Biology subjects

Flynt, A. S.

Publications and source records attributed to Flynt, A. S..

11 recordsLinked to original sources

bioRxiv2025

Recurrent expansion and rapid evolution of the Drosophilid RNAi pathway in testis

Multiple classes of selfish genetic elements, including transposable elements, meiotic drivers, and viruses, are suppressed by small interfering RNAs (siRNAs) that guide RNA interference (RNAi). These are interlocked within continually evolving genetic conflicts that are characterized by extremely rapid dynamics of change in both sequence and copy number. Indeed, it was shown that across Drosophila, the core RNAi machinery evolves under positive selection, and that the RNAi effector AGO2 has additional copies in some species. This contrasts with the core microRNA (miRNA) machinery in Drosophila, which evolves under negative selection and maintains one-to-one orthologs not only amongst flies, but even to mammals. Here, we analyze >300 long read genome assemblies of Drosophila species to generalize these attributes. Not only do we find recurrent expansion of AGO2 across [~]100 species, including lineages with ongoing amplification of AGO2, we also find dozens of species with extra copies of the other core RNAi factors, r2d2 and dicer-2. In many cases, these additional RNAi factor copies co-exist, or are nested, within a lineage bearing an ancestral expansion of AGO2. Transcriptome data provide evidence that core RNAi factors, including certain lineage-specific copies, are biased for testis expression. Finally, we use small RNA data to annotate hairpin RNAs (hpRNAs) in D. pseudoobscura, one of the species with prominent amplification of RNAi factors. We find rampant de novo hpRNA loci in this species, whose siRNAs are predominantly expressed in testis. All together, these findings highlight evolutionary plasticity of the fly RNAi pathway and affirm that it is preferentially deployed in the germline of the heterogametic sex. When considered alongside abundant genetic data for recurrent sex ratio meiotic drive against the Y chromosome, these genomic data strongly imply that a fundamental role of endogenous RNAi is to control sex chromosome conflicts.

evolutionary biology↗

bioRxiv2025

Implications of Endogenous Small Regulatory RNAs on Gene Silencing in Mollusks

Mollusks are an abundant group of animals with many economically important members that are phylogenetically distinct from nearly all genetic model organisms. This study provides clade-wide evaluation of sRNA biogenesis pathways, with emphasis on the easter oyster, Crassostrea virginica. Understanding these molecules prescribes RNAi-based gene silencing approaches, benefiting genetic investigation and biotechnology. Similar to other animal groups, mollusks have conserved microRNAs (miRNAs) with some shared with ecdysozoans and deuterostomes; however, there was no evidence of an endogenous small-interfering RNA (siRNA) pathway. These results suggest that long double-stranded RNA (dsRNA)-based RNAi is not appropriate for gene silencing in Mollusks as well as other members of the broader Lophotrochozoan clade. The study also finds an abundance of piwi-interacting RNAs (piRNAs) in both soma and gonads. Differences are also found in piRNA biology. Many invertebrates exhibit somatic piRNAs; however, mollusk piRNAs appear to be restricted to a subset of cells, limiting the potential of piRNA-based RNAi. Further, individual animals also express a unique collection of piRNAs that seem to be only partially determined through inheritance from both parents. Together this work defines the RNAi mechanisms in mollusks, which represent 23% of animals, and provides insights into the phenotypic diversity seen in this group. Significance StatementThis study provides an extensive, clade-wide evaluation of small RNA (sRNA) biogenesis pathways in mollusks. Our findings reveal that, unlike ecdysozoans and deuterostomes, mollusks lack a functional siRNA pathway, which fundamentally changes expectations around RNA interference (RNAi) applications in Mollusca. Instead, we find the expected microRNAs and an assortment of piwi-interacting RNAs (piRNAs). We show that piRNA biology in mollusks is highly cell-type specific and genetically individualized. We further demonstrate that piRNA expression is likely linked to stem-like, quiescent cells, suggesting a critical role in genomic maintenance. This work offers insight into RNAi potential in mollusks, the second largest animal phylum, and has significant implications for both basic biology and applied sciences such as pest control and aquaculture.

