression in the embryonic soma to evade silencing in germ cells.

RNF8 is not required for histone-to-protamine exchange in spermiogenesis

While an E3 ubiquitin ligase, RNF8, was initially reported to be required for histone-to-protamine exchange in spermiogenesis, we subsequently demonstrated that RNF8 is not involved in this process. Nevertheless, reflecting a lingering misunderstanding in the field, a growing number of studies have continued to postulate a requirement for RNF8 in the histone-to-protamine exchange. For example, a recent study claimed that a mouse PIWI protein, MIWI, controls RNF8-mediated histone-to-protamine exchange. Here, confirming our earlier conclusions, we show that RNF8 is required neither for the establishment of histone H4K16 acetylation, which is an initial step in histone removal during spermiogenesis, nor for the incorporation of two protamine proteins, PRM1 and PRM2. Thus, whereas RNF8 mediates ubiquitination of H2A on the sex chromosomes in meiosis, during the prior stage of spermatogenesis, our genetic evidence underscores that RNF8 is not involved in histone-to-protamine exchange.

Maternally inherited piRNAs direct transient heterochromatin formation at active transposons during early Drosophila embryogenesis

The PIWI-interacting RNA (piRNA) pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. In Drosophila, piRNAs are intergenerationally inherited through the maternal lineage, and this has demonstrated importance in the specification of piRNA source loci and in silencing of I- and P-elements in the germ cells of daughters. Maternally inherited Piwi protein enters somatic nuclei in early embryos prior to zygotic genome activation and persists therein for roughly half of the time required to complete embryonic development. To investigate the role of the piRNA pathway in the embryonic soma, we created a conditionally unstable Piwi protein. This enabled maternally deposited Piwi to be cleared from newly laid embryos within 30 minutes and well ahead of the activation of zygotic transcription. Examination of RNA and protein profiles over time, and correlation with patterns of H3K9me3 deposition, suggests a role for maternally deposited Piwi in attenuating zygotic transposon expression in somatic cells of the developing embryo. In particular, robust deposition of piRNAs targeting roo, an element whose expression is mainly restricted to embryonic development, results in the deposition of transient heterochromatic marks at active roo insertions. We hypothesize that roo, an extremely successful mobile element, may have adopted a lifestyle of expression in the embryonic soma to evade silencing in germ cells.

Terminal Modification, Sequence, and Length Determine Small RNA Stability in Animals

ABSTRACTIn animals, piRNAs, siRNAs, and miRNAs silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3′ terminal trimming and 2′-O-methylation. Both trimming and methylation influence piRNA stability. Here, we report that trimming and methylation protect mouse piRNAs from different decay mechanisms. In the absence of 2′-O-methylation, mouse piRNAs with extensive complementarity to long RNAs become unstable. In flies, 2′-O-methylation similarly protects both piRNAs and siRNAs from complementarity-dependent destabilization. Animal miRNAs are unmethylated, and complementarity-dependent destabilization helps explain differences in miRNA decay rates in both mice and flies. In contrast, trimming protects mouse piRNAs from a separate degradation pathway unaffected by target complementarity but sensitive to the 3′ terminal, untrimmed sequence. Because distinct sets of mouse piRNAs are protected by each of these mechanisms, loss of both trimming and 2′-O-methylation causes the piRNA pathway to collapse, demonstrating that these two small RNA modifications collaborate to stabilize piRNAs.Highlights2′-O-methylation protects mouse and fly piRNAs from complementarity-dependent decay2′-O-methylation protects fly siRNAs with extensive complementarity to long RNAsComplementarity to long RNAs predicts the half-life of fly and mouse miRNAsMouse pre-piRNA decay reflects both pre-piRNA sequence and PIWI protein identity

Expression and localization of PIWI proteins in testis and ovary of domestic sheep

The piRNA-PIWI protein complex plays crucial roles in safeguarding the genome against inordinate transposon mobilization and regulation of embryonic development. A previous study indicated the presence of piRNA in sheep reproductive organs. However, the tissue distribution and cellular localization of PIWI proteins in sheep remains unclear. Therefore the present study aimed to explore the expression profiles of mRNAs of mammalian PIWI proteins (PIWIL1, PIWIL2, PIWIL4 and AGO3) in 9 tissues derived from adult male and female sheep. Results showed the expression of PIWIL1, PIWIL2, and PIWIL4 was significantly higher in the testis and ovary than in the other tissues. Immunohistochemistry analysis of testes indicated that each of the 4 proteins had specific cellular localizations, and some of the localizations were different from those of other species. All the proteins were mainly localized in the primary spermatocytes, suggesting that they are crucial for silencing of transposon to guarantee the integrity of the gamete genome during early stage of spermatogenesis. In the ovaries, the PIWI proteins were mainly localized in oocytes from antral follicles and leukocytes in ovarian blood. Our results provide insights to better understand the functions of PIWI proteins during spermatogenesis, oogenesis and immune defence in sheep.

Extensive nuclear gyration and pervasive non-genic transcription during primordial germ cell development in zebrafish

SUMMARYPrimordial germ cells (PGCs) are the precursors of germ cells, which migrate to the genital ridge during early development. Relatively little is known about PGCs after their migration. We studied this post-migratory stage using microscopy and sequencing techniques, and found that many PGC-specific genes, including genes known to induce PGC fate in the mouse, are only activated several days after migration. At this same timepoint, PGC nuclei become extremely gyrated, displaying general opening of chromatin and high levels of transcription. This is accompanied by changes in nuage morphology, expression of large loci, named PERLs, enriched for retro-transposons and piRNAs, and a rise in piRNA biogenesis signatures. Interestingly, no nuclear Piwi protein could be detected at any timepoint, indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post-migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.