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.

A DNA virus-encoded immune antagonist fully masks the potent antiviral activity of RNAi in Drosophila.

Coevolution of viruses and their hosts may lead to viral strategies to avoid, evade, or suppress antiviral immunity. An example is antiviral RNA interference (RNAi) in insects: the host RNAi machinery processes viral double-stranded RNA into small interfering RNAs (siRNAs) to suppress viral replication, whereas insect viruses encode suppressors of RNAi, many of which inhibit viral small interfering RNA (vsiRNA) production. Yet, many studies have analyzed viral RNAi suppressors in heterologous systems, due to the lack of experimental systems to manipulate the viral genome of interest, raising questions about in vivo functions of RNAi suppressors. To address this caveat, we generated an RNAi suppressor-defective mutant of invertebrate iridescent virus 6 (IIV6), a large DNA virus in which we previously identified the 340R protein as a suppressor of RNAi. Loss of 340R did not affect vsiRNA production, indicating that 340R binds siRNA duplexes to prevent RNA-induced silencing complex assembly. Indeed, vsiRNAs were not efficiently loaded into Argonaute 2 during wild-type IIV6 infection. Moreover, IIV6 induced a limited set of mature microRNAs in a 340R-dependent manner, most notably miR-305-3p, which we attribute to stabilization of the miR-305-5p:3p duplex by 340R. The IIV6 340R deletion mutant did not have a replication defect in cells, but was strongly attenuated in adult Drosophila This in vivo replication defect was completely rescued in RNAi mutant flies, indicating that 340R is a bona fide RNAi suppressor, the absence of which uncovers a potent antiviral immune response that suppresses virus accumulation ∼100-fold. Together, our work indicates that viral RNAi suppressors may completely mask antiviral immunity.

The Tudor protein Veneno assembles the ping-pong amplification complex that produces viral piRNAs in Aedes mosquitoes.

PIWI-interacting RNAs (piRNAs) comprise a class of small RNAs best known for suppressing transposable elements in germline tissues. The vector mosquito Aedes aegypti encodes seven PIWI genes, four of which are somatically expressed. This somatic piRNA pathway generates piRNAs from viral RNA during infection with cytoplasmic RNA viruses through ping-pong amplification by the PIWI proteins Ago3 and Piwi5. Yet, additional insights into the molecular mechanisms mediating non-canonical piRNA production are lacking. TUDOR-domain containing (Tudor) proteins facilitate piRNA biogenesis in Drosophila melanogaster and other model organisms. We thus hypothesized that Tudor proteins are required for viral piRNA production and performed a knockdown screen targeting all A. aegypti Tudor genes. Knockdown of the Tudor genes AAEL012437, Vreteno, Yb, SMN and AAEL008101-RB resulted in significantly reduced viral piRNA levels, with AAEL012437-depletion having the strongest effect. This protein, which we named Veneno, associates directly with Ago3 in an sDMA-dependent manner and localizes in cytoplasmic foci reminiscent of piRNA processing granules of Drosophila. Veneno-interactome analyses reveal a network of co-factors including the orthologs of the Drosophila piRNA pathway components Vasa and Yb, which in turn interacts with Piwi5. We propose that Veneno assembles a multi-protein complex for ping-pong dependent piRNA production from viral RNA.

Histone-derived piRNA biogenesis depends on the ping-pong partners Piwi5 and Ago3 in Aedes aegypti.

The piRNA pathway is of key importance in controlling transposable elements in most animal species. In the vector mosquito Aedes aegypti, the presence of eight PIWI proteins and the accumulation of viral piRNAs upon arbovirus infection suggest additional functions of the piRNA pathway beyond genome defense. To better understand the regulatory potential of this pathway, we analyzed in detail host-derived piRNAs in A. aegypti Aag2 cells. We show that a large repertoire of protein-coding genes and non-retroviral integrated RNA virus elements are processed into genic piRNAs by different combinations of PIWI proteins. Among these, we identify a class of genes that produces piRNAs from coding sequences in an Ago3- and Piwi5-dependent fashion. We demonstrate that the replication-dependent histone gene family is a genic source of ping-pong dependent piRNAs and that histone-derived piRNAs are dynamically expressed throughout the cell cycle, suggesting a role for the piRNA pathway in the regulation of histone gene expression. Moreover, our results establish the Aag2 cell line as an accessible experimental model to study gene-derived piRNAs.

Low heme oxygenase-1 levels in patients with systemic sclerosis are associated with an altered Toll-like receptor response: another role for CXCL4?