evolutionary biology↗

bioRxiv2025

Processing of Genomic RNAs by Dicer in Bat Cells Limits SARS-CoV-2 Replication

Bats are reservoirs for numerous viruses that cause serious diseases in other animals and humans. Several mechanisms are proposed to contribute to the tolerance of bats to these pathogens. This study investigates the response of bat cells to double-stranded RNA generated by SARS-CoV-2 replication. Here, we found the involvement of Dicer in the processing of viral genomic RNAs during SARS-CoV-2 infection. Examining RNA sequencing of infected cells, small-interfering RNA (siRNA)-like fragments were found derived from viral RNAs. Depletion of Dicer showed a reduction in these RNAs and an increase in viral loads suggesting unlike other mammals, bats may use Dicer to limit viral replication. This prompted the exploration of key dsRNA sensors in bat cells. Our analysis showed significant upregulation of OAS1 and MX1 in response to dsRNA, while PKR levels remained low, suggesting alternative dsRNA-response mechanisms are present that eschew the common PKR-based system. These results further show how bats employ distinct strategies for antiviral defense that may contribute to tolerating viral infections. They suggest the involvement of Dicer in antiviral mechanisms in bats, a function not observed in other mammals. This highlights a mechanism for bat originating viruses to evolve features that in other animals could cause extreme antiviral responses such as is seen with SARS-CoV-2. GRAPHICAL SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/632425v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1755d68org.highwire.dtl.DTLVardef@475cb8org.highwire.dtl.DTLVardef@1185813org.highwire.dtl.DTLVardef@13e847a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

MiSiPi-Rna: an integrated tool for characterizing small regulatory RNA processing

RNA interference (RNAi) is mediated by small (20-30 nucleotide) RNAs that are produced by complex processing pathways. In animals, three main classes are recognized: microRNAs (miRNAs), small-interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs). Understanding of small RNA pathways has benefited from genetic models where key enzymatic events were identified that lead to stereotypical positioning of small RNAs relative to precursor transcripts. Increasingly there is interest in using RNAi in non-model systems due to ease of generating synthetic small RNA precursors for research and biotechnology. Unfortunately, small RNAs are often rapidly evolving, requiring investigation of a species endogenous small RNAs prior to deploying an RNAi approach. This can be accomplished through small non-coding RNA sequencing followed by applying various computational tools; however, the complexity and separately maintained packages lead to significant challenges for annotating global small RNA populations. To address this need, we developed a simple and efficient R package (MiSiPi-Rna) which can be used to characterize pre-selected loci with plots and statistics, aiding researchers understanding RNAi biology specific to their target species. Additionally, MiSiPi-Rna pioneers several computational approaches to identifying Dicer processing to assist annotation of miRNA and siRNA.

bioinformatics↗

bioRxiv2023

Gene Expression and Evolution in the Smalltooth Sawfish, Pristis Pectinata

Sawfishes (Pristidae) are large, highly threatened rays named for their tooth-studded rostrum, which is used for prey sensing and capture. Of all five species, the smalltooth sawfish, Pristis pectinata, has experienced the greatest decline in range, currently found in only [~]20% of its historic range. To better understand the genetic underpinnings of these taxonomically and morphologically unique animals, we collected transcriptomic data from several tissue types, mapped them to the recently completed reference genome and contrasted the patterns observed with comparable data from other elasmobranchs. Evidence of positive selection was detected in 79 genes in P. pectinata, several of which are involved in growth factor/receptor tyrosine kinase signaling and specification of organ symmetry, suggesting a role in morphogenesis. Data acquired also allow for examination of the molecular components of P. pectinata electrosensory systems, which are highly developed in sawfishes and have likely been influential in their evolutionary success.

evolutionary biology↗

bioRxiv2022

Endogenous RNAi silences a burgeoning sex chromosome arms race

Although the biological utilities of endogenous RNAi (endo-RNAi) have been largely elusive, recent studies reveal its critical role in the non-model fruitfly Drosophila simulans to suppress selfish genes, whose unchecked activities can severely impair spermatogenesis. In particular, hairpin RNA (hpRNA) loci generate endo-siRNAs that suppress evolutionary novel, X-linked, meiotic drive loci. The consequences of deleting even a single hpRNA (Nmy) in males are profound, as such individuals are nearly incapable of siring male progeny. Here, comparative genomic analyses of D. simulans and D. melanogaster mutants of the core RNAi factor dcr-2 reveal a substantially expanded network of recently-emerged hpRNA-target interactions in the former species. The de novo hpRNA regulatory network in D. simulans bears compelling signatures of sex chromosome conflict and provides insight into molecular strategies that underlie hpRNA emergence. In particular, our data support the existence of ongoing rapid evolution of Nmy/Dox-related networks, recurrent targeting of testis HMG Box loci by hpRNAs, and connections to the piRNA pathway. Importantly, the impact of the endo-RNAi network on gene expression flips the convention for regulatory networks, since we observe strong derepression of targets of the youngest hpRNAs, but only mild effects on the targets of the oldest hpRNAs. These data suggest that endo-RNAi are especially critical during incipient stages of intrinsic sex chromosome conflicts, and that continual cycles of distortion and resolution may contribute to the segregation of species.

