Abundant primary piRNAs, endo-siRNAs, and microRNAs in a <i>Drosophila</i> ovary cell line

Lau, Nelson C.; Robine, Nicolas; Martı́n, Raquel; Chung, Wei Jen; Niki, Yuzo; Berezikov, Eugène; Lai, Eric C.

doi:10.1101/gr.094896.109

Cited by 170 publications

(190 citation statements)

References 48 publications

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“…The above conclusion is based on the fact that DUO mutation showed no effect on piRNAs at the population level, which does not rule out the possibility that DUO mutations may affect piRNA biogenesis at specific loci. To test this possibility, we examined whether the slicer activity of Piwi is involved in the biogenesis of 17 known piRNA clusters, which comprise 12 somatic-enriched, two germ-line-enriched, and three ubiquitous clusters (16,24,25). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Cluster analysis. We selected 17 piRNA clusters from different scientific groups, which covers clusters that were expressed preferentially in germ-line, somatic cells, together with the clusters that showed no preference: 15 piRNA clusters reported by Hannon and coworkers (24) to produce the greatest number of piRNAs; 1 piRNA cluster (Traffic Jam) from Siomi and coworkers (16); and 1 piRNA cluster (OSS2) located in Chr2R: 936,722-943,706 from Lau, Lai, and coworkers (25). For this analysis, we only considered uniquely mapped small RNAs, defined as mapping perfectly to only one position in the D. melanogaster Release 5 genome.…”

Section: Methodsmentioning

confidence: 99%

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Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity

Darricarrère

Liu

Watanabe

et al. 2013

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

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The Piwi protein subfamily is essential for Piwi-interacting RNA (piRNA) biogenesis, transposon silencing, and germ-line development, all of which have been proposed to require Piwi endonuclease activity, as validated for two cytoplasmic Piwi proteins in mice. However, recent evidence has led to questioning of the generality of this mechanism for the Piwi members that reside in the nucleus. Drosophila offers a distinct opportunity to study the function of nuclear Piwi proteins because, among three Drosophila Piwi proteins-called Piwi, Aubergine, and Argonaute 3-Piwi is the only member of this subfamily that is localized in the nucleus and expressed in both germ-line and somatic cells in the gonad, where it is responsible for piRNA biogenesis and regulatory functions essential for fertility. In this study, we demonstrate beyond doubt that the slicer activity of Piwi is not required for any known functions in vivo. We show that, in transgenic flies with the DDX catalytic triad of PIWI mutated, neither primary nor secondary piRNA biogenesis is detectably affected, transposons remain repressed, and fertility is normal. Our observations demonstrate that the mechanism of Piwi is independent of its in vitro endonuclease activity. Instead, it is consistent with the alternative mode of Piwi function as a molecule involved in the piRNA-directed guidance of epigenetic factors to chromatin.small RNA | RNase H fold | transposable element | sterility

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity

Darricarrère

Liu

Watanabe

et al. 2013

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

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“…Ovaries were counterstained with DAPI (blue). our libraries, we used an anion exchange chromatography strategy to purify protein-associated small RNAs as previously described (see Supplemental Methods; Lau et al 2009). This approach depletes RNA degradation products and decreases the amount of 2S rRNA, the main Drosophila contaminant, in sequenced libraries (Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

piRNA-mediated transgenerational inheritance of an acquired trait

et al. 2012

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The maintenance of genome integrity is an essential trait to the successful transmission of genetic information. In animal germ cells, piRNAs guide PIWI proteins to silence transposable elements (TEs) in order to maintain genome integrity. In insects, most TE silencing in the germline is achieved by secondary piRNAs that are produced by a feed-forward loop (the pingpong cycle), which requires the piRNA-directed cleavage of two types of RNAs: mRNAs of functional euchromatic TEs and heterochromatic transcripts that contain defective TE sequences. The first cleavage that initiates such an amplification loop remains poorly understood. Taking advantage of the existence of strains that are devoid of functional copies of the LINE-like I-element, we report here that in such Drosophila ovaries, the initiation of a ping-pong cycle is exclusively achieved by secondary I-element piRNAs that are produced in the ovary and deposited in the embryonic germline. This unusual secondary piRNA biogenesis, detected in the absence of functional I-element copies, results from the processing of sense and antisense transcripts of several different defective I-element. Once acquired, for instance after ancestor aging, this capacity to produce heterochromatic-only secondary piRNAs is partially transmitted through generations via maternal piRNAs. Furthermore, such piRNAs acting as ping-pong initiators in a chromatin-independent manner confer to the progeny a high capacity to repress the I-element mobility. Our study explains, at the molecular level, the basis for epigenetic memory of maternal immunity that protects females from hybrid dysgenesis caused by transposition of paternally inherited functional I-element.

