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.

The Small RNA Universe of Capitella teleta.

RNAi is an evolutionarily fluid mechanism with dramatically different activities across animal phyla. One major group where there has been little investigation is annelid worms. Here, the small RNAs of the polychaete developmental model Capitella teleta are profiled across development. As is seen with nearly all animals, nearly 200 microRNAs were found with 58 high-confidence novel species. Greater miRNA diversity was associated with later stages consistent with differentiation of tissues. Outside miRNA, a distinct composition of other small RNA pathways was found. Unlike many invertebrates, an endogenous siRNA pathway was not observed, indicating pathway loss relative to basal planarians. No processively generated siRNA-class RNAs could be found arising from dsRNA precursors. This has a significant impact on RNAi technology development for this group of animals. Unlike the apparent absence of siRNAs, a significant population of piRNAs was observed. For many piRNAs, phasing and ping-pong biogenesis pathways were identified. Interestingly, piRNAs were found to be highly expressed during early development, suggesting a potential role in regulation in metamorphosis. Critically, the configuration of RNAi factors in C. teleta is found in other annelids and mollusks, suggesting that similar biology is likely to be present in the wider clade. This study is the first in providing comprehensive analysis of small RNAs in annelids.

Interleukin-17A Facilitates Chikungunya Virus Infection by Inhibiting IFN-α2 Expression.

Interferons (IFNs) are the key components of innate immunity and are crucial for host defense against viral infections. Here, we report a novel role of interleukin-17A (IL-17A) in inhibiting IFN-α2 expression thus promoting chikungunya virus (CHIKV) infection. CHIKV infected IL-17A deficient (Il17a-/- ) mice expressed a higher level of IFN-α2 and developed diminished viremia and milder footpad swelling in comparison to wild-type (WT) control mice, which was also recapitulated in IL-17A receptor-deficient (Il17ra-/- ) mice. Interestingly, IL-17A selectively blocked IFN-α2 production during CHIKV, but not West Nile virus (WNV) or Zika virus (ZIKV), infections. Recombinant IL-17A treatment inhibited CHIKV-induced IFN-α2 expression and enhanced CHIKV replication in both human and mouse cells. We further found that IL-17A inhibited IFN-α2 production by modulating the expression of Interferon Regulatory Factor-5 (IRF-5), IRF-7, IFN-stimulated gene 49 (ISG-49), and Mx1 expression during CHIKV infection. Neutralization of IL-17A in vitro leads to the increase of the expression of these antiviral molecules and decrease of CHIKV replication. Collectively, these results suggest a novel function of IL-17A in inhibiting IFN-α2-mediated antiviral responses during CHIKV infection, which may have broad implications in viral infections and other inflammatory diseases.

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.

Blocking Zebrafish MicroRNAs with Morpholinos.

Antisense morpholino oligonucleotides have been commonly used in zebrafish to inhibit mRNA function, either by inhibiting pre-mRNA splicing or by blocking translation initiation. Even with the advent of genome editing by CRISP/Cas9 technology, morpholinos provide a useful and rapid tool to knockdown gene expression. This is especially true when dealing with multiple alleles and large gene families where genetic redundancy can complicate knockout of all family members. miRNAs are small noncoding RNAs that are often encoded in gene families and can display extensive genetic redundancy. This redundancy, plus their small size which can limit targeting by CRISPR/Cas9, makes morpholino-based strategies particularly attractive for inhibition of miRNA function. We provide the rationale, background, and methods to inhibit miRNA function with antisense morpholinos during early development and in the adult retina in zebrafish.