Ars2 regulates both miRNA- and siRNA- dependent silencing and suppresses RNA virus infection in Drosophila.

Intrinsic immune responses autonomously inhibit viral replication and spread. One pathway that restricts viral infection in plants and insects is RNA interference (RNAi), which targets and degrades viral RNA to limit infection. To identify additional genes involved in intrinsic antiviral immunity, we screened Drosophila cells for modulators of viral infection using an RNAi library. We identified Ars2 as a key component of Drosophila antiviral immunity. Loss of Ars2 in cells, or in flies, increases susceptibility to RNA viruses. Consistent with its antiviral properties, we found that Ars2 physically interacts with Dcr-2, modulates its activity in vitro, and is required for siRNA-mediated silencing. Furthermore, we show that Ars2 plays an essential role in miRNA-mediated silencing, interacting with the Microprocessor and stabilizing pri-miRNAs. The identification of Ars2 as a player in these small RNA pathways provides new insight into the biogenesis of small RNAs that may be extended to other systems.

Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.

Here we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation.

A conserved virus-induced cytoplasmic TRAMP-like complex recruits the exosome to target viral RNA for degradation.

RNA degradation is tightly regulated to selectively target aberrant RNAs, including viral RNA, but this regulation is incompletely understood. Through RNAi screening in Drosophila cells, we identified the 3'-to-5' RNA exosome and two components of the exosome cofactor TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex, dMtr4 and dZcchc7, as antiviral against a panel of RNA viruses. We extended our studies to human orthologs and found that the exosome as well as TRAMP components hMTR4 and hZCCHC7 are antiviral. While hMTR4 and hZCCHC7 are normally nuclear, infection by cytoplasmic RNA viruses induces their export, forming a cytoplasmic complex that specifically recognizes and induces degradation of viral mRNAs. Furthermore, the 3' untranslated region (UTR) of bunyaviral mRNA is sufficient to confer virus-induced exosomal degradation. Altogether, our results reveal that signals from viral infection repurpose TRAMP components to a cytoplasmic surveillance role where they selectively engage viral RNAs for degradation to restrict a broad range of viruses.

RNase III nucleases from diverse kingdoms serve as antiviral effectors.

In contrast to the DNA-based viruses in prokaryotes, the emergence of eukaryotes provided the necessary compartmentalization and membranous environment for RNA viruses to flourish, creating the need for an RNA-targeting antiviral system. Present day eukaryotes employ at least two main defence strategies that emerged as a result of this viral shift, namely antiviral RNA interference and the interferon system. Here we demonstrate that Drosha and related RNase III ribonucleases from all three domains of life also elicit a unique RNA-targeting antiviral activity. Systemic evolution of ligands by exponential enrichment of this class of proteins illustrates the recognition of unbranched RNA stem loops. Biochemical analyses reveal that, in this context, Drosha functions as an antiviral clamp, conferring steric hindrance on the RNA-dependent RNA polymerases of diverse positive-stranded RNA viruses. We present evidence for cytoplasmic translocation of RNase III nucleases in response to virus in diverse eukaryotes including plants, arthropods, fish, and mammals. These data implicate RNase III recognition of viral RNA as an antiviral defence that is independent of, and possibly predates, other known eukaryotic antiviral systems.

Dogma derailed: the many influences of RNA on the genome.

Epigenetic control of gene expression is a critical component of transcriptional regulation. Remarkably, the deposition of epigenetic modifications is often guided by noncoding RNAs. Although noncoding RNAs have been most often implicated in posttranscriptional gene silencing, these molecules are now emerging as critical regulators of gene expression and genomic stability at the transcriptional level. Here, we review recent efforts to understand the mechanisms by which RNA controls the expression or content of DNA. We discuss the role of both small RNAs and long noncoding RNAs in directing chromatin changes through histone modifications and DNA methylation. Furthermore, we highlight the function of RNA in mediating DNA cleavage during genome rearrangements and pathogen defense. In understanding the mechanisms of RNA control over DNA, the power of RNA may one day be harnessed to impact gene expression in a therapeutic setting.

Virus-induced translational arrest through 4EBP1/2-dependent decay of 5'-TOP mRNAs restricts viral infection.

