37 research outputs found
Regulated dicing of pre-mir-144 via reshaping of its terminal loop.
Although the route to generate microRNAs (miRNAs) is often depicted as a linear series of sequential and constitutive cleavages, we now appreciate multiple alternative pathways as well as diverse strategies to modulate their processing and function. Here, we identify an unusually profound regulatory role of conserved loop sequences in vertebrate pre-mir-144, which are essential for its cleavage by the Dicer RNase III enzyme in human and zebrafish models. Our data indicate that pre-mir-144 dicing is positively regulated via its terminal loop, and involves the ILF3 complex (NF90 and its partner NF45/ILF2). We provide further evidence that this regulatory switch involves reshaping of the pre-mir-144 apical loop into a structure that is appropriate for Dicer cleavage. In light of our recent findings that mir-144 promotes the nuclear biogenesis of its neighbor mir-451, these data extend the complex hierarchy of nuclear and cytoplasmic regulatory events that can control the maturation of clustered miRNAs
Essential and recurrent roles for hairpin RNAs in silencing \u3ci\u3ede novo sex\u3c/i\u3e chromosome conflict in \u3ci\u3eDrosophila simulans\u3c/i\u3e
Meiotic drive loci distort the normally equal segregation of alleles, which benefits their own transmission even in the face of severe fitness costs to their host organism. However, relatively little is known about the molecular identity of meiotic drivers, their strategies of action, and mechanisms that can suppress their activity. Here, we present data from the fruitfly Drosophila simulans that address these questions. We show that a family of de novo, protamine- derived X-linked selfish genes (the Dox gene family) is silenced by a pair of newly emerged hairpin RNA (hpRNA) small interfering RNA (siRNA)-class loci, Nmy and Tmy. In the w[XD1] genetic background, knockout of nmy derepresses Dox and MDox in testes and depletes male progeny, whereas knockout of tmy causes misexpression of PDox genes and renders males sterile. Importantly, genetic interactions between nmy and tmy mutant alleles reveal that Tmy also specifically maintains male progeny for normal sex ratio. We show the Dox loci are functionally polymorphic within D. simulans, such that both nmy-associated sex ratio bias and tmy-associated sterility can be rescued by wild-type X chromosomes bearing natural deletions in different Dox family genes. Finally, using tagged transgenes of Dox and PDox2, we provide the first experimental evidence Dox family genes encode proteins that are strongly derepressed in cognate hpRNA mutants. Altogether, these studies support a model in which protamine-derived drivers and hpRNA suppressors drive repeated cycles of sex chromosome conflict and resolution that shape genome evolution and the genetic control of male gametogenesis
Gene flow mediates the role of sex chromosome meiotic drive during complex speciation
During speciation, sex chromosomes often accumulate interspecific genetic incompatibilities faster than the rest of the genome. The drive theory posits that sex chromosomes are susceptible to recurrent bouts of meiotic drive and suppression, causing the evolutionary build- up of divergent cryptic sex-linked drive systems and, incidentally, genetic incompatibilities. To assess the role of drive during speciation, we combine high-resolution genetic mapping of X-linked hybrid male sterility with population genomics analyses of divergence and recent gene flow between the fruitfly species, Drosophila mauritiana and D. simulans. Our findings reveal a high density of genetic incompatibilities and a corresponding dearth of gene flow on the X chromosome. Surprisingly, we find that a known drive element recently migrated between species and, rather than contributing to interspecific divergence, caused a strong reduction in local sequence divergence, undermining the evolution of hybrid sterility. Gene flow can therefore mediate the effects of selfish genetic elements during speciation
Evolutionary genomics of piRNA mediated transposon silencing in Drosophila
Thesis (Ph. D.)--University of Rochester. Department of Biology, 2016.Transposable elements (TEs) are abundant throughout the genomes of most
living organisms. A recently identified piwi-interacting RNA (piRNA) pathway
has been shown to defend against TEs in the Drosophila germline. This
dissertation research is an evolutionary genomic analysis of the piRNA pathway
in closely related Drosophila species. In Chapter 1, we characterize
the rate of evolution for proteins affecting 26 RNAi knockdown phenotypes,
which include three phenotypes related to regulation of transposon integration.
When RNAi phenotypes are grouped into categories according to cellular
function, we find that genes involved in the greatest number of phenotypic
categories are also significantly more likely to have a history of rapid protein
evolution. Defining pleiotropy using phenotypic categories yields different
results than studies that define pleiotropy based on physical interactions.
In Chapter 2, we characterize the expression diversity of several families
of TEs using piRNAs. Comparative genomic analysis of piRNA expression
finds dynamic changes in expression levels of several families of TEs
in D. melanogaster and the D. simulans clade. Measuring ping-pong activity—
a signature of piRNA amplification finds higher mean piRNA amplification
in D. simulans and D. mauritiana, suggesting increased activity
of several TEs in these species compared to D. melanogaster. Furthermore,
McDonald-Kreitman tests identify three genes in the piRNA pathway as
having experienced recent positive selection in D. simulans, and one gene
each in D. melanogaster and D. mauritiana. All four genes are components
of nuage, which is a macromolecular complex that mediate protein-protein
and protein-RNA interactions. Our results are consistent with a model of
antagonistic co-evolution between TEs and the piRNA pathway.
