PIWI-interacting RNA Biogenesis Research Articles

The absence of core piRNA biogenesis factors does not impact efficient transposon silencing in Drosophila.

Organisms require mechanisms to distinguish self and non-self-RNA. This distinction is crucial to initiate the biogenesis of Piwi-interacting RNAs (piRNAs). In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the 2 known mechanisms to licence an RNA for piRNA biogenesis in the germline and the soma, respectively. Both the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing transposons. However, we find that species closely related to Drosophila melanogaster have lost the yb gene, as well as the PIWI gene Ago3. We show that the precursor RNA is still selected in the absence of Yb to abundantly generate transposon antisense piRNAs in the soma. We further demonstrate that Drosophila eugracilis, which lacks Ago3, is completely devoid of ping-pong piRNAs and exclusively produces phased piRNAs in the absence of slicing. Thus, core piRNA pathway genes can be lost in evolution while still maintaining efficient transposon silencing.

Post-transcriptional regulation in spermatogenesis: all RNA pathways lead to healthy sperm.

Multiple RNA pathways are required to produce functional sperm. Here, we review RNA post-transcriptional regulation during spermatogenesis with particular emphasis on the role of 3' end modifications. From early studies in the 1970s, it became clear that spermiogenesis transcripts could be stored for days only to be translated at advanced stages of spermatid differentiation. The transition between the translationally repressed and active states was observed to correlate with the shortening of the transcripts' poly(A) tail, establishing a link between RNA 3' end metabolism and male germ cell differentiation. Since then, numerous RNA metabolic pathways have been implicated not only in the progression through spermatogenesis, but also in the maintenance of genomic integrity. Recent studies have characterized the elusive 3' biogenesis of Piwi-interacting RNAs (piRNAs), identified a critical role for messenger RNA (mRNA) 3' uridylation in meiotic progression, established the mechanisms that destabilize transcripts with long 3' untranslated regions (3'UTRs) in post-mitotic cells, and defined the physiological relevance of RNA exonucleases and deadenylases in male germ cells. In this review, we discuss RNA processing in the male germline in the light of the most recent findings. A brief recollection of different RNA-processing events will aid future studies exploring post-transcriptional regulation in spermatogenesis.

Disruption of Tdrd5 decouples the stepwise processing of long precursor transcripts during pachytene PIWI-interacting RNA biogenesis.

Disruption of Tdrd5 decouples the stepwise processing of long precursor transcripts during pachytene PIWI-interacting RNA biogenesis.

PNLDC1 is essential for piRNA 3\u2032 end trimming and transposon silencing during spermatogenesis in mice

Piwi-interacting RNAs are small regulatory RNAs with key roles in transposon silencing and regulation of gametogenesis. The production of mature piwi-interacting RNAs requires a critical step of trimming piwi-interacting RNA intermediates to achieve optimally sized piwi-interacting RNAs. The poly(A)-specific ribonuclease family deadenylase PNLDC1 is implicated in piwi-interacting RNA trimming in silkworms. The physiological function of PNLDC1 in mammals remains unknown. Using Pnldc1-deficient mice, here we show that PNLDC1 is required for piwi-interacting RNA biogenesis, transposon silencing, and spermatogenesis. Pnldc1 mutation in mice inhibits piwi-interacting RNA trimming and causes accumulation of untrimmed piwi-interacting RNA intermediates with 3′ end extension, leading to severe reduction of mature piwi-interacting RNAs in the testis. Pnldc1 mutant mice exhibit disrupted LINE1 retrotransposon silencing and defect in spermiogenesis. Together, these results define PNLDC1 as a mammalian piwi-interacting RNA biogenesis factor that protects the germline genome and ensures normal sperm production in mice.

Increasing cell density globally enhances the biogenesis of Piwi-interacting RNAs in Bombyx mori germ cells

Piwi proteins and their bound Piwi-interacting RNAs (piRNAs) are predominantly expressed in the germline and play crucial roles in germline development by silencing transposons and other targets. Bombyx mori BmN4 cells are culturable germ cells that equip the piRNA pathway. Because of the scarcity of piRNA-expressing culturable cells, BmN4 cells are being utilized for the analyses of piRNA biogenesis. We here report that the piRNA biogenesis in BmN4 cells is regulated by cell density. As cell density increased, the abundance of Piwi proteins and piRNA biogenesis factors was commonly upregulated, resulting in an increased number of perinuclear nuage-like granules where Piwi proteins localize. Along with these phenomena, the abundance of mature piRNAs also globally increased, whereas levels of long piRNA precursor and transposons decreased, suggesting that increasing cell density promotes piRNA biogenesis pathway and that the resultant accumulation of mature piRNAs is functionally significant for transposon silencing. Our study reveals a previously uncharacterized link between cell density and piRNA biogenesis, designates cell density as a critical variable in piRNA studies using BmN4 cell system, and suggests the alteration of cell density as a useful tool to monitor piRNA biogenesis and function.

Identification and expression of piwil2 in turbot Scophthalmus maximus, with implications of the involvement in embryonic and gonadal development.

