piRNA Clusters Research Articles

The composition of piRNA clusters in Drosophila melanogaster deviates from expectations under the trap model

BackgroundIt is widely assumed that the invasion of a transposable element (TE) in mammals and invertebrates is stopped when a copy of the TE jumps into a piRNA cluster (i.e., the trap model). However, recent works, which for example showed that deletion of three major piRNA clusters has no effect on TE activity, cast doubt on the trap model.ResultsHere, we test the trap model from a population genetics perspective. Our simulations show that the composition of regions that act as transposon traps (i.e., potentially piRNA clusters) ought to deviate from regions that have no effect on TE activity. We investigated TEs in five Drosophila melanogaster strains using three complementary approaches to test whether the composition of piRNA clusters matches these expectations. We found that the abundance of TE families inside and outside of piRNA clusters is highly correlated, although this is not expected under the trap model. Furthermore, the distribution of the number of TE insertions in piRNA clusters is also much broader than expected.ConclusionsWe found that the observed composition of piRNA clusters is not in agreement with expectations under the simple trap model. Dispersed piRNA producing TE insertions and temporal as well as spatial heterogeneity of piRNA clusters may account for these deviations.

Impact of heat stress on Transposable Element expression and derived small RNAs in Drosophila subobscura.

Global warming is forcing insect populations to move and adapt, triggering adaptive genetic responses. Thermal stress is known to alter gene expression, repressing the transcription of active genes, and inducing others, such as those encoding heat shock proteins. It has also been related to the activation of some specific Transposable Element (TE) families. However, the actual magnitude of this stress on the whole genome and the factors involved in these genomic changes are still unclear. We studied mRNAs and small RNAs in gonads of two Drosophila subobscura populations, considered a good model to study adaptation to temperature changes. In control conditions, we found that a few genes and TE families were differentially expressed between populations, pointing out their putative involvement in the adaptation of populations to their different environments. Under heat stress, sex specific changes in gene expression together with a trend towards overexpression, mainly of heat shock response-related genes were observed. We did not observe large changes of TE expression nor small RNA production due to stress. Only population and sex-specific expression changes of some TE families (mainly retrotransposons), or the amounts of siRNAs and piRNAs, derived from specific TE families were observed, as well as the piRNA production from some piRNA clusters. Changes in small RNA amounts and TE expression could not be clearly correlated, indicating that other factors as chromatin modulation, could also be involved. This work provides the first whole transcriptomic study including genes, TEs and small RNAs after a heat stress in D. subobscura.

The impact of paramutations on the invasion dynamics of transposable elements.

According to the prevailing view, the trap model, the activity of invading transposable elements (TEs) is greatly reduced when a TE copy jumps into a piRNA cluster, which triggers the emergence of piRNAs that silence the TE. One crucial component in the host defence are paramutations. Mediated by maternally deposited piRNAs, paramutations convert TE insertions into piRNA producing loci, thereby transforming selfish TEs into agents of the host defence. Despite this significant effect, the impact of paramutations on the dynamics of TE invasions remains unknown. To address this issue, we performed extensive forward simulations of TE invasions with piRNA clusters and paramutations. We found that paramutations significantly affect TE dynamics, by accelerating the silencing of TE invasions, reducing the number of insertions accumulating during the invasions and mitigating the fitness cost of TEs. We also demonstrate that piRNA production induced by paramutations, an epigenetically inherited trait, may be positively selected. Finally, we show that paramutations may account for three important open problems with the trap model. Firstly, paramutated TE insertions may compensate for the insufficient number of insertions in piRNA clusters observed in previous studies. Secondly, paramutations may explain the discrepancy between the observed and the expected abundance of different TE families in Drosophila melanogaster. Thirdly, piRNA clusters may be crucial to trigger the host defence, but paramutations render the clusters dispensable once the defence has been established. This could account for the lack of TE activation when three major piRNA clusters were deleted in a previous study.

The NSL complex is required for piRNA production from telomeric clusters.