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“…DCV and Nora virus belong to different +RNA virus families and both share similarities with picornaviruses. A common source of virus contamination for the two cell lines might be extracts from infected flies used in cell culture (37). BLASTX searches of the remaining assembled contigs did not identify additional viruses.…”

Section: Detection and Assembly Of Virus-derived Pirnas In Drosophilamentioning

confidence: 99%

“…We further carried out assembly of the small RNAs sequenced recently from a Drosophila ovary somatic sheet (OSS) cell line (37). Unlike S2 cells isolated originally from late embryonic stages, which do not express any of the three PIWI subfamily members, OSS cells produce abundant primary piRNAs of 24-30 nucleotides in addition to siRNAs and miRNAs because of the expression of the PIWI protein (37)(38)(39).…”

Section: Detection and Assembly Of Virus-derived Pirnas In Drosophilamentioning

confidence: 99%

Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs

Luo

et al. 2010

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

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433

411

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In response to infection, invertebrates process replicating viral RNA genomes into siRNAs of discrete sizes to guide virus clearance by RNA interference. Here, we show that viral siRNAs sequenced from fruit fly, mosquito, and nematode cells were all overlapping in sequence, suggesting a possibility of using siRNAs for viral genome assembly and virus discovery. To test this idea, we examined contigs assembled from published small RNA libraries and discovered five previously undescribed viruses from cultured Drosophila cells and adult mosquitoes, including three with a positive-strand RNA genome and two with a dsRNA genome. Notably, four of the identified viruses exhibited only low sequence similarities to known viruses, such that none could be assigned into an existing virus genus. We also report detection of virus-derived PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster that have not been previously described in any other host species and demonstrate viral genome assembly from viral piRNAs in the absence of viral siRNAs. Thus, this study provides a powerful culture-independent approach for virus discovery in invertebrates by assembling viral genomes directly from host immune response products without prior virus enrichment or amplification. We propose that invertebrate viruses discovered by this approach may include previously undescribed human and vertebrate viral pathogens that are transmitted by arthropod vectors.arboviruses | piRNAs | siRNAs | viral immunity | massively parallel sequencing T he Dicer family of host immune receptors mediates antiviral immunity in fungi, plants, and invertebrate animals by RNA interference (RNAi) or RNA silencing (1-3). In this immunity, a viral dsRNA is recognized by Dicer and diced into siRNAs. These virus-derived siRNAs are then loaded into an RNA silencing complex to act as specificity determinants and to guide slicing of the target viral RNAs by an Argonaute protein (AGO) present in the complex. Dicer proteins typically contain an RNA helicase domain, a PAZ domain shared with AGOs, and two tandem type III endoribonuclease (RNase III) domains. Dicer cleaves dsRNA with a simple preference toward a terminus of dsRNA, producing duplex small RNA fragments of discrete sizes progressively from the terminus (4).In addition to siRNAs, micro-RNAs (miRNAs) and PIWIinteracting RNAs (piRNAs) guide RNA silencing in similar complexes but with distinct AGOs (4-6). In Drosophila melanogaster, miRNAs and siRNAs are predominantly 22 and 21 nucleotides in length, dependent on Dicer-1 (DCR1) and DCR2 for their biogenesis, and act in silencing complexes containing AGO1 and AGO2 in the AGO subfamily, respectively (4-6). In contrast, ∼24-30-nt piRNAs are Dicer-independent and require AGO3, Aubergine (AUB), and PIWI in the PIWI subfamily for their biogenesis (4-6). Genetic analyses (7-10) have clearly demonstrated a role for D. melanogaster DCR2 in the immunity and biogenesis of viral siRNAs targeting diverse positive-strand (+) RNA viruses, including Flock house virus (FHV), cricket par...

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Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line

Cited by 170 publications

References 48 publications

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity

piRNA-mediated transgenerational inheritance of an acquired trait

Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs

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