A tailed mirtron promotes longevity in Drosophila.

Thousands of atypical microRNAs (miRNAs) have been described in the genomes of animals; however, it is unclear if many of these non-canonical miRNAs can measurably influence phenotypes. Mirtrons are the largest class of non-canonical miRNAs that are produced from hairpins excised by splicing, which after debranching become substrates for Dicer and load into RISC. Most mirtrons require additional processing after splicing to remove 'tail' residues interposed between one of the host intron splice sites and base of the hairpin precursor structure. Despite most mirtrons requiring tail removal no function has been elucidated for a tailed species, indeed for all mirtrons identified function has only been assigned to a single species. Here we study miR-1017, a mirtron with a 3' tail, which is well expressed and conserved in Drosophila species. We found that miR-1017 can extend lifespan when ectopically expressed in the neurons, which seems partly due to this miRNA targeting its host transcript, acetylcholine receptor Dα2. Unexpectedly we found that not only did miR-1017 function in trans but also in cis by affecting splicing of Dα2. This suggests a mechanism for mirtron evolution where initial roles of structural elements in splicing lead to secondary acquisition of trans-regulatory function.

Regulatory logic of endogenous RNAi in silencing de novo genomic conflicts.

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 provides insight into molecular strategies that underlie hpRNA emergence and their potential roles in sex chromosome conflict. In particular, our data support the existence of ongoing rapid evolution of Nmy/Dox-related networks, and recurrent targeting of testis HMG-box loci by hpRNAs. 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 speciation.

Postpolymerization Modification of Poly(2-vinyl-4,4-dimethyl azlactone) as a Versatile Strategy for Drug Conjugation and Stimuli-Responsive Release.

Postpolymerization modification of highly defined "scaffold" polymers is a promising approach for overcoming the existing limitations of controlled radical polymerization such as batch-to-batch inconsistencies, accessibility to different monomers, and compatibility with harsh synthesis conditions. Using multiple physicochemical characterization techniques, we demonstrate that poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) scaffolds can be efficiently modified with a coumarin derivative, doxorubicin, and camptothecin small molecule drugs. Subsequently, we show that coumarin-modified PVDMA has a high cellular biocompatibility and that coumarin derivatives are liberated from the polymer in the intracellular environment for cytosolic accumulation. In addition, we report the pharmacokinetics, biodistribution, and antitumor efficacy of a PVDMA-based polymer for the first time, demonstrating unique accumulation patterns based on the administration route (i.e., intravenous vs oral), efficient tumor uptake, and tumor growth inhibition in 4T1 orthotopic triple negative breast cancer (TNBC) xenografts. This work establishes the utility of PVDMA as a versatile chemical platform for producing polymer-drug conjugates with a tunable, stimuli-responsive delivery.

A molecular chemodosimeter to probe "closed shell" ions in kidney cells.

Two quinidine-functionalized coumarin molecular probes have been synthesized and have been found to bind metal cations (Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Ni2+, and Zn2+) with high affinity in organic-aqueous media (DMSO-HEPES). The chemodosimeters coordinate with the Zn2+ ions in a two-to-one ratio (molecular probe : Zn2+) with a log ß of 10.0 M-2. Upon the addition of the closed-shell metal ions studied, a fluorescence turn-on via an excimer formation is seen at 542 nm due to the quinaldine moiety adopting a syn arrangement when coordinated to the metal Zn2+ ions. Confocal microscopy monitored free Zn2+ ions in the Human Embryonic Kidney cell line HEK293 by coordinating with the chemodosimter.

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 few 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. As such, 254 miRNAs were identified across all datasets, 25 of which were high confidence and 51 low confidence known miRNAs. Further, 23 miRNAs were differentially expressed in uninfected and infected salivary glands: 11 were upregulated and 12 were downregulated upon pathogen infection. Gene ontology and network analysis of target genes of differentially expressed miRNAs predicted roles in metabolic, cellular, development, cellular component biogenesis, and biological regulation processes. Several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including sphingolipid metabolism; valine, leucine and isoleucine degradation; lipid transport and metabolism; exosome biogenesis and secretion; and phosphate-containing compound metabolic processes, were predicted as targets of differentially expressed miRNAs. A qRT-PCR assay was utilized to validate the differential expression of miRNAs. This study provides new insights into the miRNAs expressed in I. scapularis salivary glands and paves the way for their functional manipulation to prevent or treat B. burgdorferi infection.

Environmental RNAi pathways in the two-spotted spider mite.