The NSL complex is a transcriptional activator. Germline-specific knockdown of NSL complex subunits NSL1, NSL2, and NSL3 results in reduced piRNA production from a subset of bidirectional piRNA clusters, accompanied by widespread transposon derepression. The piRNAs most transcriptionally affected by NSL2 and NSL1 RNAi map to telomeric piRNA clusters. At the chromatin level, these piRNA clusters also show decreased levels of H3K9me3, HP1a, and Rhino after NSL2 depletion. Using NSL2 ChIP-seq in ovaries, we found that this protein specifically binds promoters of telomeric transposons HeT-A, TAHRE, and TART Germline-specific depletion of NSL2 also led to a reduction in nuclear Piwi in nurse cells. Our findings thereby support a role for the NSL complex in promoting the transcription of piRNA precursors from telomeric piRNA clusters and in regulating Piwi levels in the Drosophila female germline.

Modeling early germline immunization after horizontal transfer of transposable elements reveals internal piRNA cluster heterogeneity

BackgroundA fraction of all genomes is composed of transposable elements (TEs) whose mobility needs to be carefully controlled. In gonads, TE activity is repressed by PIWI-interacting RNAs (piRNAs), a class of small RNAs synthesized by heterochromatic loci enriched in TE fragments, called piRNA clusters. Maintenance of active piRNA clusters across generations is secured by maternal piRNA inheritance providing the memory for TE repression. On rare occasions, genomes encounter horizontal transfer (HT) of new TEs with no piRNA targeting them, threatening the host genome integrity. Naïve genomes can eventually start to produce new piRNAs against these genomic invaders, but the timing of their emergence remains elusive.ResultsUsing a set of TE-derived transgenes inserted in different germline piRNA clusters and functional assays, we have modeled a TE HT in Drosophila melanogaster. We have found that the complete co-option of these transgenes by a germline piRNA cluster can occur within four generations associated with the production of new piRNAs all along the transgenes and the germline silencing of piRNA sensors. Synthesis of new transgenic TE piRNAs is linked to piRNA cluster transcription dependent on Moonshiner and heterochromatin mark deposition that propagates more efficiently on short sequences. Moreover, we found that sequences located within piRNA clusters can have different piRNA profiles and can influence transcript accumulation of nearby sequences.ConclusionsOur study reveals that genetic and epigenetic properties, such as transcription, piRNA profiles, heterochromatin, and conversion efficiency along piRNA clusters, could be heterogeneous depending on the sequences that compose them. These findings suggest that the capacity of transcriptional signal erasure induced by the chromatin complex specific of the piRNA cluster can be incomplete through the piRNA cluster loci. Finally, these results have revealed an unexpected level of complexity that highlights a new magnitude of piRNA cluster plasticity fundamental for the maintenance of genome integrity.

Methylation of lysine 36 on histone H3 is required to control transposon activities in somatic cells.

Transposable elements constitute a substantial portion of most eukaryotic genomes and their activity can lead to developmental and neuronal defects. In the germline, transposon activity is antagonized by the PIWI-interacting RNA pathway tasked with repression of transposon transcription and degrading transcripts that have already been produced. However, most of the genes required for transposon control are not expressed outside the germline, prompting the question: what causes deleterious transposons activity in the soma and how is it managed? Here, we show that disruptions of the Histone 3 lysine 36 methylation machinery led to increased transposon transcription in Drosophila melanogaster brains and that there is division of labour for the repression of transposable elements between the different methyltransferases Set2, NSD, and Ash1. Furthermore, we show that disruption of methylation leads to somatic activation of key genes in the PIWI-interacting RNA pathway and the preferential production of RNA from dual-strand piRNA clusters.

The histone demethylase Kdm3 prevents auto-immune piRNAs production in Drosophila.

