The role of piRNA and Piwi proteins in regulation of germline development

A new group of small noncoding RNAs of 24-30 nucleotides in length, piRNAs, are mainly expressed in germline cells. They form complexes with Piwi proteins, members of the Argonaute family and unlike other small RNAs they are created without RNase Dicer participation. They are present in male and female germinal cells of numerous animals, from flies to humans. The piRNA biogenesis mechanism is unknown, however, it is postulated that they are formed from long single-stranded RNA precursors coded by repetitive sequences occurring in the genome. A large part of piRNA corresponds to retrotranspozon sequences, which may indicate their participation in silencing the mobile elements and maintaining genome integrity of germinal cells. However, disruption of the piRNA biosynthesis pathway and mutations genes encoding Piwi proteins cause the activation of transpozons and a number of defects in the course of gametogenesis, resulting in reproduction disturbance. In this review, the current state of knowledge on the structure, biogenesis and function of piRNA and their interactions with Piwi proteins is presented.     

Small noncoding RNAs in the germline

Small noncoding RNAs have emerged as potent regulators of gene expression, especially in the germline. We review the biogenesis and regulatory function of three major small noncoding RNA pathways in the germline: The small interfering RNA (siRNA) pathway that leads to the degradation of target mRNAs, the microRNA (miRNA) pathway that mostly represses the translation of target mRNAs, and the newly discovered Piwi-interacting RNA (piRNA) pathway that appears to have diverse functions in epigenetic programming, transposon silencing, and the regulation of mRNA translation and stability. The siRNA and miRNA pathways are present in the germline as well as many somatic tissues, whereas the piRNA pathway is predominantly confined to the germline. Investigation of the three small RNA pathways has started to reveal a new dimension of gene regulation with defining roles in germline specification and development.     

小RNAs在生殖细胞发育过程中的作用

很多动物可以产生具调节作用的小RNAs,根据产生方式和作用机制可以将它们分为三类:微小RNAs(miRNAs)、与Piwi相互作用的RNAs(piRNAs)和内源小干扰RNAs(endo-siRNAs),这些小RNAs可以在生物生殖细胞发育过程中发挥重要作用。其中miRNAs的主要作用是调节蛋白质基因的表达;piRNAs主要的作用是沉默转座因子,但piRNAs主要存在于生殖细胞中;endo-siRNAs则可能具有上述两种主要作用。该文论述了这三种小RNAs在生物生殖细胞发育过程中的作用,同时也讨论了它们在治疗生物不育及其在生物节育方面的应用前景。     

Small RNAs: regulators and guardians of the genome

Small non-coding RNAs comprise several classes and sizes, but all share a unifying function in cellular physiology: epigenetic regulation of gene expression. Here, we review the salient aspects of recent studies on the biogenesis and function of three classes of small RNAs: miRNAs, siRNAs, and piRNAs. Although the mechanisms are becoming clear by which siRNA-triggered mRNA cleavage silences genes, more studies are needed on several issues regarding miRNA-mediated translation repression. Piwi proteins have been suggested to co-operate in amplifying piRNA biogenesis to maintain transposon silencing in the germ line genome, but details of this process are still unknown as well as the functional consequences of piRNA expression at discrete genomic loci.     

Structural insights into piRNA biogenesis

Discovered two decades ago, Piwi-interacting RNAs (piRNAs) play critical roles in gene regulation, transposon element repression, and antiviral defense. Dysregulation of piRNAs has been noted in diverse human diseases including cancers. Recently, extensive studies have revealed that many more proteins are involved in piRNA biogenesis. This review will summarize the recent progress in piRNA biogenesis and functions, especially the molecular mechanisms by which piRNA biogenesis-related proteins contribute to piRNA processing.     

3' end formation of PIWI-interacting RNAs in vitro

PIWI-interacting RNAs (piRNAs) are 23-30 nucleotides small RNAs that act with PIWI proteins to silence transposon activity in animal gonads. In contrast to microRNAs and small interfering RNAs, the biogenesis of piRNAs, including how 3' ends are formed, remains largely unknown. Here, by using lysate from BmN4, a silkworm ovary-derived cell line, we have developed a cell-free system that recapitulates key steps of piRNA biogenesis: loading of long single-stranded precursor RNAs into PIWI proteins with 5'-nucleotide bias, followed by Mg(2+)-dependent 3' to 5' exonucleolytic trimming and 2'-O-methylation at 3' ends. Importantly, 3' end methylation is tightly coupled with trimming and yet is not a prerequisite for determining the mature piRNA length. Our system provides a biochemical framework for dissecting piRNA biogenesis.     

PIWI/piRNA“机器”与雄性生殖细胞发育

PIWI(P-element-induced wimpy testis)蛋白在动物生殖系细胞中特异性表达,为动物生殖细胞发育分化所必需。piRNA(PIWI-interacting RNAs)是最近在动物生殖系细胞中发现的一类非编码小分子RNA,这类小RNA特异性地与PIWI家族蛋白相互作用。PIWI/piRNA"机器"通过沉默转座元件和调控编码mRNA等方式在动物生殖细胞发育分化过程中发挥重要作用。本文围绕PIWI/piRNA"机器"的生物学功能及分子机制,对近期取得的相关研究进展进行了系统性总结。     

GPAT2, a mitochondrial outer membrane protein,in piRNA biogenesis in germline stem cells

piRNA (PIWI-interacting RNA) is a germ cell–specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.     

