Diversity of miRNAs, siRNAs, and piRNAs across 25 <i>Drosophila</i> cell lines

Wen, Jiayu; Mohammed, Jaaved; Bortolamiol-Bécet, Diane; Tsai, Harrison; Robine, Nicolas; Westholm, Jakub Orzechowski; Ladewig, Erik; Dai, Qigen; Okamura, Katsutomo; Flynt, Alex S.; Zhang, Dayu; Andrews, Justen; Cherbas, Lucy; Kaufman, Thomas C.; Cherbas, Peter; Siepel, Adam; Lai, Eric C.

doi:10.1101/gr.161554.113

Cited by 67 publications

(95 citation statements)

References 64 publications

(100 reference statements)

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“…From these annotations, we defined primary-miRNA hairpins as the Drosha-cropped pre-miRNAs, based on dominant mature and star reads, supplemented by 15 nt of sequence on either side to capture the lower stem. To identify tissue-specific miRNAs, we used in-house data (Chung et al 2008;Berezikov et al 2011;Wen et al 2014) and other public small RNA libraries from the Short Read Archive (http://www.ncbi.nlm. nih.gov/sra) (Supplemental Table 1).…”

Section: Methodsmentioning

confidence: 99%

Adaptive evolution of testis-specific, recently evolved, clustered miRNAs inDrosophila

Mohammed

Bortolamiol-Bécet

Flynt

et al. 2014

RNA

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The propensity of animal miRNAs to regulate targets bearing modest complementarity, most notably via pairing with miRNA positions ∼2-8 (the "seed"), is believed to drive major aspects of miRNA evolution. First, minimal targeting requirements have allowed most conserved miRNAs to acquire large target cohorts, thus imposing strong selection on miRNAs to maintain their seed sequences. Second, the modest pairing needed for repression suggests that evolutionarily nascent miRNAs may generally induce net detrimental, rather than beneficial, regulatory effects. Hence, levels and activities of newly emerged miRNAs are expected to be limited to preserve the status quo of gene expression. In this study, we unexpectedly show that Drosophila testes specifically express a substantial miRNA population that contravenes these tenets. We find that multiple genomic clusters of testis-restricted miRNAs harbor recently evolved miRNAs, whose experimentally verified orthologs exhibit divergent sequences, even within seed regions. Moreover, this class of miRNAs exhibits higher expression and greater phenotypic capacities in transgenic misexpression assays than do non-testis-restricted miRNAs of similar evolutionary age. These observations suggest that these testis-restricted miRNAs may be evolving adaptively, and several methods of evolutionary analysis provide strong support for this notion. Consistent with this, proof-of-principle tests show that orthologous miRNAs with divergent seeds can distinguish target sensors in a species-cognate manner. Finally, we observe that testis-restricted miRNA clusters exhibit extraordinary dynamics of miRNA gene flux in other Drosophila species. Altogether, our findings reveal a surprising tissue-directed influence of miRNA evolution, involving a distinct mode of miRNA function connected to adaptive gene regulation in the testis.

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Section: Methodsmentioning

confidence: 99%

Adaptive evolution of testis-specific, recently evolved, clustered miRNAs inDrosophila

Mohammed

Bortolamiol-Bécet

Flynt

et al. 2014

RNA

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“…Previous studies show that Drosophila tissue culture cells have amplified Tn content (Potter et al 1979;Tchurikov et al 1981;Maisonhaute et al 2007;Wen et al 2014) and hypothesize that this amplification is necessary for creating immortal cell lines (Junakovic et al 1988). Once established, Tn location and number appear stable in Drosophila Kc and S2 cell lines (Junakovic et al 1988).…”

Section: Production Of Dsrnas By Convergent Transcription Is a Novel mentioning

confidence: 99%

“…A lighter exposure ( Figure 1F, bottom "L") of the AS transcripts reveals that the 8 kb AS mdg1 transcript is most abundant, while Dm297 and blood AS RNAs are less prevalent, mirroring the RNA-seq data (Figure 1, B-D). S2 culture cells have amplified Tn content (Potter et al 1979;Junakovic et al 1988;Wen et al 2014) and RNA-seq reads cannot be mapped to the S2-specific mdg1 copies as the S2 genome has not been sequenced. Regardless, the data presented here support AS transcription of mdg1{}1720 from non-LTR tss.…”

Section: Ltr Retrotn As Transcription Initiates From Within or Near Ltrsmentioning

confidence: 99%

Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors

2015

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Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active.

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“…Notably, this approach can be applied to small RNA reads from any locus to test whether the locus produces endo-siRNAs. Even in D. melanogaster, comprehensive annotation of siRNA loci using large sequencing data sets has only been performed using cultured cell lines (Wen et al 2014), therefore, additional AGO1-IP small RNA libraries from other tissues using the same set of genotypes would facilitate accurate siRNA gene annotation.…”

Section: Two Classes Of Small Rnas From Rdnamentioning

confidence: 99%

A deeply conserved, noncanonical miRNA hosted by ribosomal DNA

Chak

Mohammed

Lai

et al. 2015

RNA

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Advances in small RNA sequencing technologies and comparative genomics have fueled comprehensive microRNA (miRNA) gene annotations in humans and model organisms. Although new miRNAs continue to be discovered in recent years, these have universally been lowly expressed, recently evolved, and of debatable endogenous activity, leading to the general assumption that virtually all biologically important miRNAs have been identified. Here, we analyzed small RNAs that emanate from the highly repetitive rDNA arrays of Drosophila. In addition to endo-siRNAs derived from sense and antisense strands of the prerRNA sequence, we unexpectedly identified a novel, deeply conserved, noncanonical miRNA. Although this miRNA is widely expressed, this miRNA was not identified by previous studies due to bioinformatics filters removing such repetitive sequences. Deep-sequencing data provide clear evidence for specific processing with precisely defined 5 ′ and 3 ′ ends. Furthermore, we demonstrate that the mature miRNA species is incorporated in the effector complexes and has detectable trans regulatory activity. Processing of this miRNA requires Dicer-1, whereas the Drosha-Pasha complex is dispensable. The miRNA hairpin sequence is located in the internal transcribed spacer 1 region of rDNA and is highly conserved among Dipteran species that were separated from their common ancestor ∼100 million years ago. Our results suggest that biologically active miRNA genes may remain unidentified even in well-studied organisms.

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Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines

Cited by 67 publications

References 64 publications

Adaptive evolution of testis-specific, recently evolved, clustered miRNAs inDrosophila

Adaptive evolution of testis-specific, recently evolved, clustered miRNAs inDrosophila

Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors

A deeply conserved, noncanonical miRNA hosted by ribosomal DNA

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