Genome integrity of the animal germline is protected from transposable element activity by PIWI-interacting RNAs (piRNAs). While piRNA biogenesis is intensively explored, little is known about the genetical determination of piRNA clusters, the genomic sources of piRNAs. Using a bimodal epigenetic state piRNA cluster (BX2), we identified the histone demethylase Kdm3 as being able to prevent a cryptic piRNA production. In the absence of Kdm3, dozens of coding gene-containing regions become genuine germline dual-strand piRNA clusters. Eggs laid by Kdm3 mutant females show developmental defects phenocopying loss of function of genes embedded into the additional piRNA clusters, suggesting an inheritance of functional ovarian "auto-immune" piRNAs. Antagonizing piRNA cluster determination through chromatin modifications appears crucial to prevent auto-immune genic piRNAs production.

A population genetics theory for piRNA-regulated transposable elements

Transposable elements (TEs) are self-reproducing selfish DNA sequences that can invade the genome of virtually all living species. Population genetics models have shown that TE copy numbers generally reach a limit, either because the transposition rate decreases with the number of copies (transposition regulation) or because TE copies are deleterious, and thus purged by natural selection. Yet, recent empirical discoveries suggest that TE regulation may mostly rely on piRNAs, which require a specific mutational event (the insertion of a TE copy in a piRNA cluster) to be activated — the so-called TE regulation “trap model”. We derived new population genetics models accounting for this trap mechanism, and showed that the resulting equilibria differ substantially from previous expectations based on a transposition–selection equilibrium. We proposed three sub-models, depending on whether or not genomic TE copies and piRNA cluster TE copies are selectively neutral or deleterious, and we provide analytical expressions for maximum and equilibrium copy numbers, as well as cluster frequencies for all of them. In the full neutral model, the equilibrium is achieved when transposition is completely silenced, and this equilibrium does not depend on the transposition rate. When genomic TE copies are deleterious but not cluster TE copies, no long-term equilibrium is possible, and active TEs are eventually eliminated after an active incomplete invasion stage. When all TE copies are deleterious, a transposition–selection equilibrium exists, but the invasion dynamics is not monotonic, and the copy number peaks before decreasing. Mathematical predictions were in good agreement with numerical simulations, except when genetic drift and/or linkage disequilibrium dominates. Overall, the trap-model dynamics appeared to be substantially more stochastic and less repeatable than traditional regulation models.

Chromatin profiling identifies transcriptional readthrough as a conserved mechanism for piRNA biogenesis in mosquitoes

The piRNA pathway in mosquitoes differs substantially from other model organisms, with an expanded PIWI gene family and functions in antiviral defense. Here, we define core piRNA clusters as genomic loci that show ubiquitous piRNA expression in both somatic and germline tissues. These core piRNA clusters are enriched for non-retroviral endogenous viral elements (nrEVEs) in antisense orientation and depend on key biogenesis factors, Veneno, Tejas, Yb, and Shutdown. Combined transcriptome and chromatin state analyses identify transcriptional readthrough as a conserved mechanism for cluster-derived piRNA biogenesis in the vector mosquitoes Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles gambiae. Comparative analyses between the two Aedes species suggest that piRNA clusters function as traps for nrEVEs, allowing adaptation to environmental challenges such as virus infection. Our systematic transcriptome and chromatin state analyses lay the foundation for studies of gene regulation, genome evolution, and piRNA function in these important vector species.