The mosquito-transmitted bunyavirus, Rift Valley fever virus (RVFV), is a highly successful pathogen for which there are no vaccines or therapeutics. Translational arrest is a common antiviral strategy used by hosts. In response, RVFV inhibits two well-known antiviral pathways that attenuate translation during infection, PKR and type I IFN signaling. Despite this, translational arrest occurs during RVFV infection by unknown mechanisms. Here, we find that RVFV infection triggers the decay of core translation machinery mRNAs that possess a 5'-terminal oligopyrimidine (5'-TOP) motif in their 5'-UTR, including mRNAs encoding ribosomal proteins, which leads to a decrease in overall ribosomal protein levels. We find that the RNA decapping enzyme NUDT16 selectively degrades 5'-TOP mRNAs during RVFV infection and this decay is triggered in response to mTOR attenuation via the translational repressor 4EBP1/2 axis. Translational arrest of 5'-TOPs via 4EBP1/2 restricts RVFV replication, and this increased RNA decay results in the loss of visible RNA granules, including P bodies and stress granules. Because RVFV cap-snatches in RNA granules, the increased level of 5'-TOP mRNAs in this compartment leads to snatching of these targets, which are translationally suppressed during infection. Therefore, translation of RVFV mRNAs is compromised by multiple mechanisms during infection. Together, these data present a previously unknown mechanism for translational shutdown in response to viral infection and identify mTOR attenuation as a potential therapeutic avenue against bunyaviral infection.

Drosha as an interferon-independent antiviral factor.

Utilization of antiviral small interfering RNAs is thought to be largely restricted to plants, nematodes, and arthropods. In an effort to determine whether a physiological interplay exists between the host small RNA machinery and the cellular response to virus infection in mammals, we evaluated antiviral activity in the presence and absence of Dicer or Drosha, the RNase III nucleases responsible for generating small RNAs. Although loss of Dicer did not compromise the cellular response to virus infection, Drosha deletion resulted in a significant increase in virus levels. Here, we demonstrate that diverse RNA viruses trigger exportin 1 (XPO1/CRM1)-dependent Drosha translocation into the cytoplasm in a manner independent of de novo protein synthesis or the canonical type I IFN system. Additionally, increased virus infection in the absence of Drosha was not due to a loss of viral small RNAs but, instead, correlated with cleavage of viral genomic RNA and modulation of the host transcriptome. Taken together, we propose that Drosha represents a unique and conserved arm of the cellular defenses used to combat virus infection.

Small creatures use small RNAs to direct antiviral defenses.

Antiviral RNA silencing has been recognized as an important defense mechanism in arthropods against RNA viruses. However, the role of this pathway in DNA virus infection remains largely unexplored. A report in this issue of the European Journal of Immunology provides new insight into the role of RNA silencing in antiviral defense against DNA viruses. Huang and Zhang [Eur. J. Immunol. 2013. 137-146] found that the dsDNA virus white spot syndrome virus, an agriculturally important pathogen of shrimp, is targeted by the shrimp RNA-silencing machinery via the production of virus-derived siRNAs. Furthermore, the authors show that the RNA-silencing pathway, and crucially, Dicer-2, is important for restricting viral infection. This study provides novel insights not only into shrimp antiviral defenses but also potentially into antiviral immunity against DNA viruses in a larger spectrum of hosts, as discussed in this Commentary. Furthermore, this study may contribute to the future development of immune-based therapeutics to combat viral pathogens, not only in aquaculture, but also in insect vectors of human diseases.

lncRNA requirements for mouse acute myeloid leukemia and normal differentiation.

A substantial fraction of the genome is transcribed in a cell-type-specific manner, producing long non-coding RNAs (lncRNAs), rather than protein-coding transcripts. Here, we systematically characterize transcriptional dynamics during hematopoiesis and in hematological malignancies. Our analysis of annotated and de novo assembled lncRNAs showed many are regulated during differentiation and mis-regulated in disease. We assessed lncRNA function via an in vivo RNAi screen in a model of acute myeloid leukemia. This identified several lncRNAs essential for leukemia maintenance, and found that a number act by promoting leukemia stem cell signatures. Leukemia blasts show a myeloid differentiation phenotype when these lncRNAs were depleted, and our data indicates that this effect is mediated via effects on the MYC oncogene. Bone marrow reconstitutions showed that a lncRNA expressed across all progenitors was required for the myeloid lineage, whereas the other leukemia-induced lncRNAs were dispensable in the normal setting.

Dicer-2 processes diverse viral RNA species.

RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double-stranded (ds)RNA precursors by Dicer and guide the RNA-induced silencing complex (RISC) to targets. Although principles governing small RNA sorting into RISC have been uncovered, the spectrum of RNA species that can be targeted by Dicer proteins, particularly the viral RNAs present during an infection, are poorly understood. Dicer-2 potently restricts viral infection in insects by generating virus-derived siRNAs from viral RNA. To better characterize the substrates of Dicer-2, we examined the virus-derived siRNAs produced during the Drosophila antiviral RNAi response to four different viruses using high-throughput sequencing. We found that each virus was uniquely targeted by the RNAi pathway; dicing substrates included dsRNA replication intermediates and intramolecular RNA stem loops. For instance, a putative intergenic RNA hairpin encoded by Rift Valley Fever virus generates abundant small RNAs in both Drosophila and mosquito cells, while repetitive sequences within the genomic termini of Vaccinia virus, which give rise to abundant small RNAs in Drosophila, were found to be transcribed in both insect and mammalian cells. Moreover, we provide evidence that the RNA species targeted by Dicer-2 can be modulated by the presence of a viral suppressor of RNAi. This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi.