evolutionary biology↗

bioRxiv2022

Identification of microRNAs in the Lyme disease vector Ixodes scapularis

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in many biological processes, including the immune pathways that control bacterial, parasitic, and viral infections. Pathogens probably modify host miRNAs to facilitate successful infection, so they might be useful targets for vaccination strategies. There are little data on differentially expressed miRNAs in the black- legged tick Ixodes scapularis after infection with Borrelia burgdorferi, the causative agent of Lyme disease in the United States. Small RNA sequencing and qRT-PCR analysis were used to identify and validate differentially-expressed I. scapularis salivary miRNAs. Small RNA-seq yielded 133,465,828 ([≥]18 nucleotides) and 163,852,135 ([≥]18 nucleotides) small RNA reads from Borrelia- infected and uninfected salivary glands for downstream analysis using the miRDeep2 algorithm. Two hundred and fifty-four miRNAs were identified across all datasets, 25 of which were high confidence and 51 low confidence known miRNAs. Twenty-three miRNAs were differentially expressed in uninfected and infected salivary glands: 11 were upregulated and 12 were downregulated upon pathogen infection. This study provides new insights into the miRNAs expressed in I. scapularis salivary glands and pave the way for their functional manipulation to prevent or treat B. burgdorferi infection.

molecular biology↗

bioRxiv2021

Human 5p-tailed Mirtrons are Processed by RNaseP

Approximately a thousand microRNAs (miRNAs) are documented from human cells. A third appear to transit non-canonical pathways that typically bypass processing by Drosha, the dedicated nuclear miRNA producing enzyme. The largest class of non-canonical miRNAs are mirtrons which eschew Drosha to mature through spliceosome activity. While mirtrons are found in several configurations, the vast majority of human mirtron species are 5-tailed. For these mirtrons, a 3 splice site defines the 3 end of their hairpin precursor while a "tail" of variable length separates the 5 base of the hairpin from the nearest splice site. How this tail is removed is not understood. Here we examine sequence motifs in 5-tailed mirtrons and interactions with RNA turnover processes to characterize biogenesis processes. Through studying the high confidence 5-tailed mirtron, hsa-miR-5010, we identify RNaseP as necessary and sufficient for "severing" the 5 tail of this mirtron. Further, depletion of RNaseP activity globally decreased 5-tailed mirtron expression implicating this endoribonuclease in biogenesis of the entire class. Moreover, as 5-tailed mirtron biogenesis appears to be connected to tRNA processing we found a strong correlation between accumulation of tRNA fragments (tRFs) and 5-tailed mirtron abundance. This suggests that dysregulation of tRNA processing seen in cancers may also impact expression of the [~]400 5-tailed mirtrons encoded in the human genome. SUMMARYAbundant non-canonical human miRNAs referred to as tailed mirtrons are processed by RNaseP, which "severs" tail nucleotides to yield a precursor hairpin suitable for Dicer processing. Biogenesis of these miRNAs is correlated with tRFs, which are also products of RNaseP processing.

molecular biology↗

bioRxiv2021

The effect of root exudates on the transcriptome of rhizosphere Pseudomonas spp.