Issue Information

Molecular EcologyVolume 32, Issue 6 p. i-ii ISSUE INFORMATIONFree Access Issue Information First published: 07 March 2023 https://doi.org/10.1111/mec.16512AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Cover Illustrations: (Left to Right): (Image 1): The Blue-faced Honeyeater (Entomyzon cyanotis) is a widespread honeyeater in Australia and exhibits classic patterns of biogeography and genetic differentiation. On p. 1248-1270, Burley et al. use short- and long-read whole genome sequence data to characterize the phylogeography of this species and report on the discovery of a neo-sex chromosome, a first for the honeyeater family (Meliphagidae); (Image 2): A golden takin dancing on the rocks; (Image 3): The hot-spring snake (Thermophis baileyi) is endemic to the Qinghai-Tibet Plateau at elevations above 3600 m; (Image 4): Ancient Daphnia pulicaria resurrected from the South Center Lake (MN, USA ) from an ephippia originally laid down circa 1418-1301 A.D.; (Image 5): Inflorescence of the mangrove species Lumitzera racemosa, a woody plant that has adapted to the extreme intertidal environments; (Image 6): Eelpout Ophthalmolycus amberensis captured in the Gerlache Strait; (Image 7): A breeding pair of Amphilophus xiloensis, one of species that make up the Midas cichlid adaptive radiation. In the picture the female (smaller) is gold colocea. The genetic basis of both troits were uncovered through long- read sequencing. The genetic cause of sex determination is reported in this issue; (Image 8): Morphology of the chasmogamous (CH) and cleistogamous (CL) flowers and petals. Scale bars, 1 cm; (Image 9): Lycaeides butterfly from the Dubois hybrid zone perched on its host plant, alfalfa; (Image 10): Male Xylocopa sonorina on a flower; (Image 11): piRNA clusters are evolving rapidly in Drosophila; (Image 12): Dotplot of the self-alignment of an array of satellite DNA; (Image 13): Haplotype blocks span through time and genomic space; (Image 14): Adults of two sympatric species of Lake Whitefish, Coregonus clupeaformis spp. Normal & Dwarf from Cliff Lake, Maine, USA. Photo Credit: (Left to Right): (Image 1): Angus Emmott; (Image 2): Naxun Zhao; (Image 3): Yu-Fan Wang; (Image 4): Dagmar Frisch; (Image 5): Cairong Zhong; (Image 6): Thomas Desvignes; (Image 7): Ad konings; (Image 8): Congcong Dong; (Image 9): Lauren K. Lucas; (Image 10): Kathy Keatley Garvey; (Image 11): Filip Wierzbicki, Robert Kofler, Sarah Signor; (Image 12): Valentina Peona; (Image 13): N/A; (Image 14): Illustration by Paul Vecsei for Louis Bernatchez. Volume32, Issue6Special Issue: ECOLOGICAL AND EVOLUTIONARY INFERENCES FROM LONG-READ SEQUENCINGMarch 2023Pages i-ii RelatedInformation

Structural insights into the chromodomain of Oxpecker in complex with histone H3 lysine 9 trimethylation reveal a transposon silencing mechanism by heterodimerization

Oxpecker, the homolog of Rhino/HP1D, exclusively expressed in Drosophila ovaries, belongs to the Heterochromatin Protein 1 family, as does Rhino. Rhi recognizes piRNA clusters enriched with the heterochromatin marker H3K9me3 via its N-terminal chromodomain and recruits Deadlock via its C-terminal chromoshadow domain, further recruits Moonshiner, a paralog of the TATA box-binding protein-related factor 2 large subunits, to promote transcription of piRNA precursors, thereby protecting the genome. Despite Oxp possessing only the chromodomain, its loss leads to the upregulation of transposons in the female germline. In this study, we solved the crystal structure of the Oxp chromodomain in complex with the histone H3K9me3 peptide. As the Oxp chromodomain dimerizes, two H3K9me3 peptides bind to the Oxp chromodomain in an antiparallel manner. ITC experiments and site-directed mutagenesis experiments showed that E44 determines Oxp's five-fold stronger binding ability to H3K9me3 than that of Rhi. In addition, we found that Oxp and Rhi can form a heterodimer, which may shed light on the molecular mechanism by which Oxp regulates transposon silencing in the absence of CSD.

The piRNA cluster torimochi is an expanding transposon in cultured silkworm cells.

PIWI proteins and PIWI-interacting RNAs (piRNAs) play a central role in repressing transposable elements in animal germ cells. It is thought that piRNAs are mainly produced from discrete genomic loci named piRNA clusters, which often contain many "dead" transposon remnants from past invasions and have heterochromatic features. In the genome of silkworm ovary-derived cultured cells called BmN4, a well-established model for piRNA research, torimochi was previously annotated as a unique and specialized genomic region that can capture transgenes and produce new piRNAs bearing a trans-silencing activity. However, the sequence identity of torimochi has remained elusive. Here, we carefully characterized torimochi by utilizing the updated silkworm genome sequence and the long-read sequencer MinION. We found that torimochi is in fact a full-length gypsy-like LTR retrotransposon, which is exceptionally active and has massively expanded its copy number in BmN4 cells. Many copies of torimochi in BmN4 cells have features of open chromatin and the ability to produce piRNAs. Therefore, torimochi may represent a young, growing piRNA cluster, which is still "alive" and active in transposition yet capable of trapping other transposable elements to produce de novo piRNAs.

