Functional Analysis of Individual piRNAs in Aedes aegypti Cells and Embryos Using Antisense Oligonucleotides.

In insects, PIWI-interacting (pi)RNAs fulfill versatile regulatory functions inside and outside the germline, including posttranscriptional repression of transposable elements and regulation of gene expression. Canonically, piRNAs act-and have been studied-as a conglomerate of several thousand sequences that cooperatively silence target RNAs. Interestingly, however, an increasing number of studies have demonstrated that individual piRNAs can have profound biological activity as a unique piRNA sequence. Prime examples are the tapiR1 and 2 piRNAs, which mediate target RNA degradation in the developing embryo of Aedes mosquitoes. To study such outstanding individual piRNA species, we describe here a method to interfere with RNA target silencing using antisense oligonucleotides in cell culture as well as in mosquito pre-blastoderm embryos. Although the method has been established for Aedes mosquitoes, it can likely be adapted for use in other invertebrate species as well.

A piRNA-lncRNA regulatory network initiates responder and trailer piRNA formation during mosquito embryonic development.

PIWI-interacting (pi)RNAs are small silencing RNAs that are crucial for the defense against transposable elements in germline tissues of animals. In Aedes aegypti mosquitoes, the piRNA pathway also contributes to gene regulation in somatic tissues, illustrating additional roles for piRNAs and PIWI proteins besides transposon repression. Here, we identify a highly abundant endogenous piRNA (propiR1) that associates with both Piwi4 and Piwi5. PropiR1-mediated target silencing requires base-pairing in the seed region with supplemental base-pairing at the piRNA 3' end. Yet, propiR1 represses a limited set of targets, among which is the lncRNA AAEL027353 (lnc027353). Slicing of lnc027353 initiates production of responder and trailer piRNAs from the cleavage fragment. Expression of propiR1 commences early during embryonic development and mediates degradation of maternally provided lnc027353 Both propiR1 and its lncRNA target are conserved in the closely related Aedes albopictus mosquito, underscoring the importance of this regulatory network for mosquito development.

Zooming in on targets of mosquito small RNAs.

Small RNAs are crucial for the regulation of basic cellular processes and protection against viruses and transposons in mosquitoes. Rozen-Gagnon et al. established CLIP (cross-linking and immunoprecipitation) for Argonaute proteins in Aedes aegypti. Their study sheds light on small RNA-target interactions in mosquitoes and provides an important resource for further study.

Population genomics in the arboviral vector Aedes aegypti reveals the genomic architecture and evolution of endogenous viral elements.

Horizontal gene transfer from viruses to eukaryotic cells is a pervasive phenomenon. Somatic viral integrations are linked to persistent viral infection whereas integrations into germline cells are maintained in host genomes by vertical transmission and may be co-opted for host functions. In the arboviral vector Aedes aegypti, an endogenous viral element from a nonretroviral RNA virus (nrEVE) was shown to produce PIWI-interacting RNAs (piRNAs) to limit infection with a cognate virus. Thus, nrEVEs may constitute a heritable, sequence-specific mechanism for antiviral immunity, analogous to piRNA-mediated silencing of transposable elements. Here, we combine population genomics and evolutionary approaches to analyse the genomic architecture of nrEVEs in A. aegypti. We conducted a genome-wide screen for adaptive nrEVEs and searched for novel population-specific nrEVEs in the genomes of 80 individual wild-caught mosquitoes from five geographical populations. We show a dynamic landscape of nrEVEs in mosquito genomes and identified five novel nrEVEs derived from two currently circulating viruses, providing evidence of the environmental-dependent modification of a piRNA cluster. Overall, our results show that virus endogenization events are complex with only a few nrEVEs contributing to adaptive evolution in A. aegypti.

Improved reference genome of the arboviral vector Aedes albopictus.

BACKGROUND: The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes. RESULTS: We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. CONCLUSION: The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.

A satellite repeat-derived piRNA controls embryonic development of Aedes.

