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Long first exons and epigenetic marks distinguish conserved pachytene piRNA clusters from other mammalian genes

ABSTRACT: In the male germ cells of eutherian animals, 26–30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. These pachytene piRNAs derive from ~100 discrete autosomal loci resemble canonical protein-coding genes and long non-coding RNA-producing genes—they are transcribed by RNA polymerase II, bearing 5´ caps and 3´ poly(A) tails, and their transcripts often contain introns that are removed before nuclear export and processing into piRNAs. However, it is unclear which genic and epigenetic features distinguish pachytene piRNA genes from other types of genes and dictate their germline-specific expression. We report that an unusually long first exon (≥ 10 kb) or a long gene absent of introns altogether is highly correlated with both the germline-specific production of piRNA precursor transcripts from mouse pachytene piRNA loci. We also found that these precursor transcripts are enriched in the binding by THOC1 (also known as HPR1) and THOC2, subunits of the THO complex critical for transcription elongation and nuclear export of mRNAs. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA loci. We further found that a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA loci.

ORGANISM(S): Mus musculus Macaca mulatta

PROVIDER: GSE147724 | GEO | 2020/10/24

REPOSITORIES: GEO

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A heterochromatin-specific RNA export pathway facilitates piRNA production

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2019-03-28 | GSE126578 | GEO

Severe sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA

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2019-08-30 | GSE136603 | GEO

The RNA Polymerase II subunit RPB-9 directs transcriptional elongation at piRNA loci in C. elegans

Project description:PIWI-interacting RNAs (piRNAs) are genomically-encoded small RNAs that regulate germ cell development and guarantee germline integrity. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. To date, piRNA biogenesis mechanisms are still unclear. Here, we show that the RNA Polymerase II subunit RPB-9 is required to promote transcription elongation at piRNA loci. Through genetic and biochemical experiments, we demonstrate that rpb-9-mediated piRNA production is needed to repress two DNA transposon families and a subset of somatic genes in the C. elegans germline.

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Project description:In the fetal mouse testis, PIWI Interacting RNAs (piRNAs) guide PIWI proteins to silence transposons, but after birth, most post-pubertal pachytene piRNAs map to genome uniquely and are thought to regulate genes required for male fertility. In human males, the developmental classes, precise genomic origins, and transcriptional regulation of post-natal piRNAs remain undefined. Here, we demarcate the genes and transcripts that produce post-natal piRNAs in human juvenile and adult testes. As in mouse, A‑MYB in humans drives transcription of both pachytene piRNA precursor transcripts and the mRNAs encoding piRNA biogenesis factors. Although human piRNA genes are syntenic to those in other placental mammals, their sequences are poorly conserved. In fact, pachytene piRNA loci are rapidly diverging even among modern humans. Our findings suggest that during mammalian evolution, pachytene piRNA genes are under fewer selective constraints. We speculate that pachytene piRNA diversity may provide a hitherto unrecognized driver of reproductive isolation.

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Specialization of the Drosophila nuclear export family protein, Nxf3, for piRNA precursor export

Project description:The piRNA pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilisation. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use non-canonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and polyA tails. mRNA processing is important for general mRNA export mediated by Nuclear export factor 1. Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila Nuclear export factor family protein, Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We find that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters bypass canonical mRNA features to be specifically exported via Nxf3, ensuring proper piRNA production

2019-07-29 | PXD014472 | Pride

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Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. In Drosophila ovaries, piRNAs are produced from discrete genomic loci, called piRNA clusters, which are composed of inactive transposon copies and fragments and thus constitute a genetically encoded memory of past transposon challenges. Two types of piRNA clusters exist in flies: dual-strand clusters, expressed only in the germline via a highly specialised machinery, and uni-strand cluster, which are predominantly expressed in the somatic follicle cells. Flamenco (flam) is the major uni-strand piRNA cluster in Drosophila, giving rise to the majority of somatic piRNAs. Flam resembles a canonical RNA polymerase II transcriptional unit, nonetheless it can be specifically recognised by the piRNA pathway and directed to the biogenesis machinery. Recent work has implicated the RNA helicase Yb in the licensing of somatic piRNA production, however a detailed understanding of the molecular mechanisms underlying flam export and specification is still lacking. Here, we show that flam export triggers the assembly of peri-nuclear condensates of Yb and provide evidence that piRNA production from flam specifically requires subunits of the Nuclear Pore Complex (NPC). In the absence of some NPC subunits, transposons become de-silenced and piRNA biogenesis is compromised exclusively from flam. We also show that Yb transiently associates with the NPC to promote flam export. Taken together, our data shed light on how the export of uni-strand cluster transcripts is achieved and suggest the evolution of a specialised machinery that couples transcription, nuclear export and piRNA production.

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Ricochet transcription of pachytene piRNA promoters in mammals

Project description:The origin and stability of germline noncoding RNAs remain largely uncharacterized in mammals. Here, we demonstrate that the vast majority of pachytene piRNAs originate from less than 100 bi- and unidirectionally transcribed loci in the mouse genome. These loci show features characteristic of transcribed protein-coding genes, including primary transcription by RNA polymerase II, H3K4 trimethylation marking the transcription initiation site, and H3K36 trimethylation present at elongation regions in a highly tissue-specific manner. We identified MYBL1, a spermatocyte-enriched transcription factor, as a potential driver of piRNA precursor transcripts. By similarly mapping piRNA clusters in testes of dog and opossum, we revealed that the regulatory architectures generating piRNAs appear to be highly conserved in mammals. Finally, we discovered that apparently neutral chromosomal rearrangements in evolution can act as an unexpected mechanism to generate novel piRNA clusters in diverse mammals. Thus, our results demonstrate how an integrated, multi-species approach can help define the genomic features and evolutionary mechanisms underlying mammalian germline noncoding RNAs.

2015-09-01 | E-MTAB-1265 | biostudies-arrayexpress

Transcriptome analysis during mouse spermatogenic cell development

Project description:In animal germline cells, Piwi-interacting RNAs (piRNAs) silence retrotransposons through post-transcriptional and transcriptional mechanisms. However, little is known, especially in mammals, about the functions of piRNAs beyond retrotransposon suppression1-5. In mammalian spermatocytes, piRNAs are known to be abundantly expressed6-10. Here, we show that a subset of coding and noncoding RNAs in mouse spermatocytes is degraded by the piRNA pathway. By analyzing the germline trasnscriptome of mice deficient in piRNA biogenesis, we identify hundreds of mRNAs as direct targets of piRNAs. Remarkably, the 3' untranslated region (UTR) of the mRNAs up-regulated in the piRNA pathway mutants are highly enriched with retrotransposon sequenes, implying that these sequences serve as regulatory elements for piRNA-mediated regulation. Furthermore, deficiencies of piRNAs derived from pseudogenes result in increased mRNA levels of their cognate genes, indicating that pseudogenes regulate their functional cognates via piRNAs. Moreover, we identify a large population of testis-enriched long intergenic noncoding RNAs (lincRNAs), some of which are also degraded by the piRNA pathway. Collectively, our results reveal that the piRNA pathway regulates the expression of both mRNAs and lincRNAs in addition to retrotransposon RNAs during meiosis and the key role of retrotransposons and pseudogenes, two major types of genomic sequences, in this regulation by acting as piRNA sources and/or regulatory elements in target RNAs. mRNAs in leptotene/zygotene spermatocytes, early-pachytene spermatocytes, mid-pachytene spermatocytes, late pachytene/diplotene spermatocytes and round spermatids were analyzed by deep sequencing using Illumina HiSeq.

2014-11-20 | E-GEOD-41976 | biostudies-arrayexpress

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