In Chapter 3, we use single-molecule sequencing to perform a detailed
comparative analysis of the flamenco piRNA cluster in both D. melanogaster
and D. mauritiana. Our analysis reveals that there is very little sequence
homology of flamenco between the two species, indicating rapid turnover
of TE sequence. Furthermore, spatial heterogeneity in piRNA abundance
in the flamenco region identifies two putative piRNA clusters adjacent to
flamenco that may be functionally distinct. This study is the first practical
demonstration of re-constructing the genome sequence of highly repetitive
piRNA clusters in closely related Drosophila species
Additional file 1 of The effects of natural selection across molecular pathways in Drosophila melanogaster
Supplementary Tables. Table S1. The 26 RNAi knockdown phenotypes surveyed in this study. We identify phenotypes that are enriched for proteins that significantly deviate from the genome average in their direction of selection (DoS). Table S2. Details of the 15 Drosophila melanogaster strains used in this study, including database accession numbers and identifiers [51], percent of the reference genome with reads mapped from that strain, and the average read depth across the entire genome assembly. (PDF 103 kb
Evolution of genome structure in the Drosophila simulans species complex
The rapid evolution of repetitive DNA sequences, including satellite DNA, tandem duplications, and transposable elements, underlies phenotypic evolution and contributes to hybrid incompatibilities between species. However, repetitive genomic regions are fragmented and misassembled in most contemporary genome assemblies. We generated highly contiguous de novo reference genomes for the Drosophila simulans species complex (D. simulans, D. mauritiana, and D. sechellia), which speciated ∼250,000 yr ago. Our assemblies are comparable in contiguity and accuracy to the current D. melanogaster genome, allowing us to directly compare repetitive sequences between these four species. We find that at least 15% of the D. simulans complex species genomes fail to align uniquely to D. melanogaster owing to structural divergence—twice the number of single-nucleotide substitutions. We also find rapid turnover of satellite DNA and extensive structural divergence in heterochromatic regions, whereas the euchromatic gene content is mostly conserved. Despite the overall preservation of gene synteny, euchromatin in each species has been shaped by clade- and species-specific inversions, transposable elements, expansions and contractions of satellite and tRNA tandem arrays, and gene duplications. We also find rapid divergence among Y-linked genes, including copy number variation and recent gene duplications from autosomes. Our assemblies provide a valuable resource for studying genome evolution and its consequences for phenotypic evolution in these genetic model species
Rapid evolutionary diversification of the flamenco locus across simulans clade Drosophila species.
Suppression of transposable elements (TEs) is paramount to maintain genomic integrity and organismal fitness. In D. melanogaster, the flamenco locus is a master suppressor of TEs, preventing the mobilization of certain endogenous retrovirus-like TEs from somatic ovarian support cells to the germline. It is transcribed by Pol II as a long (100s of kb), single-stranded, primary transcript, and metabolized into ~24-32 nt Piwi-interacting RNAs (piRNAs) that target active TEs via antisense complementarity. flamenco is thought to operate as a trap, owing to its high content of recent horizontally transferred TEs that are enriched in antisense orientation. Using newly-generated long read genome data, which is critical for accurate assembly of repetitive sequences, we find that flamenco has undergone radical transformations in sequence content and even copy number across simulans clade Drosophilid species. Drosophila simulans flamenco has duplicated and diverged, and neither copy exhibits synteny with D. melanogaster beyond the core promoter. Moreover, flamenco organization is highly variable across D. simulans individuals. Next, we find that D. simulans and D. mauritiana flamenco display signatures of a dual-stranded cluster, with ping-pong signals in the testis and/or embryo. This is accompanied by increased copy numbers of germline TEs, consistent with these regions operating as functional dual-stranded clusters. Overall, the physical and functional diversity of flamenco orthologs is testament to the extremely dynamic consequences of TE arms races on genome organization, not only amongst highly related species, but even amongst individuals
The hpRNA/RNAi Pathway Is Essential to Resolve Intragenomic Conflict in the Drosophila Male Germline
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Gene flow mediates the role of sex chromosome meiotic drive during complex speciation
During speciation, sex chromosomes often accumulate interspecific genetic incompatibilities faster than the rest of the genome. The drive theory posits that sex chromosomes are susceptible to recurrent bouts of meiotic drive and suppression, causing the evolutionary build- up of divergent cryptic sex-linked drive systems and, incidentally, genetic incompatibilities. To assess the role of drive during speciation, we combine high-resolution genetic mapping of X-linked hybrid male sterility with population genomics analyses of divergence and recent gene flow between the fruitfly species, Drosophila mauritiana and D. simulans. Our findings reveal a high density of genetic incompatibilities and a corresponding dearth of gene flow on the X chromosome. Surprisingly, we find that a known drive element recently migrated between species and, rather than contributing to interspecific divergence, caused a strong reduction in local sequence divergence, undermining the evolution of hybrid sterility. Gene flow can therefore mediate the effects of selfish genetic elements during speciation