Tandem repeat elements such as the diverse class of satellite repeats occupy large parts of eukaryotic chromosomes, mostly at centromeric, pericentromeric, telomeric and subtelomeric regions1. However, some elements are located in euchromatic regions throughout the genome and have been hypothesized to regulate gene expression in cis by modulating local chromatin structure, or in trans via transcripts derived from the repeats2-4. Here we show that a satellite repeat in the mosquito Aedes aegypti promotes sequence-specific gene silencing via the expression of two PIWI-interacting RNAs (piRNAs). Whereas satellite repeats and piRNA sequences generally evolve extremely quickly5-7, this locus was conserved for approximately 200 million years, suggesting that it has a central function in mosquito biology. piRNA production commenced shortly after egg laying, and inactivation of the more abundant piRNA resulted in failure to degrade maternally deposited transcripts in the zygote and developmental arrest. Our results reveal a mechanism by which satellite repeats regulate global gene expression in trans via piRNA-mediated gene silencing that is essential for embryonic development.

Mosquito-specific and mosquito-borne viruses: evolution, infection, and host defense.

Recent virus discovery programs have identified an extensive reservoir of viruses in arthropods. It is thought that arthropod viruses, including mosquito-specific viruses, are ancestral to vertebrate-pathogenic arboviruses. Mosquito-specific viruses are restricted in vertebrate cells at multiple levels, including entry, RNA replication, assembly, and by the inability to replicate at high temperatures. Moreover, it is likely that the vertebrate immune system suppresses replication of these viruses. The evolution from single to dual-host tropism may also require changes in the course of infection in the mosquito host. In this review we explore the adaptive changes required for a switch from a mosquito-specific to a mosquito-borne transmission cycle.

Clinical and molecular implications of MED15 in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) progression depends on various dysregulated pathways. Regulation of diverse pathways is mediated by the mediator complex. The mediator subunit MED15 is essential for transforming growth factor (TGF)-ß signaling and involved in breast and prostate cancers. We investigated the implication of MED15 in HNSCC. IHC for MED15 was performed on 324 tissue samples, and TGF-ß assessed the use of Ki-67 and pSMAD3 as markers. MED15 knockdown followed by proliferation and migration assays, as well as TGF-ß1 treatment followed by MED15 analysis, was also performed. MED15 was overexpressed in 35% of primary tumors, 30% of lymph node metastases, and 70% of recurrences in contrast to no or low expression in benign tumors. MED15 overexpression in primary tumors from patients who developed recurrences was associated with higher mortality rates and occurred at highest frequency in oral cavity or oropharyngeal tumors. Furthermore, MED15 expression correlated between primary tumors and corresponding lymph node metastases. MED15 correlated with proliferation in tissues, and MED15 knockdown reduced proliferation and migration. We observed an association between MED15 and TGF-ß activity in tissues because TGF-ß activation led to increased MED15 expression and reduced pSMAD3 on MED15 knockdown. Taken together, our results implicate MED15 in HNSCC and hint that MED15 overexpression is a clonal event during HNSCC progression. MED15 may serve as a prognostic marker for recurrence and as a therapeutic target.

MED12 overexpression is a frequent event in castration-resistant prostate cancer.

In a recent effort to unravel the molecular basis of prostate cancer (PCa), Barbieri and colleagues using whole-exome sequencing identified a novel recurrently mutated gene, MED12, in 5.4% of primary PCa. MED12, encoding a subunit of the Mediator complex, is a transducer of Wnt/ß-catenin signaling, linked to modulation of hedgehog signaling and to the regulation of transforming growth factor beta (TGFß)-receptor signaling. Therefore, these studies prompted us to investigate the relevance of MED12 in PCa. Expression of MED12, SMAD3 phosphorylation, and proliferation markers was assessed by immunohistochemistry on tissue microarrays from 633 patients. siRNA-mediated knockdown of MED12 was carried out on PCa cell lines followed by cellular proliferation assays, cell cycle analysis, apoptosis assays, and treatments with recombinant TGFß3. We found nuclear overexpression of MED12 in 40% (28/70) of distant metastatic castration-resistant prostate cancer (CRPC(MET)) and 21% (19/90) of local-recurrent CRPC (CRPC(LOC)) in comparison with frequencies of less than 11% in androgen-sensitive PCa, and no overexpression in benign prostatic tissues. MED12 expression was significantly correlated with high proliferative activity in PCa tissues, whereas knockdown of MED12 decreased proliferation, reduced G1- to S-phase transition, and increased the expression of the cell cycle inhibitor p27. TGFß signaling activation associates with MED12 nuclear overexpression in tissues and results in a strong increase in MED12 nuclear expression in cell lines. Furthermore, MED12 knockdown reduced the expression of the TGFß target gene vimentin. Our findings show that MED12 nuclear overexpression is a frequent event in CRPC in comparison with androgen-sensitive PCa and is directly implicated in TGFß signaling.