Exposure to elevated temperature affects the expression of PIWI-interacting RNAs and associated transcripts in mouse testes.

Exposure to heat waves could result in adverse effects on human health, especially in male testicles. PIWI-interacting RNA (piRNA) is a novel type of small non-coding RNA, which can notably impact mRNA turnover and preserve germline maintenance in germline cells. However, piRNA's expression status when adapting to testicular heat stress remains largely unclear. To investigate the function and mechanisms of relevant piRNAs during testicular heat stress. In this study, a mouse testicular heat stress model was constructed, and the testes were removed for piRNA-sequencing. Bioinformatics analysis was used to discover the differential expressed piRNAs, piRNA clusters, and enriched pathways. A cell heat stress model was constructed to validate the top five upregulated piRNAs. Proliferation and apoptosis assays were utilized to validate the function of selected piRNA. Bioinformatics prediction, western blotting, and immunohistochemistry were used to illustrate the downstream mechanisms. Through the bioinformatics analysis, we identified the differential expression profile and enriched pathways of piRNAs and piRNA clusters during testicular hyperthermia. Besides, piR-020492 was proved to be upregulated in heat stress mouse testes and a germ cell model. A series of in vitro assays illustrated that an overexpression of piR-020492 could restrain the proliferation and promote the apoptosis of mouse germ cells. Kyoto Encyclopedia of Genes and Genomes analysis of piRNA-generating genes in the testicular heat stress model and piR-020492 targeting genes showed that the overlap pathways are adenosine monophosphate-activated protein kinase (AMPK) and insulin pathways. Validation experiments demonstrated that the key genes of AMPK and insulin pathway exhibit differential expression after an overexpression of piR-020492 or testicular heat stress. In conclusion, our findings revealed the expression profile of piRNAs in testicular heat stress and illustrated the function and mechanisms of piR-020492 in germ cells, which could provide novel insights into the mechanism of hyperthermia-induced testicular injury.

Intermitochondrial cement (IMC) harbors piRNA biogenesis machinery and exonuclease domain-containing proteins EXD1 and EXD2 in mouse spermatocytes.

Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3'-5' domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.

Identification and functional characterization of developmental-stage-dependent piRNAs in Tibetan sheep testes.

The core function of the testes is to produce sperms, which is the prerequisite for maintaining male fertility. PIWI-interacting RNAs (piRNAs) are a class of non-coding small RNAs that are mainly enriched in the reproductive organ and play a key role in germ cell development and spermatogenesis. However, the expression and function of piRNAs in the testes of Tibetan sheep, a domestic animal endemic to the Tibetan Plateau, remain unknown. In this study, we evaluated the sequence structure, expression profile, and potential function of piRNAs in testicular tissues from Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years of age, respectively) by small RNA sequencing. Of the identified piRNAs, the sequence lengths of 24-26 nt and 29 nt dominate. Most piRNA sequences begin with uracil and have a distinct ping-pong structure which mainly distributes in exons, repeat regions, introns, and other unannotated regions of the genome. The piRNAs in the repeat region are primarily derived from the retrotransposons: long terminal repeats, long interspersed nuclear elements, and short interspersed elements. These piRNAs constitute 2,568 piRNA clusters, which mainly distribute on chromosomes 1, 2, 3, 5, 11, 13, 14, and 24, and of these clusters, a total of 529 piRNA clusters were differentially expressed in at least two age groups. Most of the piRNAs were expressed in a low abundance in the testes of developing Tibetan sheep. A total of 41,552 and 2,529 differential piRNAs were identified in testes from 3 months vs. 1 year, and 1 year vs. 3 years, respectively, presenting significantly increased abundance for most piRNAs in 1 year and 3 years compared with 3 months. The functional evaluation of the target genes showed that the differential piRNAs are mainly involved in regulating gene expression, transcription, protein modification, and cell development during spermatogenesis and testicular development. In conclusion, this study focused on the sequence structure and expression characteristics of piRNAs in the testis of Tibetan sheep and provided new insights into the functional mechanism of piRNAs in testicular development and spermatogenesis of sheep.

