Fly Antiviral RNA Silencing and miRNA Biogenesis Claim ARS2
2009; Cell Press; Volume: 6; Issue: 2 Linguagem: Inglês
10.1016/j.chom.2009.08.002
ISSN1934-6069
Autores Tópico(s)CRISPR and Genetic Engineering
ResumoIn plants and invertebrates, small silencing RNAs function in antiviral defense and developmental patterning through pathways that were so far considered genetically distinct. In a recent issue of Cell, Sabin and colleagues report the identification of Drosophila Ars2, a protein required for both these small RNA-mediated functions. In plants and invertebrates, small silencing RNAs function in antiviral defense and developmental patterning through pathways that were so far considered genetically distinct. In a recent issue of Cell, Sabin and colleagues report the identification of Drosophila Ars2, a protein required for both these small RNA-mediated functions. Viruses are obligate intracellular pathogens of all life forms, and organisms have evolved diverse mechanisms to combat their aggressions. One mechanism, RNA silencing, involves small RNAs, 19–30 nucleotides in length, that repress gene expression by annealing to target RNA or DNA. Silencing small RNAs include small-interfering RNAs (siRNAs) and microRNAs (miRNAs), which are both processed from double-stranded (ds)RNA by the RNaseIII Dicer. siRNAs derive from perfectly-to-near-perfectly base-paired dsRNAs of endogenous or exogenous origin, including viruses. miRNAs originate from primary miRNA transcripts (pri-miRNA) produced from independent transcription units found in intergenic or intronic nuclear DNA. pri-miRNAs contain imperfect intramolecular stem loops, which, in animals, are first excised in the nucleus by the Microprocessor complex, primarily composed of the Dicer-like enzyme Drosha and binding partner, Pasha. Dicer then cytoplasmically converts the resulting precursor miRNA (pre-miRNA) into a single, mature miRNA (Figure 1A). miRNAs are important cell-fate determinants; consequently, miRNA-deficient organisms display severe-to-lethal developmental defects. miRNAs and siRNAs incorporate RNA-induced silencing complexes (RISCs), assembled upon loading of one selected small RNA strand into an Argonaute (Ago) protein family member. siRNA- and miRNA-programmed RISCs then repress target gene expression through endonucleolysis or translational repression of fully or partially complementary mRNAs (Figures 1A and 1B). In Drosophila, miRNAs and siRNAs are produced by two separate Dicers, Dcr1 and Dcr2—and also distinctly effected by Ago1 and Ago2, respectively. The fact that many viruses infect and are transmitted by insects was probably an important driving force leading to the separation of siRNA and miRNA pathways in arthropods (Obbard et al., 2006Obbard D.J. Jiggins F.M. Halligan D.L. Little T.J. Curr. Biol. 2006; 16: 580-585Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Several studies have now established that Dcr2 generates siRNAs from viral dsRNA, a common replication intermediate of most viruses. siRNA-loaded Ago2 then executes antiviral silencing by targeting viral RNAs with sequences complementary to the siRNAs (Figure 1B). Accordingly, Drosophila dcr2 and ago2 mutants are developmentally normal, but hypersusceptible to viruses (reviewed in Ding and Voinnet, 2007Ding S.W. Voinnet O. Cell. 2007; 130: 413-426Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar). Sabin and colleagues now report the isolation of Ars2, a novel, general antiviral silencing factor in Drosophila. Ars2 was isolated in a small-throughput RNAi-based genetic screen aimed at identifying virus-restricting host factors in cultured S2 cells (Sabin et al., 2009Sabin L.R. Zhou R. Gruber J.J. Lukinova N. Bambina S. Berman A. Lau C.K. Thompson C.B. Cherry S. Cell. 2009; 138: 340-351Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The screen employed Vesicular Stomatitis virus, modified to contain a GFP reporter gene. Among ∼100 individual gene knockdowns tested, Ars2 depletion consistently caused a 5-fold increase in the number of GFP-positive cells. Several additional viruses displayed enhanced infection rates that did not result from increased internalization of viral antigens (measuring viral entry into cells), but rather from improved viral RNA replication possibly inherent to defects in the siRNA pathway, because the viruses tested were previously characterized as being susceptible to RNA silencing. Ars2-depleted cells were indeed less competent to silence expression of sensor transgenes reporting the activity of Dcr2-dependent siRNAs produced from exogenous or endogenous dsRNA. The effect was, however, bypassed if siRNAs were delivered directly into cells, suggesting that Ars2 acts upstream of RISC. Accordingly, Ars2 