The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells

The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used. Using either synthetic siRNAs or a short hairpin RNA construct, NS3 was able to significantly suppress the RNAi-mediated silencing of luciferase expression in both monkey (Vero) and human (HEK293) cells. These results support the proposed mode of action of NS3 to act by sequestering siRNAs, the key molecules of the RNAi pathway conserved in all eukaryotes. The possible applications of this protein in modulating RNAi and investigating the proposed antiviral RNAi response in mammalian cell systems are discussed

New insights into control of arbovirus replication and spread by insect RNA interference pathways

Arthropod-borne (arbo) viruses are transmitted by vectors, such as mosquitoes, to susceptible vertebrates. Recent research has shown that arbovirus replication and spread in mosquitoes is not passively tolerated but induces host responses to control these pathogens. Small RNA-mediated host responses are key players among these antiviral immune strategies. Studies into one such small RNA-mediated antiviral response, the exogenous RNA interference (RNAi) pathway, have generated a wealth of information on the functions of this mechanism and the enzymes which mediate antiviral activities. However, other small RNA-mediated host responses may also be involved in modulating antiviral activity. The aim of this review is to summarize recent research into the nature of small RNA-mediated antiviral responses in mosquitoes and to discuss future directions for this relatively new area of research

Control of chicken CR1 retrotransposons is independent of Dicer-mediated RNA interference pathway

BACKGROUND: Dicer is an RNase III-ribonuclease that initiates the formation of small interfering RNAs as a defence against genomic parasites such as retrotransposons. Despite intensive characterization in mammalian species, the biological functions of Dicer in controlling retrotransposable elements of the non-mammalian vertebrate are poorly understood. In this report, we examine the role of chicken Dicer in controlling the activity of chicken CR1 retrotransposable elements in a chicken-human hybrid DT40 cell line employing a conditional lossof- Dicer function. RESULTS: Retrotransposition is detrimental to host genome stability and thus eukaryotic cells have developed mechanisms to limit the expansion of retrotransposons by Dicer-mediated RNAi silencing pathways. However, the mechanisms that control the activity and copy numbers of transposable elements in chicken remain unclear. Here, we describe how the loss of Dicer in chicken cells does not reactivate endogenous chicken CR1 retrotransposons with impaired RNAi machinery, suggesting that the control of chicken CR1 is independent of Dicer-induced RNAi silencing. In contrast, upon introduction of a functionally active human L1 retrotransposable element that contains an active 5' UTR promoter, the Dicer-deficient chicken cells show a strong increase in the accumulation of human L1 transcripts and retrotransposition activity, highlighting a major difference between chicken CR1 and other mammalian L1 retrotransposons. CONCLUSION: Our data provide evidence that chicken CR1 retrotransposons, unlike their mammalian L1 counterparts, do not undergo retrotransposition because most CR1 retrotransposons are truncated or mutated at their 5'UTR promoters and thus are not subjected to Dicer-mediated RNAi-silencing control

Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs.

Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors.IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from human RIG-I can substitute for the corresponding domains of DRH-1 to mediate antiviral RNAi in C. elegans, suggesting an analogous role for DRH-1 as an intracellular dsRNA sensor to initiate antiviral RNAi. Here, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in C. elegans Characterization of four distinct drh-1 mutants obtained from the screen revealed that DRH-1 did not function to initiate antiviral RNAi. We show that DRH-1 acted in a downstream step to enhance Dicer-dependent biogenesis of viral siRNAs in C. elegans As mammals produce Dicer-dependent viral siRNAs to target RNA viruses, our findings suggest a possible role for mammalian RLRs and interferon signaling in the biogenesis of viral siRNAs

Induction of Stress Granule Assembly is Essential for the Orchestration of DNA Damage Response