Plants live in association with microorganisms that positively influence plant development, vigor, and fitness in response to pathogens and abiotic stressors. The bulk of the plant microbiome is concentrated belowground at the plant root-soil interface. Plant roots secrete carbon-rich rhizodeposits containing primary and secondary low molecular-weight metabolites, lysates, and mucilages. These exudates provide nutrients for soil microorganisms and modulate their affinity to host plants, but molecular details of this process are largely unresolved. We addressed this gap by focusing on the molecular dialogue between eight well-characterized beneficial strains of the Pseudomonas fluorescens group and Brachypodium distachyon, a model for economically important food, feed, forage, and biomass crops of the grass family. We collected and analyzed root exudates of B. distachyon and demonstrated the presence of multiple carbohydrates, amino acids, organic acids and phenolic compounds. The subsequent screening of bacteria by Biolog Phenotype MicroArrays revealed that many of these metabolites provide carbon and energy for the Pseudomonas strains. RNA-seq profiling of bacterial cultures amended with root exudates revealed changes in the expression of genes encoding numerous catabolic and anabolic enzymes, transporters, transcriptional regulators, stress response, and conserved hypothetical proteins. Almost half of the differentially expressed genes mapped to the variable part of the strains pangenome, reflecting the importance of the variable gene content in the adaptation of P. fluorescens to the rhizosphere lifestyle. Our results collectively reveal the diversity of cellular pathways and physiological responses underlying the establishment of mutualistic interactions between these beneficial rhizobacteria and their plant hosts.

microbiology↗

bioRxiv2019

Assessing Environmental RNAi in a Non-Model Organism

RNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation in invertebrates and has applications that includes control of pests. Successful RNAi technologies are dependent on delivery method. The most convenient method is likely feeding which is effective in some animals while others are insensitive. Thus, there is a need to develop RNAi technology on a per-species basis, which will require a comprehensive approach for assessing small RNA production from synthetic nucleic acids. Using a biochemical and sequencing approaches we investigated the metabolism of ingested RNAs using the two-spotted spider mite, Tetranychus urticae, as a model for RNAi insensitivity. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. To identify RNAi substrates in T. urticae we characterized processing of synthetic RNAs and those derived from plant transcripts ingested during feeding. Through characterization of read size length and overlaps of small RNA reads, visualization methods were developed that facilitate distinguish trans-acting small RNAs from degradation fragments. Using a strategy that delineates small RNA classes, we found a variety of RNA species are gated into spider mite RNAi pathways, however, potential mature trans-acting RNAs appear very unstable and rare. This suggests spider mite RNAi pathway products that originate as ingested materials may be preferentially metabolized instead of converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers.

bioinformatics↗

bioRxiv2017

Deep experimental profiling of microRNA diversity, deployment, and evolution across the Drosophila genus

Comparative genomic analyses of microRNAs (miRNAs) have yielded myriad insights into their biogenesis and regulatory activity. While miRNAs have been deeply annotated in a small cohort of model organisms, evolutionary assessments of miRNA flux are clouded by the functional uncertainty of orthologs in related species, and insufficient data regarding the extent of species-specific miRNAs. We address this by generating a comparative small RNA (sRNA) catalog of unprecedented breadth and depth across the Drosophila genus, extending our extant deep analyses of D. melanogaster with sRNA data from multiple tissues of 11 other fly species. Aggregate analysis of several billion sRNA reads permits curation of accurate and holistic compendia of miRNAs across this genus, providing abundant opportunities to identify species- and clade-specific variation in miRNA identity, abundance, and processing. Amongst well-conserved miRNAs, we observe unexpected cases of clade-specific variation in 5' end precision, occasional antisense loci, and some putatively non-canonical loci. We also employ strict criteria to identify a massive set (649) of novel, evolutionarily-restricted miRNAs. Amongst the bulk collection of species-restricted miRNAs, two notable subpopulations of rapidly-evolving miRNAs are splicing-derived mirtrons and testis-restricted, clustered (TRC) canonical miRNAs. We quantify rates of miRNA birth and death using our annotation and a phylogenetic model for estimating rates of miRNA turnover in the presence of annotation uncertainty. We show striking differences in birth and death rates across miRNA classes defined by biogenesis pathway, genomic clustering, and tissue restriction, and even identify variation heterogeneity amongst Drosophila clades. In particular, distinct molecular rationales underlie the distinct evolutionary behavior of different miRNA classes. We broaden observations made from D. melanogaster as Drosophilid-wide principles for opposing evolutionary viewpoints for miRNA maintenance. Mirtrons are associated with a high rate of 3' untemplated addition, a mechanism that impedes their biogenesis, whereas TRC miRNAs appear to evolve under positive selection. Altogether, these data reveal miRNA diversity amongst Drosophila species and permit future discoveries in understanding their emergence and evolution.

genomics↗