was required for endogenous siRNA accumulation and was coimmunoprecipitated with Dcr2. Moreover, extracts of Ars2-depleted cells produced less siRNAs from labeled dsRNA compared to extracts of control cells. Thus, Ars2 apparently facilitates Dcr2 action, possibly through direct protein-protein interactions. These findings are relevant to authentic infections because adult flies in which Ars2 expression was conditionally suppressed succumbed to viruses more rapidly than control flies. Ars2-depleted flies were not easily engineered, though: transposon insertions or constitutive RNAi were embryonic lethal. This was unexpected, since ago2 or dcr2 knockouts are innocuous to Drosophila, unlike miRNA pathway mutations. The closest Ars2 homolog, the Arabidopsis C2H2-Zinc-finger protein SERRATE (SE), is required for pri-to-pre-miRNA processing, and this hinted at the possibility that Ars2 could be similarly required in the fly miRNA pathway, in addition to its antiviral function. Ars2-depleted cells indeed exhibited compromised miRNA functions, owing to reduced pri-miRNA accumulation. This suggested that Ars2 might stabilize pri-miRNAs to facilitate their Microprocessor-dependent nuclear conversion into pre-miRNAs (Figure 1A). Coimmunoprecipitation of Ars2 and Pasha in S2 cells further supported this idea. A report from Gruber et al., in the same issue of Cell, directly implicates human Ars2 in pri-miRNA processing, and documents its interaction with CBP80, a subunit of the nuclear Cap-binding complex (CBC) (Gruber et al., 2009Gruber J.J. Zatechka D.S. Sabin L.R. Yong J. Lum J.J. Kong M. Zong W.X. Zhang Z. Lau C.K. Rawlings J. et al.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Remarkably, Arabidopsis with mutations in ABH1/CBP80 and CBP20 also show impaired pri-to-pre-miRNA processing and exhibit developmental defects overlapping with those of se mutants (Gregory et al., 2008Gregory B.D. O'Malley R.C. Lister R. Urich M.A. Tonti-Filippini J. Chen H. Millar A.H. Ecker J.R. Dev. Cell. 2008; 14: 854-866Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, Laubinger et al., 2008Laubinger S. Sachsenberg T. Zeller G. Busch W. Lohmann J.U. Ratsch G. Weigel D. Proc. Natl. Acad. Sci. USA. 2008; 105: 8795-8800Crossref PubMed Scopus (293) Google Scholar). Sabin and colleagues confirmed that the fly Cbp20 immunoprecipitates overexpressed Ars2, and that Cbp20 or Cbp80 depletion compromises silencing by miRNAs, and by exogenous and endogenous siRNAs. Also as in Ars2-depleted cells, pri-miRNA levels were reduced in CBC-depleted S2 cells, which, furthermore, supported greater replication of at least two viruses tested in the siRNA pathway experiments described above. The work of Sabin et al. put forward a tangible link between two fly silencing pathways so far considered as largely distinct: Ars2 could well represent a key factor that permitted the evolutionary transition from a gene-regulatory pathway operated by miRNAs to an innate immune pathway operated by siRNAs, (or vice versa, as it is presently impossible to state confidently which arose first). Unlike Dcr1 and Ago1, Dcr2, and Ago2 are among the fastest evolving 3% of all Drosophila genes (Obbard et al., 2006Obbard D.J. Jiggins F.M. Halligan D.L. Little T.J. Curr. Biol. 2006; 16: 580-585Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), and it will be interesting to investigate the extent to which this signature of host pathogens arms race is found in natural Ars2 variants among arthropods. Besides, the study raises many questions. The most pressing point is to clarify whether the roles of Ars2 in antiviral defense and pri-to-pre-miRNA processing entail similar mechanisms. The first uncertainty pertains to Ars2 expression and subcellular localization. Hence, Gruber et al. show that human Ars2 is nearly exclusively expressed in dividing cells, where it facilitates the differential accumulation of miRNAs specifying totipotency. Likewise, the role of SERRATE was discovered through the defects exhibited by se mutants in meristems, the stem cell niches of plant apexes. However, the fat body cells that normally accumulate viruses in infected adult flies are nonproliferating. Human Ars2 and Arabidopsis SERRATE are also very predominantly nuclear localized, yet many viruses tested by Sabin et al. are exclusively cytoplasmic. One testable way to reconcile the data is to propose that Ars2 is not only induced, but also cytoplasmically relocalized upon infection (Figure 1B). Second, plant and animal pri-miRNA are produced by PolII and, thus, are 5′-capped. This, evidently, provides an ideal scenario as to how Ars2 might recruit the Microprocessor to pri-miRNA through its interaction with nuclear CBC components (Figure 