Small RNA profiling and characterization of piRNA clusters in the adult testes of the common marmoset,a model primate

Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.     

Heterotypic piRNA Ping-Pong requires qin, a protein with both E3 ligase and Tudor domains

piRNAs guide PIWI proteins to silence transposons in animal germ cells. Reciprocal cycles of piRNA-directed RNA cleavage--catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila melanogaster--expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. We show that qin, a piRNA pathway gene whose protein product contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, a perinuclear structure implicated in transposon silencing. In qin mutants, less Ago3 binds Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, and DNA damage accumulates in nurse cells and oocytes. We propose that Qin enforces heterotypic Ping-Pong between Aub and Ago3, ensuring that transposons are silenced and maintaining the integrity of the germline genome.     

piRNA：一类新的非编码小RNA

piRNA（Piwi-interacting RNA）是从哺乳动物生殖细胞中分离得到的一类长度约为30nt的小RNA,并且这种小RNA与PIWI蛋白家族成员相结合才能发挥它的调控作用。目前,越来越多的文献表明piRNA在生殖细胞的生长发育中的调控是由于Piwi-piRNA复合物引起的基因沉默导致的,但由于对piRNA的研究尚处于初级阶段,它的一些具体的功能和生源论尚在研究当中。本文主要综述了piRNA的最新研究进展。     

Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI‐interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl‐arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain‐containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor‐like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon‐derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality‐controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.     

The interplay of transposon silencing genes in the Drosophila melanogaster germline

Complexes of Piwi proteins and Piwi-interacting RNAs (piRNAs) carry out the repression of transposable elements in animal gonads. The Piwi protein clade is represented in D. melanogaster by three members: Piwi, Aub and Ago3. Piwi protein functions in the nuclei of somatic and germinal ovarian cells, whereas Aub and Ago3 are cytoplasmic proteins of germinal cells. Aub and Ago3 interact with each other in the perinuclear nuage organelle to perform piRNA amplification via the ping-pong mechanism. Previously, derepression of several transposable elements as a result of mutations in the piRNA silencing system was shown. Here we quantify the increase in expression level of an enlarged number of retrotransposons due to the mutations in the piwi gene, nuage components coding aub, mael and spn-E genes and the RNA helicase armi gene mutation that impairs Piwi nuclear localization, but not the ping-pong cycle. We reveal that piwi, armi, aub, spn-E and mael genes participate together in the repression of several transposons (HMS-Beagle, Gate and HeT-A), whereas silencing of land G elements requires the same genes except piwi. We suggest that Armi has other functions besides the localizing of Piwi protein in the nuclei. Our data suggest also a role of cytoplasmic Aub, Spn-E and Mael nuage proteins in Piwi-mediated repression of Gate and HMS-Beagle transposons in the germline nuclei. As a whole, our results corroborate the idea that genome stabilization in the germline is realized by different silencing strategies specific for different transposable elements. At the same time, our data suggest the existence of yet unknown mechanisms of interplay between nuclear and cytoplasmic components of the piRNA machinery in the germline.     

Characterization of Sus scrofa Small Non-Coding RNAs Present in Both Female and Male Gonads

Small non-coding RNAs (sncRNAs) are indispensable for proper germ cell development, emphasizing the need for greater elucidation of the mechanisms of germline development and regulation of this process by sncRNAs. We used deep sequencing to characterize three families of small non-coding RNAs (piRNAs, miRNAs, and tRFs) present in Sus scrofa gonads and focused on the small RNA fraction present in both male and female gonads. Although similar numbers of reads were obtained from both types of gonads, the number of unique RNA sequences in the ovaries was several times lower. Of the sequences detected in the testes, 2.6% of piRNAs, 9% of miRNAs, and 10% of tRFs were also present in the ovaries. Notably, the majority of the shared piRNAs mapped to ribosomal RNAs and were derived from clustered loci. In addition, the most abundant miRNAs present in the ovaries and testes are conserved and are involved in many biological processes such as the regulation of homeobox genes, the control of cell proliferation, and carcinogenesis. Unexpectedly, we detected a novel sncRNA type, the tRFs, which are 30–36-nt RNA fragments derived from tRNA molecules, in gonads. Analysis of S. scrofa piRNAs show that testes specific piRNAs are biased for 5′ uracil but both testes and ovaries specific piRNAs are not biased for adenine at the 10^th nucleotide position. These observations indicate that adult porcine piRNAs are predominantly produced by a primary processing pathway or other mechanisms and secondary piRNAs generated by ping-pong mechanism are absent.     

Interplay of transposon-silencing genes in the germline of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Complexes of Piwi family proteins with short piRNAs (Piwi-interacting RNAs) are responsible for silencing transposable elements in animal reproductive organs. In Drosophila melanogaster, three proteins (Piwi, Aub, and Ago3) are members of the Piwi family. Piwi is the nuclear protein of somatic and germinal ovarian cells, whereas Aub and Ago3 are cytoplasmic proteins involved in piRNA amplification in perinuclear granules that constitute special organelles of germinal cells called nuage. Mutations in genes of the piRNA system are known to cause derepression of several transposable elements. In this study, we compared quantitatively changes in expression of a larger number of elements in the case of mutations in the piwi gene, genes aub, mael, and spn-E, which encode proteins of nuage granules, and armi gene coding an RNA helicase, the lack of which does not interfere with cytoplasmic piRNA amplification but disturbs nuclear localization of Piwi protein. We found that the genes piwi, armi, aub, spn-E, and mael interact to induce silencing of some retrotransposons (HMS-Beagle, Gate and HeT-A); the same genes, except piwi, are involved in repression of I and G elements. We propose that Armi is involved in control of not only nuclear Piwi localization. Our data suggest the relation of nuage proteins Aub, Spn-E, and Mael to Piwi-mediated silencing of retrotransposons Gate and HMS-Beagle in the nucleus. In general, our results corroborate the idea of genome stabilization by means of various silencing strategies specific to different transposable elements. At the same time, our data suggest the existence of yet unknown mechanisms of interplay between nuclear and cytoplasmic components of the piRNA machinery in germinal cells.