Transcriptional pausing controls a rapid antiviral innate immune response in Drosophila.

Innate immune responses are characterized by precise gene expression whereby gene subsets are temporally induced to limit infection, although the mechanisms involved are incompletely understood. We show that antiviral immunity in Drosophila requires the transcriptional pausing pathway, including negative elongation factor (NELF) that pauses RNA polymerase II (Pol II) and positive elongation factor b (P-TEFb), which releases paused Pol II to produce full-length transcripts. We identify a set of genes that is rapidly transcribed upon arbovirus infection, including components of antiviral pathways (RNA silencing, autophagy, JAK/STAT, Toll, and Imd) and various Toll receptors. Many of these genes require P-TEFb for expression and exhibit pausing-associated chromatin features. Furthermore, transcriptional pausing is critical for antiviral immunity in insects because NELF and P-TEFb are required to restrict viral replication in adult flies and vector mosquito cells. Thus, transcriptional pausing primes virally induced genes to facilitate rapid gene induction and robust antiviral responses.

Degradation of host microRNAs by poxvirus poly(A) polymerase reveals terminal RNA methylation as a protective antiviral mechanism.

The life cycle of several viruses involves host or virally encoded small noncoding RNAs, which play important roles in posttranscriptional regulation. Small noncoding RNAs include microRNAs (miRNAs), which modulate the transcriptome, and small interfering RNAs (siRNAs), which are involved in pathogen defense in plants, worms, and insects. We show that insect and mammalian poxviruses induce the degradation of host miRNAs. The virally encoded poly(A) polymerase, which polyadenylates viral transcripts, also mediates 3' polyadenylation of host miRNAs, resulting in their degradation by the host machinery. In contrast, siRNAs, which are protected by 2'O-methylation (2'OMe), were not targeted by poxviruses. These findings suggest that poxviruses may degrade host miRNAs to promote replication and that virus-mediated small RNA degradation likely contributed to 2'OMe evolution.

Global analysis of RNA secondary structure in two metazoans.

The secondary structure of RNA is necessary for its maturation, regulation, processing, and function. However, the global influence of RNA folding in eukaryotes is still unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach to identify the paired (double-stranded RNA [dsRNA]) and unpaired (single-stranded RNA [ssRNA]) components of the Drosophila melanogaster and Caenorhabditis elegans transcriptomes, which allows us to identify conserved features of RNA secondary structure in metazoans. From this analysis, we find that ssRNAs and dsRNAs are significantly correlated with specific epigenetic modifications. Additionally, we find key structural patterns across protein-coding transcripts that indicate that RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in animals. Finally, we identify and characterize 546 mRNAs whose folding pattern is significantly correlated between these metazoans, suggesting that their structure has some function. Overall, our findings provide a global assessment of RNA folding in animals.

The exoribonuclease Nibbler controls 3' end processing of microRNAs in Drosophila.

MicroRNAs (miRNAs) are endogenous noncoding small RNAs with important roles in many biological pathways; their generation and activity are under precise regulation [1-3]. Emerging evidence suggests that miRNA pathways are precisely modulated with controls at the level of transcription [4-8], processing [9-11], and stability [12, 13], with miRNA deregulation linked with diseases [14] and neurodegenerative disorders [15]. In the Drosophila miRNA biogenesis pathway, long primary miRNA transcripts undergo sequential cleavage [16-18] to release the embedded miRNAs. Mature miRNAs are then loaded into Argonaute1 (Ago1) within the RNA-induced silencing complex (RISC) [19, 20]. Intriguingly, we found that Drosophila miR-34 displays multiple isoforms that differ at the 3' end, suggesting a novel biogenesis mechanism involving 3' end processing. To define the cellular factors responsible, we performed an RNA interference (RNAi) screen and identified a putative 3'→5' exoribonuclease CG9247/nibbler essential for the generation of the smaller isoforms of miR-34. Nibbler (Nbr) interacts with Ago1 and processes miR-34 within RISC. Deep sequencing analysis revealed a larger set of multi-isoform miRNAs that are controlled by nibbler. These findings suggest that Nbr-mediated 3' end processing represents a critical step in miRNA maturation that impacts miRNA diversity.

Innate antiviral immunity in Drosophila.

The study of Drosophila, and other genetically tractable insects, has expanded our understanding of innate immunity and more recently antiviral innate mechanisms. The Drosophila antiviral program includes inflammatory signaling cascades as well as antiviral RNA silencing and autophagy. This review will highlight the recent discoveries in antiviral immunity in insects and will reveal some of the lessons learned.

RNAi screening for host factors involved in Vaccinia virus infection using Drosophila cells.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system (1). Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance (2,3,4). Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication (5). Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses (4,6,7,8, 9). Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.