InteractiveFly: GeneBrief

Ars2: Biological Overview | References

BIOLOGICAL OVERVIEW

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, Drosophila cells were screened for modulators of viral infection using an RNAi library. Ars2 was identified 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, it was found that Ars2 physically interacts with Dcr-2, modulates its activity in vitro, and is required for siRNA-mediated silencing. Furthermore, Ars2 plays an essential role in miRNA-mediated silencing, interacting with the Microprocessor and stabilizing primary miRNA (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 (Sabin, 2009).

Innate immunity is the most ancient line of defense against pathogens. In mammals, the innate immune system provides the initial response to infection and primes the adaptive immune response. In contrast, invertebrates and plants lack adaptive immunity and therefore rely solely on innate mechanisms to combat infections. Recent studies have identified RNA interference (RNAi) as an ancient, cell-intrinsic immune mechanism that controls RNA viruses in plants and insects. RNAi is one of several small RNA-dependent silencing pathways that control gene expression in a sequence-specific manner in plants and animals. Small RNA-driven silencing is initiated by an RNase III enzyme Dicer, which produces RNA duplexes of approximately 21 nucleotides through the cleavage of longer precursor molecules. Once generated, the duplex is incorporated into the multiprotein RNA-induced silencing complex (RISC) where one strand of the duplex is preferentially retained. This guide strand directs RISC to a homologous target, where an Argonaute protein (Ago) mediates posttranscriptional gene silencing (Sabin, 2009).

In Drosophila, small-interfering RNAs (siRNAs), which are derived from exogenous or endogenous sources of double-stranded RNA (dsRNA), are generated by Dicer-2 (Dcr-2) and incorporated into an Ago2-dependent RISC, leading to the degradation of complementary mRNAs or viral RNAs. In contrast, endogenous primary miRNA transcripts are processed into pre-miRNAs in the nucleus by the Microprocessor, which includes the RNase III enzyme Drosha and its binding partner Pasha. Next, the pre-miRNAs are exported and further processed by cytoplasmic Dicer-1 (Dcr-1) into mature miRNAs. Mature miRNAs are incorporated into an Ago1-dependent RISC and mediate translational inhibition of target transcripts (Sabin, 2009).

Studies using mutants in the known components of the classical siRNA pathway, including Dcr-2, r2d2, and AGO2, revealed the essential antiviral role that RNAi plays against positive-stranded RNA viruses in Drosophila (Galiana-Arnoux, 2006; van Rij, 2006; Wang, 2006). This study set out to identify additional cellular components of the Drosophila intrinsic antiviral arsenal. An unbiased screen for factors that, when lost, led to increased viral replication identified Ars2 (CG7843). Ars2 is a poorly characterized gene that is highly conserved and required for development in Arabidopsis, zebrafish, and mice. The best characterized Ars2 homolog is Arabidopsis SERRATE, which recently emerged as a component of the miRNA biogenesis pathway (Lobbes, 2006; Yang, 2006). In plants, Ars2 genetically interacts with the nuclear cap-binding complex (CBC) components ABH1/CBP80 and CBP20 (Laubinger, 2008). Both thus study and the work in the accompanying manuscript now reveal a physical interaction between Ars2 and the CBC in Drosophila and mammals (Gruber, 2009). It is further demonstrated that Ars2, along with the CBC, plays a role in antiviral immunity against a battery of RNA viruses and is required for both siRNA- and miRNA-mediated silencing, controlling the biogenesis of small regulatory RNAs (Sabin, 2009).

To identify novel genes that control viral replication, a small-scale screen was performed for cellular factors that allow increased viral replication in cells when depleted by RNAi. A Drosophila cell culture system was used for these studies due to the high potency of RNAi, low genomic redundancy, and lack of a complicating interferon response. To screen for such host factors, Drosophila cells with dsRNA were treated, incubated the cells for 3 days to allow for loss-of-function phenotypes, and the cells were challenged with the mammalian virus Vesicular Stomatitis Virus (VSV). VSV is an enveloped, negative-stranded RNA virus that is naturally transmitted to mammals from insects. To monitor infection, a recombinant virus was used that expresses the reporter GFP upon replication. Treatment with dsRNA against GFP is used as a positive control for silencing, as it is expressed by the virus upon replication. Luciferase dsRNA is used as a nontargeting control, as it is not expressed in this system. Using a fluorescence assay it was found that VSV readily infects Drosophila cells, and that RNAi-mediated depletion of GFP can be monitored by microscopy. Using this strategy approximately 100 genes were screened and CG7843, the Drosophila homolog of mammalian Ars2, which led to an increase in the percentage of VSV-infected cells when depleted by RNAi, was identified. To validate that the increase in infection was due to a depletion of Ars2 rather than an off-target effect, an independent dsRNA to Ars2 was generated and a similar increase in VSV infection was observed. Effective knockdown of Ars2 was verified by northern blot analysis (Sabin, 2009).