Diurnal small RNA expression and post-transcriptional regulation in young and old Drosophila melanogaster heads

Background: MicroRNAs are a class of small (~22nt) endogenous RNAs that regulate target transcript expression post-transcriptionally. Previous studies characterized age-related changes in diurnal transcript expression but it is not understood how these changes are regulated, and whether they may be attributed in part to changes in microRNA expression or activity with age. Diurnal small RNA expression changes with age were not previously studied. Methods: To interrogate changes in small RNA expression with age, we collected young (5 day) and old (55 day) Drosophila melanogaster around-the-clock and performed deep sequencing on size-selected RNA from whole heads. Results: We found several microRNAs with changes in rhythmicity after aging, and we investigated microRNAs which are differentially expressed with age. We found that predicted targets of differentially expressed microRNAs have RNA-binding and transcription factor activity. We used a previously published method to identify mRNA transcripts which show evidence of microRNA targeting that is altered after aging, and found several that are involved in muscle development and maintenance. Finally, we identified novel microRNAs using the random-forest-based method miRWoods, which surprisingly also discovered transfer RNA-derived fragments. Conclusions: We showed a decrease in global microRNA expression and a corresponding increase in piRNA expression during aging. We also found an increase in rhythmicity of Drosophila small RNAs during aging, including microRNAs, piRNA clusters, and novel transfer RNA-derived fragments. To our knowledge this is the first study examining diurnal small RNA expression around the clock in young and old Drosophila, and as such it paves the way for future research on changes in small RNA regulatory molecules in the context of aging.

Characteristics of piRNAs and their comparative profiling in testes of sheep with different fertility.

As a novel class of small RNAs, piRNAs are highly expressed in the animal gonads and their main known role is to inhibit transposon activity for ensuring the correctness and integrity of genome. In order to explore the characteristics of piRNAs in sheep testis and their possible regulatory roles on male reproduction, deep sequencing technology was used to sequence small RNAs and identify piRNAs in testes of sheep. The length of piRNAs in sheep testes showed a unimodal distribution between 26 and 31nt, with a peak at 29nt. These piRNAs exhibited obvious ping-pong signature and strand specificity. In the genome, they were mainly aligned to CDS, intron, repetitive sequence regions and unannotated regions. Furthermore, in transposon analysis, piRNAs were aligned predominantly to LINE, SINE, and LTR types of retrotransposon in sheep testes, and the piRNAs derived from each type showed obvious ping-pong signature. The piRNA clusters identified in sheep testes were mainly distributed on chromosomes 3, 7, 15, 17, 18 and 20. The results combining semen determination with pathway enrichment analysis implied that differentially expressed piRNAs between the testes of rams with different fertility might participate in spermatogenesis by regulating multiple pathways closely related to stabilization of blood-testis barrier and renewal and differentiation of spermatogonial stem cell. Taken together, the study provided new insights into the characteristics, origin and expression patterns of piRNAs in sheep testes tissue, which would help us better understand the role of piRNAs in sheep reproduction.