DNA damage provokes several responses including DNA repair, cell cycle regulation and apoptosis that collectively represent the DNA damage response (DDR). Here, we demonstrate that the DDR incorporates the activation of stress granule (SG) formation pathway as a mechanism to process destabilized RNAs. UV irradiation induced the assembly of SGs during the G2 phase and newly formed SGs appeared exclusively in the early G1 phase. SG assembly pathway was activated within the first hours after DNA damage, suggesting that the processing of destabilized RNAs is activated at an early stage. The induction of SGs and RNAi effector protein Argonaute 2 recruitment after UV exposure was independent of ATM and ATR signaling cascades. Apoptosis occurred only in SG-negative cells indicating that SGs promote cell survival after genotoxic stress. Analysis of several DNA damage/repair deficient MEFs revealed that the SG accumulation remained unaltered after UV exposure. Our results show that SGs are an essential component of DDR that is activated in parallel to the DNA damage kinase response networks

Arboviruses and the challenge to establish systemic and persistent infections in competent mosquito vectors : the interaction with the RNAi mechanism

Arboviruses are capable to establish long-term persistent infections in mosquitoes that do not affect significantly the physiology of the insect vectors. Arbovirus infections are controlled by the RNAi machinery via the production of viral siRNAs and the formation of RISC complexes targeting viral genomes and mRNAs. Engineered arboviruses that contain cellular gene sequences can therefore be transformed to "viral silencing vectors" for studies of gene function in reverse genetics approaches. More specifically, "ideal" viral silencing vectors must be competent to induce robust RNAi effects while other interactions with the host immune system should be kept at a minimum to reduce non-specific effects. Because of their inconspicuous nature, arboviruses may approach the "ideal" viral silencing vectors in insects and it is therefore worthwhile to study the mechanisms by which the interactions with the RNAi machinery occur. In this review, an analysis is presented of the antiviral RNAi response in mosquito vectors with respect to the major types of arboviruses (alphaviruses, flaviviruses, bunyaviruses, and others). With respect to antiviral defense, the exo-RNAi pathway constitutes the major mechanism while the contribution of both miRNAs and viral piRNAs remains a contentious issue. However, additional mechanisms exist in mosquitoes that are capable to enhance or restrict the efficiency of viral silencing vectors such as the amplification of RNAi effects by DNA forms, the existence of incorporated viral elements in the genome and the induction of a non-specific systemic response by Dicer-2. Of significance is the observation that no major "viral suppressors of RNAi" (VSRs) seem to be encoded by arboviral genomes, indicating that relatively tight control of the activity of the RNA-dependent RNA polymerase (RdRp) may be sufficient to maintain the persistent character of arbovirus infections. Major strategies for improvement of viral silencing vectors therefore are proposed to involve engineering of VSRs and modifying of the properties of the RdRp. Because of safety issues (pathogen status), however, arbovirus-based silencing vectors are not well suited for practical applications, such as RNAi-based mosquito control. In that case, related mosquito-specific viruses that also establish persistent infections and may cause similar RNAi responses may represent a valuable alternative solution

Role of RNA Interference (RNAi) in the Moss Physcomitrella patens

RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species

Functional Analysis of MicroRNA Pathway Genes in the Somatic Gonad and Germ Cells During Ovulation in \u3cem\u3eC. Elegans\u3c/em\u3e

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in animal development and physiology, though functions for most miRNAs remain unknown. Worms with reduced miRNA biogenesis due to loss of Drosha or Pasha/DGCR8 activity are sterile and fail to ovulate, indicating that miRNAs are required for the process of oocyte maturation and ovulation. Starting with this penetrant sterile phenotype and using new strains created to perform tissue specific RNAi, we characterized the roles of the C. elegans Pasha, pash-1, and two miRNA-specific Argonautes, alg-1 and alg-2, in somatic gonad cells and in germ cells in the regulation of ovulation. Conditional loss of pash-1activity resulted in a reduced rate of ovulation and in basal and ovulatory sheath contractions. Similarly, knockdown of miRNA-specific Argonautes in the cells of the somatic gonad by tissue-specific RNAi results in a reduction of the ovulation rate and in basal and ovulatory sheath contractions. Reduced miRNA pathway gene activity resulted in a range of defects, including oocytes that were pinched upon entry of the oocyte into the distal end of the spermatheca in about 42% of the ovulation events observed following alg-1 RNAi. This phenotype was not observed on worms exposed to control RNAi. In contrast, knockdown of alg-1 and alg-2 in germ cells results in few defects in oocyte maturation and ovulation. These data identify specific steps in the process of ovulation that require miRNA pathway gene activity in the somatic gonad cells