BACKGROUND: 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 and has applications that includes arthropod pest control. RNAi technologies are dependent on delivery method with the most convenient likely being feeding, which is effective in some animals while others are insensitive. The two-spotted spider mite, Tetranychus urticae, is prime candidate for developing RNAi approaches due to frequent occurrence of conventional pesticide resistance. Using a sequencing-based approach, the fate of ingested RNAs was explored to identify features and conditions that affect small RNA biogenesis from external sources to better inform RNAi design. RESULTS: Biochemical and sequencing approaches in conjunction with extensive computational assessment were used to evaluate metabolism of ingested RNAs in T. urticae. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. Processing of synthetic and plant host RNAs ingested during feeding were evaluated to identify active substrates for spider mite RNAi pathways. Through cataloging characteristics of biochemically purified RNA from these sources, trans-acting small RNAs could be distinguished from degradation fragments and their origins documented. CONCLUSIONS: Using a strategy that delineates small RNA processing, we found many transcripts have the potential to enter spider mite RNAi pathways, however, trans-acting RNAs appear very unstable and rare. This suggests potential RNAi pathway substrates from ingested materials are mostly degraded and infrequently 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 for T.urticae.

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

To assess miRNA evolution across the Drosophila genus, we analyzed several billion small RNA reads across 12 fruit fly species. These data permit comprehensive curation of species- and clade-specific variation in miRNA identity, abundance, and processing. Among well-conserved miRNAs, we observed unexpected cases of clade-specific variation in 5' end precision, occasional antisense loci, and putatively noncanonical loci. We also used strict criteria to identify a large set (649) of novel, evolutionarily restricted miRNAs. Within the bulk collection of species-restricted miRNAs, two notable subpopulations are splicing-derived mirtrons and testes-restricted, recently evolved, clustered (TRC) canonical miRNAs. We quantified miRNA birth and death using our annotation and a phylogenetic model for estimating rates of miRNA turnover. We observed striking differences in birth and death rates across miRNA classes defined by biogenesis pathway, genomic clustering, and tissue restriction, and even identified flux heterogeneity among Drosophila clades. In particular, distinct molecular rationales underlie the distinct evolutionary behavior of different miRNA classes. Mirtrons are associated with high rates 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 among Drosophila species and principles underlying their emergence and evolution.

Rewired RNAi-mediated genome surveillance in house dust mites.

House dust mites are common pests with an unusual evolutionary history, being descendants of a parasitic ancestor. Transition to parasitism is frequently accompanied by genome rearrangements, possibly to accommodate the genetic change needed to access new ecology. Transposable element (TE) activity is a source of genomic instability that can trigger large-scale genomic alterations. Eukaryotes have multiple transposon control mechanisms, one of which is RNA interference (RNAi). Investigation of the dust mite genome failed to identify a major RNAi pathway: the Piwi-associated RNA (piRNA) pathway, which has been replaced by a novel small-interfering RNA (siRNA)-like pathway. Co-opting of piRNA function by dust mite siRNAs is extensive, including establishment of TE control master loci that produce siRNAs. Interestingly, other members of the Acari have piRNAs indicating loss of this mechanism in dust mites is a recent event. Flux of RNAi-mediated control of TEs highlights the unusual arc of dust mite evolution.

siRNAs and piRNAs collaborate for transposon control in the two-spotted spider mite.

RNAi has revolutionized genetic research, and is being commercialized as an insect pest control technology. Mechanisms exploited for this purpose are antiviral and therefore rapidly evolving. Ideally, RNAi will also be used for noninsect pests; however, differences in RNAi biology make this uncertain. Tetranychus urticae (two-spotted spider mite) is a destructive noninsect pest, which has a proclivity to develop pesticide resistance. Here we provide a comprehensive study of the endogenous RNAi pathways of spider mites to inform design of exogenous RNAi triggers. This effort revealed unexpected roles for small RNAs and novel genome surveillance pathways. Spider mites have an expanded RNAi machinery relative to insects, encoding RNA dependent RNA polymerase (Rdrp) and extra Piwi-class effectors. Through analyzing T. urticae transcriptome data we explored small RNA biogenesis, and discovered five siRNA loci that appear central to genome surveillance. These RNAs are expressed in the gonad, which we hypothesize to trigger production of piRNAs for control of transposable elements (TEs). This work highlights the need to investigate endogenous RNAi biology as lessons from model organisms may not hold in other species, impacting development of an RNAi strategy.

Water-Soluble NIR Absorbing and Emitting Indolizine Cyanine and Indolizine Squaraine Dyes for Biological Imaging.