Virome and nrEVEome diversity of Aedes albopictus mosquitoes from La Reunion Island and China

BackgroundAedes albopictus is a public health threat for its worldwide spread and ability to transmit arboviruses. Understanding mechanisms of mosquito immunity can provide new tools to control arbovirus spread. The genomes of Aedes mosquitoes contain hundreds of nonretroviral endogenous viral elements (nrEVEs), which are enriched in piRNA clusters and produce piRNAs, with the potential to target cognate viruses. Recently, one nrEVE was shown to limit cognate viral infection through nrEVE-derived piRNAs. These findings suggest that nrEVEs constitute an archive of past viral infection and that the landscape of viral integrations may be variable across populations depending on their viral exposure.MethodsWe used bioinformatics and molecular approaches to identify known and novel (i.e. absent in the reference genome) viral integrations in the genome of wild collected Aedes albopictus mosquitoes and characterize their virome.ResultsWe showed that the landscape of viral integrations is dynamic with seven novel viral integrations being characterized, but does not correlate with the virome, which includes both viral species known and unknown to infect mosquitoes. However, the small RNA coverage profile of nrEVEs and the viral genomic contigs we identified confirmed an interaction among these elements and the piRNA and siRNA pathways in mosquitoes.ConclusionsMosquitoes nrEVEs have been recently described as a new form of heritable, sequence-specific mechanism of antiviral immunity. Our results contribute to understanding the dynamic distribution of nrEVEs in the genomes of wild Ae. albopictus and their interaction with mosquito viruses.

Identification and Characterization of PIWI-Interacting RNAs in Spinyhead Croakers (Collichthys lucidus) by Small RNA Sequencing

PIWI-interacting RNAs (piRNAs) are an emerging class of small RNAs which protect the animal germline genome against deleterious transposable elements. Nevertheless, the characteristics and sex-related expression patterns of piRNA in Collichthys lucidus remain unknown. In this study, we first performed systematic next-generation high-throughput sequencing in C. lucidus ovaries and testes. We identified 3,027,834 piRNAs across six gonad libraries. Of these, 2225 piRNAs were differently expressed between testes and ovaries; 1195 were upregulated and 1030 downregulated in the testes. Interestingly, the potential target genes of 208 differentially expressed piRNAs had sex-related functions, including germ cell development, gonad development, ovarian follicle development, gamete generation, spermatid development, and spermatogenesis. Moreover, these target genes are involved in the TGF-β, Wnt, MAPK, mTOR, VEGF, and PI3K-Akt pathways. Further, 10 piRNAs were derived from Nectin2 and Mea1, which play important roles in sexual reproduction, male gamete generation, and germ cell development. We also identified 5482 piRNA clusters across the gonads, among which 139 piRNA clusters were uniquely expressed in the testes and 98 in the ovaries. The expression of core sex-related piRNA was validated by real-time PCR. Overall, our findings provide significant insights into C. lucidus’ sex-related piRNAs.

Temperature-dependent Small RNA Expression Depends on Wild Genetic Backgrounds of Caenorhabditis briggsae.

Geographically distinct populations can adapt to the temperature conditions of their local environment, leading to temperature-dependent fitness differences between populations. Consistent with local adaptation, phylogeographically distinct Caenorhabditis briggsae nematodes show distinct fitness responses to temperature. The genetic mechanisms underlying local adaptation, however, remain unresolved. To investigate the potential role of small noncoding RNAs in genotype-specific responses to temperature, we quantified small RNA expression using high-throughput sequencing of C. briggsae nematodes from tropical and temperate strain genotypes reared under three temperature conditions (14 °C, 20 °C, and 30 C). Strains representing both tropical and temperate regions showed significantly lower expression of PIWI-interacting RNAs (piRNAs) at high temperatures, primarily mapping to a large ∼7 Mb long piRNA cluster on chromosome IV. We also documented decreased expression of 22G-RNAs antisense to protein-coding genes and other genomic features at high rearing temperatures for the thermally-intolerant temperate strain genotype, but not for the tropical strain genotype. Reduced 22G-RNA expression was widespread along chromosomes and among feature types, indicative of a genome-wide response. Targets of the EGO-1/CSR-1 22G-RNA pathway were most strongly impacted compared with other 22G-RNA pathways, implicating the CSR-1 Argonaute and its RNA-dependent RNA polymerase EGO-1 in the genotype-dependent modulation of C. briggsae 22G-RNAs under chronic thermal stress. Our work suggests that gene regulation via small RNAs may be an important contributor to the evolution of local adaptations.