1A). A similar scenario cannot, however, apply to the antiviral Dcr2 complex, because at least one virus tested by Sabin et al. replicates via uncapped RNAs. The Sabin and Gruber studies therefore propose that Ars2 is a general, direct facilitator of cytoplasmic and nuclear RNase III activities across species. While work with SERRATE in Arabidopsis extracts supports the above hypothesis (Dong et al., 2008Dong Z. Han M.H. Fedoroff N. Proc. Natl. Acad. Sci. USA. 2008; 105: 9970-9975Crossref PubMed Scopus (314) Google Scholar), it remains possible that the mechanisms of Ars2-mediated antiviral defense and Ars2-mediated miRNA biogenesis overlap only partially. First, quantitative and/or qualitative profiling of viral small RNAs in wild-type (wt) versus Ars2-depleted S2 cells should provide stronger ground to the proposed role of Ars2 as a direct modulator of Dcr2-mediated processing of viral dsRNA (Figure 1B). Such analyses might also unravel specific viral RNA features resembling those of pri-miRNAs, which could preferentially attract Ars2 and Dicer-like enzymes on both types of templates. Limited changes in viral siRNA profiles between the two cell types, on the other hand, could point to more indirect contributions of Ars2 to antiviral silencing. Noteworthy, the induction of Vago, a cystein-rich polypeptide with antiviral activity in adult flies, requires the function of Dcr2 through as-yet-unspecified mechanisms (Deddouche et al., 2008Deddouche S. Matt N. Budd A. Mueller S. Kemp C. Galiana-Arnoux D. Dostert C. Antoniewski C. Hoffmann J.A. Imler J.L. Nat. Immunol. 2008; 9: 1425-1432Crossref Scopus (261) Google Scholar) (Figure 1C, left). Possible alterations in the levels of Vago (or other innate immune factors unrelated to silencing) in Ars2-depeleted cells thus warrant consideration. miRNAs also regulate innate and adaptive immunity across kingdoms, and their frequent dismissal as possible contributors to fly antiviral defense is mostly attributable to the unavailability of true dcr1 or ago1 hypomorphic mutations in Drosophila (Ding and Voinnet, 2007Ding S.W. Voinnet O. Cell. 2007; 130: 413-426Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar). Based on these premises, mysregulation of miRNA biogenesis in Ars2-deficient cells might also contribute indirectly to altered antiviral immunity. An important role for Ars2 in antiviral silencing also predicts that it will be targeted by one or several viral suppressors of RNA silencing (VSR), which are commonly deployed by insect and plant viruses against critical steps of the machinery (Ding and Voinnet, 2007Ding S.W. Voinnet O. Cell. 2007; 130: 413-426Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar). The B2 protein of flock-house virus (FHV) is a strong candidate because it binds FHV replication complexes to directly inhibit viral siRNA production by Dcr2 (Aliyari et al., 2008Aliyari R. Wu Q. Li H.W. Wang X.H. Li F. Green L.D. Han C.S. Li W.X. Ding S.W. Cell Host Microbe. 2008; 4: 387-397Abstract Full Text Full Text PDF Scopus (198) Google Scholar) (Figure 1B, right). Targeting of Ars2 by B2 might actually explain the relatively modest effects of Ars2 depletion observed by Sabin et al. on FHV replication. However, it is of note that expression of B2, or indeed of any VSR studied so far in Drosophila, incurs no apparent defects in the miRNA pathway, yet RNAi is usually strongly suppressed in S2 cells and adult flies (Berry et al., 2009Berry B. Deddouche S. Kirschner D. Imler J.L. Antoniewski C. PLoS ONE. 2009; 4: e5866Crossref Scopus (55) Google Scholar). Ultimately, genetic studies might clarify this question, particularly in flies, which are highly amenable to forward and reverse approaches. Use of available sensor transgene reporting miRNA as well as siRNA activities should facilitate the identification of point mutation alleles of Ars2 that could possibly uncouple its involvements in the two pathways. Meanwhile, the study of Sabin and colleagues prompts an evaluation of the antiviral role of SERRATE in plants, which has eluded characterization so far. Based on the findings of Gruber and colleagues, use of meristem-infecting viruses or geminiviruses known to induce plant cell proliferation might constitute a reasonable starting point in this endeavor. Ars2 Regulates Both miRNA- and siRNA- Dependent Silencing and Suppresses RNA Virus Infection in DrosophilaSabin et al.CellJuly 23, 2009In BriefIntrinsic 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. Full-Text PDF Open ArchiveArs2 Links the Nuclear Cap-Binding Complex to RNA Interference and Cell ProliferationGruber et al.CellJuly 23, 2009In BriefHere 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. Full-Text PDF Open Archive
Referência(s)