Piwil2, a member of the Argonaute family, is involved in the biogenesis of PIWI-interacting RNAs (piRNAs) and plays an important role in regulating gametogenesis. In the present study, we identified turbot Scophthalmus maximus piwil2 gene, named Smpiwil2, which contained a PAZ domain and a PIWI domain. Sequence comparison, genomic structure and phylogenetic analyses showed that Smpiwil2 is homologous to that of teleosts and tetrapods. The Smpiwil2 transcript showed higher expression in the ovary than in the testis, demonstrating a sexually dimorphic gene expression pattern. In situ hybridization (ISH) showed that Smpiwil2 was expressed in the oogonia and all the stages of oocytes in the ovary as well as in spermatogonia and spermatocytes in the testis. Embryonic expression profile revealed that Smpiwil2 was maternally inherited, and its level was higher from the zygote to the blastula stage and subsequently decreased until hatching. Moreover, a CpG island was predicted to locate in the 5'-flanking region of Smpiwil2 gene, and its methylation levels detected by sodium bisulfite sequencing showed significant disparity between females and males, implying that the sexually dimorphic expression of Smpiwil2 might be regulated by methylation. These results indicated that Smpiwil2 had potentially vital functions in embryonic and gonadal development in this species. In addition, the temporal and sex differences in Smpiwil2 expression indicated that this gene may play different roles in gonadal development of different sexes.

TEtools facilitates big data expression analysis of transposable elements and reveals an antagonism between their activity and that of piRNA genes.

Over recent decades, substantial efforts have been made to understand the interactions between host genomes and transposable elements (TEs). The impact of TEs on the regulation of host genes is well known, with TEs acting as platforms of regulatory sequences. Nevertheless, due to their repetitive nature it is considerably hard to integrate TE analysis into genome-wide studies. Here, we developed a specific tool for the analysis of TE expression: TEtools. This tool takes into account the TE sequence diversity of the genome, it can be applied to unannotated or unassembled genomes and is freely available under the GPL3 (https://github.com/l-modolo/TEtools). TEtools performs the mapping of RNA-seq data obtained from classical mRNAs or small RNAs onto a list of TE sequences and performs differential expression analyses with statistical relevance. Using this tool, we analyzed TE expression from five Drosophila wild-type strains. Our data show for the first time that the activity of TEs is strictly linked to the activity of the genes implicated in the piwi-interacting RNA biogenesis and therefore fits an arms race scenario between TE sequences and host control genes.

Respective Functions of Two Distinct Siwi Complexes Assembled during PIWI-Interacting RNA Biogenesis in Bombyx Germ Cells

PIWI-interacting RNA (piRNA) biogenesis consists of two sequential steps: primary piRNA processing and the ping-pong cycle that depends on reciprocal Slicer-mediated RNA cleavage by PIWI proteins. However, the molecular functions of the factors involved remain elusive. Here, we show that RNAs cleaved by a Bombyx mori PIWI, Siwi, remain bound to the protein upon cleavage but are released by a DEAD box protein BmVasa. BmVasa copurifies with Siwi but not another PIWI BmAgo3. A lack of BmVasa does not affect primary piRNA processing but abolishes the ping-pong cycle. Siwi also forms a complex with BmSpn-E and BmQin. This complex is physically separable from the Siwi/BmVasa complex. BmSpn-E, unlike BmVasa, is necessary for primary piRNA production. We propose a model for piRNA biogenesis, where the BmSpn-E/BmQin dimer binds Siwi to function in primary piRNA processing, whereas BmVasa, by associating with Siwi, ensures target RNA release upon cleavage to facilitate the ping-pong cycle.

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

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.

PIWI-interacting RNAs: from generation to transgenerational epigenetics

Small-RNA-guided gene regulation is a recurring theme in biology. Animal germ cells are characterized by an intriguing small-RNA-mediated gene-silencing mechanism known as the PIWI pathway. For a long time, both the biogenesis of PIWI-interacting RNAs (piRNAs) as well as their mode of gene silencing has remained elusive. A recent body of work is shedding more light on both aspects and implicates PIWI in the establishment of transgenerational epigenetic states. In fact, the epigenetic states imposed by PIWI on targets may actually drive piRNA production itself. These findings start to couple small RNA biogenesis with small-RNA-mediated epigenetics.

MITOPLD Is a Mitochondrial Protein Essential for Nuage Formation and piRNA Biogenesis in the Mouse Germline

MITOPLD is a member of the phospholipase D superfamily proteins conserved among diverse species. Zucchini (Zuc), the Drosophila homolog of MITOPLD, has been implicated in primary biogenesis of Piwi-interacting RNAs (piRNAs). By contrast, MITOPLD has been shown to hydrolyze cardiolipin in the outer membrane of mitochondria to generate phosphatidic acid, which is a signaling molecule. To assess whether the mammalian MITOPLD is involved in piRNA biogenesis, we generated Mitopld mutant mice. The mice display meiotic arrest during spermatogenesis, demethylation and derepression of retrotransposons, and defects in primary piRNA biogenesis. Furthermore, in mutant germ cells, mitochondria and the components of the nuage, a perinuclear structure involved in piRNA biogenesis/function, are mislocalized to regions around the centrosome, suggesting that MITOPLD may be involved in microtubule-dependent localization of mitochondria and these proteins. Our results indicate a conserved role for MITOPLD/Zuc in the piRNA pathway and link mitochondrial membrane metabolism/signaling to small RNA biogenesis.