OBJECTIVE: SSc is a disease characterized by inflammation and fibrosis. Heme Oxygenase-1 (HO-1) is a haem-degrading enzyme that mediates resolution of inflammation and is induced upon mediators abundantly present in SSc. We aimed to assess whether HO-1 expression/function is disturbed in SSc patients and could therefore be contributing to the ongoing inflammation. METHODS: In total, 92 SSc patients and 48 healthy controls were included. By measuring total bilirubin in plasma in vivo, HO-activity was assessed. HO-1 expression levels were determined with western blot in monocytes before and after induction of HO-1 with cobalt protoporphyrin (CoPP) with or without CXCL4. Monocyte-derived dendritic cells (DCs) were stimulated with several Toll-like receptor (TLR) ligands with or without pre-stimulation with CoPP for 24 h. Cytokine levels were measured in the supernatants using the Luminex Bead Array. RESULTS: SSc patients have lower plasma levels of bilirubin, suggestive of an aberrant HO-1 function. We demonstrated low HO-1 expression in immune cells from SSc patients, whereas induction with CoPP was able to restore HO-1 levels in DCs from SSc patients, almost normalizing the increased TLR response observed in SSc. Co-exposure to CXCL4 completely abrogated CoPP-induced HO-1 expression, suggesting that the high CXCL4 levels present in SSc patients block the normal induction of HO-1 and its function. CONCLUSION: We demonstrate that HO activity in SSc patients is decreased and show its functional consequences. Since CXCL4 blocks the induction of HO-1 expression, neutralization of CXCL4 in SSc patients could have clinical benefits by diminishing overactivation of immune cells and other anti-inflammatory effects of HO-1.

PIWI proteomics identifies Atari and Pasilla as piRNA biogenesis factors in Aedes mosquitoes.

As in most arthropods, the PIWI-interacting RNA (piRNA) pathway in the vector mosquito Aedes aegypti is active in diverse biological processes in both soma and germline. To gain insights into piRNA biogenesis and effector complexes, we mapped the interactomes of the somatic PIWI proteins Ago3, Piwi4, Piwi5, and Piwi6 and identify numerous specific interactors as well as cofactors associated with multiple PIWI proteins. We describe the Piwi5 interactor AAEL014965, the direct ortholog of the Drosophila splicing factor pasilla. We find that Ae. aegypti Pasilla encodes a nuclear isoform and a cytoplasmic isoform, the latter of which is required for efficient piRNA production. In addition, we characterize a splice variant of the Tudor protein AAEL008101/Atari that associates with Ago3 and forms a scaffold for PIWI proteins and target RNAs to promote ping-pong amplification of piRNAs. Our study provides a useful resource for follow-up studies of somatic piRNA biogenesis, mechanism, and function in Aedes mosquitoes.

Cell-type-specific downregulation of heme oxygenase-1 by lipopolysaccharide via Bach1 in primary human mononuclear cells.

Heme oxygenase (HO)-1 is the inducible isoform of the heme-degrading enzyme HO, which is upregulated by multiple stress stimuli. HO-1 has major immunomodulatory and anti-inflammatory effects via its cell-type-specific functions in mononuclear cells. Contradictory findings have been reported on HO-1 regulation by the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) in these cells. Therefore, we reinvestigated the effects of LPS on HO-1 gene expression in human and murine mononuclear cells in vitro and in vivo. Remarkably, LPS downregulated HO-1 in primary human peripheral blood mononuclear cells (PBMCs), CD14(+) monocytes, macrophages, dendritic cells, and granulocytes, but upregulated this enzyme in primary murine macrophages and human monocytic leukemia cell lines. Furthermore, experiments with human CD14(+) monocytes revealed that activation of other TLRs including TLR1, -2, -5, -6, -8, and -9 decreased HO-1 mRNA expression. LPS-dependent downregulation of HO-1 was specific, because expression of cyclooxygenase-2, NADP(H)-quinone oxidoreductase-1, and peroxiredoxin-1 was increased under the same experimental conditions. Notably, LPS upregulated expression of Bach1, a critical transcriptional repressor of HO-1. Moreover, knockdown of this nuclear factor enhanced basal and LPS-dependent HO-1 expression in mononuclear cells. Finally, downregulation of HO-1 in response to LPS was confirmed in PBMCs from human individuals subjected to experimental endotoxemia. In conclusion, LPS downregulates HO-1 expression in primary human mononuclear cells via a Bach1-mediated pathway. As LPS-dependent HO-1 regulation is cell-type- and species-specific, experimental findings in cell lines and animal models need careful interpretation.