An endogenous siRNA pathway in Drosophila

Watson School of Biological Sciences, Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Nature (Impact Factor: 41.46). 07/2008; 453(7196):798-802. DOI: 10.1038/nature07007
Source: PubMed


Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of approximately 22 nucleotides in length, which arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes. These join Argonaute-1 to regulate gene expression. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs. Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles.

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Available from: Benjamin Czech
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    • "In the latter case, TE transcripts can be processed into a second class of piRNAs that target transcripts of piRNA clusters, thus initiating a so-called ping-pong amplification loop (Brennecke et al. 2007; Gunawardane et al. 2007). Besides piRNAs, another class of small noncoding (snc-) RNAs termed short interfering (si-) RNAs was found to play a crucial role in transposon defense in a variety of organisms (Ketting et al. 1999; Tabara et al. 1999; Sijen et al. 2003; Batista et al. 2008; Chung et al. 2008; Czech et al. 2008; Das et al. 2008; Ghildiyal et al. 2008; Hayashi et al. 2008; Tam et al. 2008; Flemr et al. 2013). In contrast to piRNAs, siRNAs arise from Dicer dependent processing of double-stranded (ds-) RNA precursors (Slotkin and Martienssen 2007). "
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    ABSTRACT: Argonaute proteins comprising Piwi-like and Argonaute-like proteins and their guiding small RNAs combat mobile DNA on the transcriptional and post-transcriptional level. While Piwi-like proteins and associated piRNAs are generally restricted to the germline, Argonaute-like proteins and siRNAs have been linked with transposon control in the germline as well as in the soma. Intriguingly, evolution has realized distinct Argonaute subfunctionalization patterns in different species but our knowledge about mammalian RNA interference pathways relies mainly on findings from the mouse model. However, mice differ from other mammals by absence of functional Piwil3 and expression of an oocyte-specific Dicer isoform. Thus, studies beyond the mouse model are required for a thorough understanding of function and evolution of mammalian RNA interference pathways. We high-throughput sequenced small RNAs from the male Tupaia belangeri germline, which represents a close outgroup to primates, hence phylogenetically links mice with humans. We identified transposon-derived piRNAs as well as siRNAs clearly contrasting the separation of piRNA- and siRNA-pathways into male and female germline as seen in mice. Genome-wide analysis of tree shrew transposons reveal that putative siRNAs map to transposon sites that form foldback secondary structures thus representing suitable Dicer substrates. In contrast piRNAs target transposon sites that remain accessible. With this we provide a basic mechanistic explanation how secondary structure of transposon transcripts influences piRNA- and siRNA-pathway utilization. Finally, our analyses of tree shrew piRNA clusters indicate A-Myb and the testis-expressed transcription factor RFX4 to be involved in the transcriptional regulation of mammalian piRNA clusters. © 2015 Rosenkranz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
    Full-text · Article · Mar 2015 · RNA
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    • "For phenotypic analysis we used (1) the null allele dcr-2[L811fsX] (Lee et al., 2004) in trans to Df[BSC45] (BL#130353), compared to rescue with a dcr-2 fosmid (Kemp et al., 2013), and (2) the null alleles ago2[321] and ago2[454] (Hain et al., 2010) in trans to Df[BSC558] (BL#25120), compared to rescues with a Flag-HA-AGO2 genomic transgene (Czech et al., 2008). To analyze hpRNA1, we generated a deletion of CG4770/hpRNA1/CG4462 by mobilizing Minos [MB01974]. "
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    ABSTRACT: Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs. Importantly, we find broad evidence for evolutionary divergences that preferentially maintain compensatory pairing between hpRNAs and targets, serving as first evidence for adaptive selection for siRNA-mediated target regulation in metazoans. We demonstrate organismal impact of hpRNA activity, since knockout of hpRNA1 derepresses its target ATP synthase-β in testes and compromises spermatogenesis and male fertility. Moreover, we reveal surprising male-specific impact of RNAi factors on germ cell development and fertility, consistent with testis-directed function of the hpRNA pathway. Finally, the collected hpRNA loci chronicle an evolutionary timeline that reflects their origins from prospective target genes, mirroring a strategy described for plant miRNAs. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Dec 2014 · Molecular cell
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    • "summarized to illustrate the compositions of small RNA samples ( Fig . S1A , B in Supplementary data ) . MiRNA is normally ~19e23 nt ( Bartel , 2004 ) , endogenous siRNA is ~21 nt ( Czech et al . , 2008 ) , and piRNA is ~27e30 nt ( Siomi et al . , 2011 ) . MiRNA and endogenous siRNA are similar lengths , however , miRNA has a tendency to begin with uracil ( Fig . S1C , D in Supplementary data ) . We analyzed the first nucleotide bias of small RNAs . The results suggested that majority of the ~19e22 nt small RNAs were miRNA rather than "
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    ABSTRACT: Pyrethroids are the major class of insecticides used for mosquito control. Excessive and improper use of insecticides, however, has resulted in pyrethroid resistance, which has become a major obstacle for mosquito control. The development of pyrethroid resistance is a complex process involving many genes, and information on post-transcription regulation of pyrethroid resistance is lacking. In this study, we extracted RNA from mosquitoes in various life stages (fourth-instar larvae, pupae, male and female adult mosquitoes) from deltamethrin-sensitive (DS) and resistant (DR) strains. Using illumina sequencing, we obtained 13760296 and 12355472 reads for DS-strains and DR-strains, respectively. We identified 100 conserved miRNAs and 42 novel miRNAs derived from 21 miRNA precursors in Culex pipiens. After normalization, we identified 28 differentially expressed miRNAs between the two strains. Additionally, we found that cpp-miR-71 was significant down regulated in female adults from the DR-strain. Based on microinjection and CDC Bottle Bioassay data, we found that cpp-miR-71 may play a contributing role in deltamethrin resistance. The present study provides the firstly large-scale characterization of miRNAs in Cu. pipiens and provides evidence of post-transcription regulation. The differentially expressed miRNAs between the two strains are expected to contribute to the development of pyrethroid resistance.
    Full-text · Article · Nov 2014 · Insect Biochemistry and Molecular Biology
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