Rapid Intraspecific Evolution of miRNA and siRNA Genes in the Mosquito Aedes aegypti

et al. (2012) Rapid Intraspecific Evolution of miRNA and siRNA Genes in the Mosquito Aedes aegypti. PLoS ONE 7(9): e44198. doi:10.1371/journal.pone.0044198 Rapid Intraspecific Evolution of miRNA and siRNA Genes in the Mosquito Aedes aegypti Scott A. Bernhardt 0 Mark P. Simmons 0 Ken E. Olson 0 Barry J. Beaty 0 Carol D. Blair 0 William C. Black 0 Basil Brooke, National Institute for Communicable Diseases/NHLS, South Africa 0 1 Department of Microbiology, Immunology and Pathology, Colorado State University , Fort Collins , Colorado, United States of America, 2 Department of Biology, Colorado State University , Fort Collins, Colorado , United States of America RNA silencing, or RNA interference (RNAi) in metazoans mediates development, reduces viral infection and limits transposon mobility. RNA silencing involves 21-30 nucleotide RNAs classified into microRNA (miRNA), exogenous and endogenous small interfering RNAs (siRNA), and Piwi-interacting RNA (piRNA). Knock-out, silencing and mutagenesis of genes in the exogenous siRNA (exo-siRNA) regulatory network demonstrate the importance of this RNAi pathway in antiviral immunity in Drosophila and mosquitoes. In Drosophila, genes encoding components for processing exo-siRNAs are among the fastest evolving 3% of all genes, suggesting that infection with pathogenic RNA viruses may drive diversifying selection in their host. In contrast, paralogous miRNA pathway genes do not evolve more rapidly than the genome average. Silencing of exosiRNA pathway genes in mosquitoes orally infected with arboviruses leads to increased viral replication, but little is known about the comparative patterns of molecular evolution among the exo-siRNA and miRNA pathways genes in mosquitoes. We generated nearly complete sequences of all exons of major miRNA and siRNA pathway genes dicer-1 and dicer-2, argonaute-1 and argonaute-2, and r3d1 and r2d2 in 104 Aedes aegypti mosquitoes collected from six distinct geographic populations and analyzed their genetic diversity. The ratio of replacement to silent amino acid substitutions was 1.4 fold higher in dicer-2 than in dicer-1, 27.4 fold higher in argonaute-2 than in argonaute-1 and similar in r2d2 and r3d1. Positive selection was supported in 32% of non-synonymous sites in dicer-1, in 47% of sites in dicer-2, in 30% of sites in argonaute-1, in all sites in argonaute-2, in 22% of sites in r3d1 and in 55% of sites in r2d2. Unlike Drosophila, in Ae. aegypti, both exo-siRNA and miRNA pathway genes appear to be undergoing rapid, positive, diversifying selection. Furthermore, refractoriness of mosquitoes to infection with dengue virus was significantly positively correlated for nucleotide diversity indices in dicer-2. - Competing Interests: The authors have declared that no competing interests exist. RNA silencing, or RNA interference (RNAi), in plants and animals mediates normal growth and development [1,2], controls or eliminates viral infection [3] and limits transposon mobility in both germ line [4] and somatic cells [5,6]. RNA silencing involves small RNAs that are 2130 nucleotides (nt) in length and are divided into three main classes: microRNAs (miRNAs), exogenous and endogenous small interfering RNAs (exo- and endo-siRNAs), and Piwi-interacting RNAs (piRNAs). Much of what we know about RNAi in insects has been elucidated in Drosophila melanogaster, where the biogenesis and regulatory functions of each of the small RNA classes have been separated into distinct pathways [7]. The exo-siRNA pathway has a central role in Drosophila antiviral immunity [8],[9] and is initiated by Dicer-2 (Dcr2). Dcr2 is an RNase III family protein that recognizes cytoplasmic long dsRNA and cleaves it into ,21 bp siRNAs [10,11]. The siRNAs, in association with Dcr2 and the dsRNA-binding protein R2D2, are loaded into a multiprotein RNA-induced silencing complex (RISC), which contains Argonaute-2 (Ago2) [12,13,14]. In the effector stage of the pathway, the RISC unwinds and degrades one of the siRNA strands and retains the other strand as a guide for recognition and sequence-specific cleavage of viral mRNA, mediated by the slicer endonuclease activity of Ago2 [15,16,17,18]. MicroRNAs (miRNAs) are 2223 nt RNA guides that regulate cellular functions such as differentiation and development and metabolic homeostasis. Although only invertebrates have siRNAs, both vertebrates and invertebrates have miRNAs, which are transcribed from the cellular genome as primary miRNAs by RNA polymerase II and are processed sequentially by two distinct endonucleases in the RNase III family, nuclear Drosha and cytoplasmic Dicer 1 (Dcr1), the only ortholog of the dcr gene family in mammals. Dcr1 processes pre-miRNA to imperfectly basepaired duplex miRNA with ,23 nt strands and acts with the dsRNA-binding protein R3D1 to load the miRNA guide strand into an Argonaute-1 (Ago1)-containing RISC [13,19]. Typically miRNAs recognize targets in the 39 non-coding region of cellular mRNAs by imperfect complementarity and inhibit their translation [20]. There is currently a great deal of interest in identifying genes that condition the ability of arthropods to transmit RNA viruses that are pathogenic to humans and domestic animals. Of particular interest is the mosquito Aedes aegypti, which is an important vector of a number of pathogenic arthropod borne viruses (arboviruses), including the dengue viruses (DENV1-4), yellow fever virus and chikungunya virus, and is also a tractable genetic system with which to identify candidate genes [21]. Aedes aegypti is distributed in all subtropical and tropical regions of the world. Most importantly, Ae. aegypti populations demonstrate a great deal of variation in their susceptibility to arboviral infection [22]. Several lines of evidence suggest the importance of the exosiRNA pathway in antiviral immunity in Drosophila and mosquitoes. Drosophila with mutations in or depletion of known exosiRNA pathway components are hypersensitive to RNA virus infections and develop a dramatically increased viral load [9,23,24]. Increases in arboviral replication occur after knockdown of one or more genes in the exo-siRNA pathway [25,26]. siRNAs derived from the infecting virus genome (viRNAs) have been discovered and characterized in infected insects [27,28,29,30]. Many insect pathogenic viruses encode suppressors of RNAi that counteract insect immunity [31]. Noting that interaction between RNA viruses that encode suppressors of RNAi and their insect hosts may lead to a coevolutionary arms race and directional selection on RNAi genes, Obbard et al. [32] undertook a comparative study of the rates of amino acid evolution in exo-siRNA and miRNA pathway components in three species of Drosophila. They showed that among Drosophila species, the ratio of replacement to silent amino acid substitutions (w = KA/KS) among the exo-siRNA genes dcr2, r2d2, and ago2 is much greater than w among their miRNApathway counterparts dcr1, r3d1, and ago1. In (...truncated)