This study demonstrates that Ars2 and the cap-binding complex (CBC) are required for miRNA- and siRNA-mediated silencing as well as antiviral defense in Drosophila. Ars2 and the CBC are required at upstream steps in both Drosophila and mammalian RNA silencing pathways, consistent with recent data from plants; the Ars2 homolog SERRATE and homologs of the CBC (ABH1 and CBP20) control pri-miRNA processing in Arabidopsis (Gregory, 2008; Laubinger, 2008; Lobbes, 2006; Yang, 2006). Ars2 functionally and biochemically interacts with the nuclear Microprocessor, as does SERRATE, which physically interacts with HYL1, a component of the Arabidopsis miRNA biogenesis pathway similar in function to Drosophila Pasha and mammalian DGCR8 (Lobbes, 2006; Yang, 2006; Sabin, 2009 and references therein).

The results of these studies, combined with the evidence from existing literature, have led to the proposal of two potential models for Ars2 function. The first is a bridging model in which Ars2 serves as a recruitment factor to guide the RNA processing machinery to the proper substrates. Under this model, Ars2 and the CBC bind pri-miRNA transcripts through recognition of their 5' cap, and Ars2 actively recruits the Microprocessor to the transcript, promoting its cleavage into a pre-miRNA. This bridging model suggests a mechanism by which Ars2 and the CBC increase the efficiency of pri-miRNA processing by acting as chaperones to stabilize and deliver the primary transcripts directly to the Microprocessor. It then follows that in the absence of Ars2 or the CBC, Drosha-directed pri-miRNA processing is impaired since the recruitment of the Microprocessor to primary transcripts is less efficient, and the unprocessed transcripts are destabilized. The finding that pri-bantam levels are reduced in Ars2 or CBC-depleted cells along with similar findings in mammalian cells (Gruber, 2009) support this model. While the bridging model provides a compelling mechanism for the Ars2 and CBC requirement in the miRNA pathway, it is less likely to be relevant for the siRNA pathway, as the substrates of the pathway (dsRNA, viral RNAs) are not necessarily 5' capped. Although it is possible that Ars2 recruits Dcr-2 to uncapped substrates through the targeting of secondary RNA structure, the data favor an RNA recognition-independent model (Sabin, 2009).

This second model proposes that Ars2 serves as a cofactor for the enzymatic activity of RNase III enzymes. Under this model, the presence of Ars2 in Drosha- or Dcr-2-containing complexes promotes robust enzymatic cleavage of RNA substrates and increases the fidelity of processing. Consistent with this model, recent work by Dong has shown that the addition of recombinant SERRATE to an in vitro pri-miRNA processing assay enhances both the activity and the accuracy of DCL1 substrate cleavage (Dong, 2008). Moreover, Gruber demonstrate in the accompanying manuscript that mammalian pri-miRNA processing is altered in the absence of Ars2 (Gruber, 2009). Functional dicing assay demonstrates that Dcr-2-mediated processing of long dsRNA is impaired in the absence of Ars2, lending support to the idea that Ars2 is an essential accessory factor for Dcr-2 activity on uncapped RNAs. Since the substrates of the cytoplasmic siRNA pathway are not necessarily capped, this further argues that the CBC may be required for the cofactor activity of Ars2 rather than for substrate recognition (Sabin, 2009).

The underlying difference between the two models is the precise step of substrate recognition and processing for which Ars2 is required; the bridging model poses that Ars2 and the CBC physically bind the RNA substrate, recruiting the proper processing activity for cleavage. Conversely, the cofactor model suggests that the binding of Ars2 and the CBC to the processing machinery allows the enzymes to execute robust and accurate substrate cleavage. Of course, these models are not mutually exclusive, and the true function of Ars2 may combine aspects of both models. Ars2 may also play distinct roles depending on its particular binding partners or intracellular localization. Ultimately, this study implicates Ars2 as a fundamental component of several modes of RNA silencing and contributes to the growing body of evidence that RNA silencing pathways are more interconnected than previously appreciated (Zhou, 2008). The RNA content of a cell is influenced by the contributions of many transcriptional and posttranscriptional regulatory pathways that have evolved to respond quickly and sensitively to the needs of the cell. The identification of novel components of these pathways, such as Ars2, aids in efforts to uncover the mechanisms by which cellular processes such as proliferation or antiviral defense are exquisitely regulated (Sabin, 2009).