Organic dyes that absorb and emit in the near-infrared (NIR) region are potentially noninvasive, high-resolution, and rapid biological imaging materials. Indolizine donor-based cyanine and squaraine dyes with water-solubilizing sulfonate groups were targeted in this study due to strong absorptions and emissions in the NIR region. As previously observed for nonwater-soluble derivatives, the indolizine group with water-solubilizing groups retains a substantial shift toward longer wavelengths for both absorption and emission with squaraines and cyanines relative to classically researched indoline donor analogues. Very high quantum yields (as much as 58%) have been observed with absorption and emission >700 nm in fetal bovine serum. Photostability studies, cell culture cytotoxicity, and cell uptake specificity profiles were all studied for these dyes, demonstrating exceptional biological imaging suitability.

RNAi in Xenopus: look before you leap.

RNAi has revolutionized reverse genetics; however, RNAi is not necessarily ubiquitous or constitutive. Lund and colleagues (pp. 1121-1131) show that microRNA (miRNA) effector Argonautes (Agos) are limiting and easily saturated during early Xenopus embryogenesis. Moreover, this stage is devoid of slicing capacity. Supplementation of Ago proteins rescued endogenous miRNA activity in the presence of exogenous siRNAs, and, excitingly, ectopic Ago2 could now support RNAi in Xenopus. These observations may potentially facilitate RNAi in other recalcitrant model organisms.

Guanidinium-Functionalized Interpolyelectrolyte Complexes Enabling RNAi in Resistant Insect Pests.

RNAi-based technologies are ideal for pest control as they can provide species specificity and spare nontarget organisms. However, in some pests biological barriers prevent use of RNAi, and therefore broad application. In this study we tested the ability of a synthetic cationic polymer, poly-[ N-(3-guanidinopropyl)methacrylamide] (pGPMA), that mimics arginine-rich cell penetrating peptides to trigger RNAi in an insensitive animal- Spodoptera frugiperda. Polymer-dsRNA interpolyelectrolyte complexes (IPECs) were found to be efficiently taken up by cells, and to drive highly efficient gene knockdown. These IPECs could also trigger target gene knockdown and moderate larval mortality when fed to S. frugiperda larvae. This effect was sequence specific, which is consistent with the low toxicity we found to be associated with this polymer. A method for oral delivery of dsRNA is critical to development of RNAi-based insecticides. Thus, this technology has the potential to make RNAi-based pest control useful for targeting numerous species and facilitate use of RNAi in pest management practices.

MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila.

microRNAs (miRNAs) are approximately 22 nucleotide regulatory RNAs derived from hairpins generated either by Drosha cleavage (canonical substrates) or by splicing and debranching of short introns (mirtrons). The 5' end of the highly conserved Drosophila mirtron-like locus mir-1017 is coincident with the splice donor, but a substantial "tail" separates its hairpin from the 3'splice acceptor. Genetic and biochemical studies define a biogenesis pathway involving splicing, lariat debranching, and RNA exosome-mediated "trimming," followed by conventional dicing and loading into AGO1 to yield a miRNA that can repress seed-matched targets. Analysis of cloned small RNAs yielded six additional candidate 3' tailed mirtrons in D. melanogaster. Altogether, these data reveal an unexpected role for the exosome in the biogenesis of miRNAs from hybrid mirtron substrates.

Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines.

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.

Adaptive evolution of testis-specific, recently evolved, clustered miRNAs in Drosophila.

The propensity of animal miRNAs to regulate targets bearing modest complementarity, most notably via pairing with miRNA positions ∼2-8 (the "seed"), is believed to drive major aspects of miRNA evolution. First, minimal targeting requirements have allowed most conserved miRNAs to acquire large target cohorts, thus imposing strong selection on miRNAs to maintain their seed sequences. Second, the modest pairing needed for repression suggests that evolutionarily nascent miRNAs may generally induce net detrimental, rather than beneficial, regulatory effects. Hence, levels and activities of newly emerged miRNAs are expected to be limited to preserve the status quo of gene expression. In this study, we unexpectedly show that Drosophila testes specifically express a substantial miRNA population that contravenes these tenets. We find that multiple genomic clusters of testis-restricted miRNAs harbor recently evolved miRNAs, whose experimentally verified orthologs exhibit divergent sequences, even within seed regions. Moreover, this class of miRNAs exhibits higher expression and greater phenotypic capacities in transgenic misexpression assays than do non-testis-restricted miRNAs of similar evolutionary age. These observations suggest that these testis-restricted miRNAs may be evolving adaptively, and several methods of evolutionary analysis provide strong support for this notion. Consistent with this, proof-of-principle tests show that orthologous miRNAs with divergent seeds can distinguish target sensors in a species-cognate manner. Finally, we observe that testis-restricted miRNA clusters exhibit extraordinary dynamics of miRNA gene flux in other Drosophila species. Altogether, our findings reveal a surprising tissue-directed influence of miRNA evolution, involving a distinct mode of miRNA function connected to adaptive gene regulation in the testis.

Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate.

Numerous microRNAs (miRNAs) have been discovered in the genomes of higher eukaryotes, and functional studies indicate that they are important during development. However, little is known concerning the function of individual miRNAs. We approached this problem in zebrafish by combining identification of miRNA expression, functional analyses and experimental validation of potential targets. We show that miR-214 is expressed during early segmentation stages in somites and that varying its expression alters the expression of genes regulated by Hedgehog signaling. Inhibition of miR-214 results in a reduction or loss of slow-muscle cell types. We show that su(fu) mRNA, encoding a negative regulator of Hedgehog signaling, is targeted by miR-214. Through regulation of su(fu), miR-214 enables precise specification of muscle cell types by sharpening cellular responses to Hedgehog.

Discovery of hundreds of mirtrons in mouse and human small RNA data.

Atypical miRNA substrates do not fit criteria often used to annotate canonical miRNAs, and can escape the notice of miRNA genefinders. Recent analyses expanded the catalogs of invertebrate splicing-derived miRNAs ("mirtrons"), but only a few tens of mammalian mirtrons have been recognized to date. We performed meta-analysis of 737 mouse and human small RNA data sets comprising 2.83 billion raw reads. Using strict and conservative criteria, we provide confident annotation for 237 mouse and 240 human splicing-derived miRNAs, the vast majority of which are novel genes. These comprise three classes of splicing-derived miRNAs in mammals: conventional mirtrons, 5'-tailed mirtrons, and 3'-tailed mirtrons. In addition, we segregated several hundred additional human and mouse loci with candidate (and often compelling) evidence. Most of these loci arose relatively recently in their respective lineages. Nevertheless, some members in each of the three mirtron classes are conserved, indicating their incorporation into beneficial regulatory networks. We also provide the first Northern validation for mammalian mirtrons, and demonstrate Dicer-dependent association of mature miRNAs from all three classes of mirtrons with Ago2. The recognition of hundreds of mammalian mirtrons provides a new foundation for understanding the scope and evolutionary dynamics of Dicer substrates in mammals.

The impact of age, biogenesis, and genomic clustering on Drosophila microRNA evolution.

The molecular evolutionary signatures of miRNAs inform our understanding of their emergence, biogenesis, and function. The known signatures of miRNA evolution have derived mostly from the analysis of deeply conserved, canonical loci. In this study, we examine the impact of age, biogenesis pathway, and genomic arrangement on the evolutionary properties of Drosophila miRNAs. Crucial to the accuracy of our results was our curation of high-quality miRNA alignments, which included nearly 150 corrections to ortholog calls and nucleotide sequences of the global 12-way Drosophilid alignments currently available. Using these data, we studied primary sequence conservation, normalized free-energy values, and types of structure-preserving substitutions. We expand upon common miRNA evolutionary patterns that reflect fundamental features of miRNAs that are under functional selection. We observe that melanogaster-subgroup-specific miRNAs, although recently emerged and rapidly evolving, nonetheless exhibit evolutionary signatures that are similar to well-conserved miRNAs and distinct from other structured noncoding RNAs and bulk conserved non-miRNA hairpins. This provides evidence that even young miRNAs may be selected for regulatory activities. More strikingly, we observe that mirtrons and clustered miRNAs both exhibit distinct evolutionary properties relative to solo, well-conserved miRNAs, even after controlling for sequence depth. These studies highlight the previously unappreciated impact of biogenesis strategy and genomic location on the evolutionary dynamics of miRNAs, and affirm that miRNAs do not evolve as a unitary class.

Utilizing Intrinsic Properties of Polyaniline to Detect Nucleic Acid Hybridization through UV-Enhanced Electrostatic Interaction.

Detection of specific RNA or DNA molecules by hybridization to "probe" nucleic acids via complementary base-pairing is a powerful method for analysis of biological systems. Here we describe a strategy for transducing hybridization events through modulating intrinsic properties of the electroconductive polymer polyaniline (PANI). When DNA-based probes electrostatically interact with PANI, its fluorescence properties are increased, a phenomenon that can be enhanced by UV irradiation. Hybridization of target nucleic acids results in dissociation of probes causing PANI fluorescence to return to basal levels. By monitoring restoration of base PANI fluorescence as little as 10(-11) M (10 pM) of target oligonucleotides could be detected within 15 min of hybridization. Detection of complementary oligos was specific, with introduction of a single mismatch failing to form a target-probe duplex that would dissociate from PANI. Furthermore, this approach is robust and is capable of detecting specific RNAs in extracts from animals. This sensor system improves on previously reported strategies by transducing highly specific probe dissociation events through intrinsic properties of a conducting polymer without the need for additional labels.