Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus

Yamamoto, Yasuhiro; Watanabe, Toshiaki; Hoki, Yuko; Shirane, Kenjirou; Li, Yufeng; Ichiiyanagi, Kenji; Kuramochi‐Miyagawa, Satomi; Toyoda, Atsushi; Fujiyama, Asao; Oginuma, Masayuki; Suzuki, Hitomi; Sado, Takashi; Nakano, Toru; Sasaki, Hidetada

doi:10.1101/gr.137224.112

Cited by 34 publications

(35 citation statements)

References 48 publications

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“…Of note, modern bornaviruses can be transmitted vertically (Okamoto et al 2003;Kerski et al 2012). EBLN integration into piRNA clusters could thus have resulted in viral silencing in germ cells, similar to the transgene silencing observed after insertion of identical sequences into piRNA clusters (Yamamoto et al 2013). As transcriptional silencing via repressive chromatin modification, rather than post-transcriptional silencing, appears the dominant mechanism of piRNA-mediated silencing, it is notable that modern bornaviruses, unlike most RNA viruses, replicate in the nucleus and interact directly with chromatin (Matsumoto et al 2012).…”

Section: Discussionmentioning

confidence: 94%

piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals

Parrish

Fujino

Shiromoto

et al. 2015

RNA

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Endogenous bornavirus-like nucleoprotein elements (EBLNs) are sequences within vertebrate genomes derived from reverse transcription and integration of ancient bornaviral nucleoprotein mRNA via the host retrotransposon machinery. While species with EBLNs appear relatively resistant to bornaviral disease, the nature of this association is unclear. We hypothesized that EBLNs could give rise to antiviral interfering RNA in the form of PIWI-interacting RNAs (piRNAs), a class of small RNA known to silence transposons but not exogenous viruses. We found that in both rodents and primates, which acquired their EBLNs independently some 25-40 million years ago, EBLNs are present within piRNA-generating regions of the genome far more often than expected by chance alone (P = 8 × 10 −3 -6 × 10 −8 ). Three of the seven human EBLNs fall within annotated piRNA clusters and two marmoset EBLNs give rise to bona fide piRNAs. In both rats and mice, at least two of the five EBLNs give rise to abundant piRNAs in the male gonad. While no EBLNs are syntenic between rodent and primate, some of the piRNA clusters containing EBLNs are; thus we deduce that EBLNs were integrated into existing piRNA clusters. All true piRNAs derived from EBLNs are antisense relative to the proposed ancient bornaviral nucleoprotein mRNA. These observations are consistent with a role for EBLN-derived piRNA-like RNAs in interfering with ancient bornaviral infection. They raise the hypothesis that retrotransposon-dependent virus-to-host gene flow could engender RNA-mediated, sequence-specific antiviral immune memory in metazoans analogous to the CRISPR/Cas system in prokaryotes.

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Section: Discussionmentioning

confidence: 94%

piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals

Parrish

Fujino

Shiromoto

et al. 2015

RNA

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“…It is worth noting that pseudogene-derived endogenous siRNAs have been found in mouse oocytes where they repress transcription from targeted loci (Tam et al 2008;Watanabe et al 2008). Furthermore, it has been shown that insertion of DNA targeting genes into piRNA clusters in flies and mice, leads to their processing into piRNAs and silencing of the targeted genes in trans (Muerdter et al 2012;Yamamoto et al 2013). Therefore, in principle, the pseudogene-derived piRNAs we have identified could target and post-transcriptionally regulate expression of their parent genes in the human male germline.…”

Section: Discussionmentioning

confidence: 99%

The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

Pantano

Jodar

Bak

et al. 2015

RNA

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At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among spermspecific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline.

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“…However, the majority of these mouse models used human L1-based transgenes or constitutively active non-L1 promoters. The current model suggests a sequence-specific interaction between a piRNA and its target for transcriptional (30,31) and posttranscriptional silencing (19,32,33). Thus, we reasoned that an L1 transgene with an endogenous mouse L1 promoter is the best way to model L1 regulation by the piRNA-DNA methylation pathway.…”

Section: Significancementioning

confidence: 99%

Intact piRNA pathway prevents L1 mobilization in male meiosis

Newkirk

Lee

Grandi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The PIWI-interacting RNA (piRNA) pathway is essential for retrotransposon silencing. In piRNA-deficient mice, L1-overexpressing male germ cells exhibit excessive DNA damage and meiotic defects. It remains unknown whether L1 expression simply highlights piRNA deficiency or actually drives the germ-cell demise. Specifically, the sheer abundance of genomic L1 copies prevents reliable quantification of new insertions. Here, we developed a codon-optimized L1 transgene that is controlled by an endogenous mouse L1 promoter. Importantly, DNA methylation dynamics of a single-copy transgene were indistinguishable from those of endogenous L1s. Analysis of Mov10l1 −/− testes established that de novo methylation of the L1 transgene required the intact piRNA pathway. Consistent with loss of DNA methylation and programmed reduction of H3K9me2 at meiotic onset, the transgene showed 1,400-fold increase in RNA expression and consequently 70-fold increase in retrotransposition in postnatal day 14 Mov10l1 −/− germ cells compared with the wildtype. Analysis of adult Mov10l1 −/− germ-cell fractions indicated a stage-specific increase of retrotransposition in the early meiotic prophase. However, extrapolation of the transgene data to endogenous L1s suggests that it is unlikely insertional mutagenesis alone accounts for the Mov10l1 −/− phenotype. Indeed, pharmacological inhibition of reverse transcription did not rescue the meiotic defect. Cumulatively, these results establish the occurrence of productive L1 mobilization in the absence of an intact piRNA pathway but leave open the possibility of processes preceding L1 integration in triggering meiotic checkpoints and germ-cell death. Additionally, our data suggest that many heritable L1 insertions originate from individuals with partially compromised piRNA defense.LINE-1 reporter transgene | meiotic arrest | PIWI-interacting RNA | retrotransposition | spermatogenesis T he bulk of mammalian genomes are made up of transposable elements, the majority of which are retrotransposons (1). Retrotransposons are classified into long-interspersed elements (LINEs), short-interspersed elements (SINEs), and LTR retrotransposons, which collectively account for 43% and 37% of the human and mouse genomes, respectively (2). Retrotransposons amplify in the genome through an RNA intermediate, a process termed retrotransposition. LINEs are autonomous elements. An intact, full-length LINE-1 (L1) encodes two ORFs (i.e., ORF1 and ORF2); both are required for L1 mobilization (3). SINEs are nonautonomous elements and rely on L1's proteins for propagation in the genome (4). LTR retrotransposons are autonomous but appear to be inactivated in the human genome (5). Retrotransposition endangers the integrity of both somatic and germline genomes through insertional mutagenesis. In somatic tissues, both elevated L1 expression and retrotransposition have been strongly associated with many types of human cancers (6, 7). In a few cases, specific retrotransposition events (i.e., insertions) have been determined to drive t...

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Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus

Cited by 34 publications

References 48 publications

piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals

piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals

The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

Intact piRNA pathway prevents L1 mobilization in male meiosis

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