Mitochondrial protein BmPAPI modulates the length of mature piRNAs

PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons in animal germlines. The molecular mechanisms and components responsible for piRNA biogenesis remain elusive. PIWI proteins contain conserved symmetrical dimethylarginines (sDMAs) that are specifically targeted by TUDOR domain-containing proteins. Here we report that the sDMAs of PIWI proteins play crucial roles in PIWI localization and piRNA biogenesis in Bombyx mori-derived BmN4 cells, which harbor fully functional piRNA biogenesis machinery. Moreover, RNAi screenings for Bombyx genes encoding TUDOR domain-containing proteins identified BmPAPI, a Bombyx homolog of Drosophila PAPI, as a factor modulating the length of mature piRNAs. BmPAPI specifically recognized sDMAs and interacted with PIWI proteins at the surface of the mitochondrial outer membrane. BmPAPI depletion resulted in 3′-terminal extensions of mature piRNAs without affecting the piRNA quantity. These results reveal the BmPAPI-involved piRNA precursor processing mechanism on mitochondrial outer membrane scaffolds.     

Molecular functions of small regulatory noncoding RNA

Recently, using large-scale genomic sequencing, a great number of small noncoding RNAs (ncRNA) has been discovered. Short ncRNAs can be classified into three major classes — small interfering RNA (siRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA). These short ncRNAs ranging from 20 to 300 nt in size are now recognized as a new paradigm of gene regulation for controlling many biological processes. In this paper, we review the biogenesis and recent research on the functions of small regulatory non-coding RNAs and aim at understanding their important functions in living organisms.     

Evolution of animal Piwi-interacting RNAs and prokaryotic CRISPRs

Piwi-interacting RNAs (piRNAs) and CRISPR RNAs (crRNAs) are two recently discovered classes of small noncoding RNA that are found in animals and prokaryotes, respectively. Both of these novel RNA species function as components of adaptive immune systems that protect their hosts from foreign nucleic acids-piRNAs repress transposable elements in animal germlines, whereas crRNAs protect their bacterial hosts from phage and plasmids. The piRNA and CRISPR systems are nonhomologous but rather have independently evolved into logically similar defense mechanisms based on the specificity of targeting via nucleic acid base complementarity. Here we review what is known about the piRNA and CRISPR systems with a focus on comparing their evolutionary properties. In particular, we highlight the importance of several factors on the pattern of piRNA and CRISPR evolution, including the population genetic environment, the role of alternate defense systems and the mechanisms of acquisition of new piRNAs and CRISPRs.     

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.     

Identification of piRNAs in the central nervous system

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs generated by a conserved pathway. Their most widely studied function involves restricting transposable elements, particularly in the germline, where piRNAs are highly abundant. Increasingly, another set of piRNAs derived from intergenic regions appears to have a role in the regulation of mRNA from early embryos and gonads. We report a more widespread expression of a limited set of piRNAs and particularly focus on their expression in the hippocampus. Deep sequencing of extracted RNA from the mouse hippocampus revealed a set of small RNAs in the size range of piRNAs. These were confirmed by their presence in the piRNA database as well as coimmunoprecipitation with MIWI. Their expression was validated by Northern blot and in situ hybridization in cultured hippocampal neurons, where signal from one piRNA extended to the dendritic compartment. Antisense suppression of this piRNA suggested a role in spine morphogenesis. Possible targets include genes, which control spine shape by a distinctive mechanism in comparison to microRNAs.     

tiRNAs: A novel class of small noncoding RNAs that helps cells respond to stressors and plays roles in cancer progression

tRNA-derived stress-induced RNAs (tiRNAs), important components of tRNA-derived fragments, are gaining popularity for their functions as small noncoding RNAs involved in cancer progression. Under cellular stress, tiRNAs are generated when mature tRNA is specifically cleaved by angiogenin and suggested to act as transducers or effectors involved in cellular stress responses. tiRNAs facilitate cells to respond to stresses mainly via reprogramming translation, inhibiting apoptosis, degrading mRNA, and generating stress granules. This review introduces the cellular biogenesis, molecular mechanisms, and biological roles of tiRNAs in stress response and disease regulation. A better understanding of their roles in regulating cancer may provide novel biomarkers or therapeutic targets for diagnosis and treatment.     

A Unique HMG-Box Domain of Mouse Maelstrom Binds Structured RNA but Not Double Stranded DNA

Piwi-interacting piRNAs are a major and essential class of small RNAs in the animal germ cells with a prominent role in transposon control. Efficient piRNA biogenesis and function require a cohort of proteins conserved throughout the animal kingdom. Here we studied Maelstrom (MAEL), which is essential for piRNA biogenesis and germ cell differentiation in flies and mice. MAEL contains a high mobility group (HMG)-box domain and a Maelstrom-specific domain with a presumptive RNase H-fold. We employed a combination of sequence analyses, structural and biochemical approaches to evaluate and compare nucleic acid binding of mouse MAEL HMG-box to that of canonical HMG-box domain proteins (SRY and HMGB1a). MAEL HMG-box failed to bind double-stranded (ds)DNA but bound to structured RNA. We also identified important roles of a novel cluster of arginine residues in MAEL HMG-box in these interactions. Cumulatively, our results suggest that the MAEL HMG-box domain may contribute to MAEL function in selective processing of retrotransposon RNA into piRNAs. In this regard, a cellular role of MAEL HMG-box domain is reminiscent of that of HMGB1 as a sentinel of immunogenic nucleic acids in the innate immune response.