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

A component of the nuclear RNA cap-binding complex (CBC), Ars2, 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 impairs miRNA-mediated repression and leads to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduces 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 (Gruber, 2009).

When Ars2 cDNAs were isolated from a murine hematopoietic cell line the clones obtained had an open reading frame coding for a predicted protein of 875 amino acids, in contrast to the 225 amino acids coded for by the open reading frame of the cDNA originally isolated (Rossman, 1999). The predicted protein contained an amino-terminal arginine-rich domain and an RNA recognition motif in addition to the carboxy-terminal zinc finger domain originally identified. When expressed in either murine fibroblasts or the human cell line K562, this clone of Ars2 resulted in no increase in resistance to arsenic trioxide compared to control transfected cells at a range of doses (Gruber, 2009).

Since the original reported Ars2 clone appeared to be a truncation of the full-length gene product, it was reasoned that it may have functioned as a dominant negative. As determined by western blot analysis, 3T3 mouse embryonic fibroblasts (MEFs) infected with shRNA to Ars2 (shArs2-1) showed effective depletion of the protein after 4 days in culture, compared to cells treated with empty vector control retrovirus. When Ars2-depleted cells were treated with arsenic trioxide for 48 hr the cells exhibited a slower rate of cell death when compared to control infected cells treated with arsenic. However, few of the cells infected with Ars2 shRNA were able to proliferate when subsequently passaged in arsenic-free medium (Gruber, 2009).

To extend these findings, 3T3 clonal cell lines were generated (hp1-11 and hp2-9) expressing shRNAs targeted to two independent sequences within the Ars2 mRNA. These stable cell line clones showed reduced levels of Ars2 in comparison to two different vector control clonal cell lines. The sensitivity of these cell lines to arsenic trioxide was assessed by colony-forming assay. Ars2 knockdown cell lines consistently formed one-half the number of colonies as control cell lines (Gruber, 2009).

The inability of viable Ars2-deleted cells to recover following treatment with arsenic trioxide suggested the possibility that these cells had a proliferative defect. 3T3 MEFs were treated with Ars2 shRNA and population doublings measured over time in culture. shArs2-1 caused a near complete loss of detectable Ars2 protein and suppressed proliferation for 4 days in culture, whereas shArs2-2 caused a lesser amount of Ars2 depletion and had a more modest effect on cell proliferation. The proliferative defect of Ars2-deficient cells was associated with a reduced ability of Ars2 knockdown cells to incorporate BrdU following serum stimulation. Ars2-deficient cells did not arrest at a particular stage in the cell cycle or exhibit a significant amount of cell death until after they had ceased progressing through the cell cycle (Gruber, 2009).

The studies reported in this paper demonstrate that the Ars2 protein plays a critical role in the ability of mammalian cells to proliferate. In the absence of Ars2, mammalian cells are incapable of maintaining proliferative expansion in vitro. Consistent with a critical role in proliferation, Ars2 is selectively expressed in proliferating cells. In the absence of Ars2, cells undergo cell-cycle slowing at all stages of the cell cycle as evidenced by impairment of proliferation without discernable changes in the cell-cycle profile. A biochemical screen for Ars2-interacting proteins yielded CBP80, a protein previously shown to assemble on 7mG-capped transcripts. Further immunoprecipitation experiments revealed that Ars2 efficiently immunoprecipitates both core components of the CBC, CBP80 and CBP20 (Gruber, 2009).

An independent line of evidence supporting Ars2-CBC interactions comes from studies of Arabidopsis. Plants deficient in SERRATE, an Ars2 homolog, partially phenocopy plants with CBP80 (ABH1; ABA HYPERSENSITIVE 1) mutations. Both plants have pleiotropic developmental defects including increased cauline leaf number and serrated leaf morphology (Bezerra, 2004). SERRATE has been genetically implicated in a nuclear step of miRNA biogenesis in plants, and SERRATE-deficient plants have reduced mature miRNA levels (Grigg, 2005; Lobbes, 2006; Yang, 2006). To examine whether Ars2, as a constituent of the nuclear CBC, might have a similar effect on miRNA-mediated gene silencing in proliferating cells the ability of Ars2 to affect let-7-mediated repression in proliferating cells was examined. Suppression of either CBP80 or Ars2 was sufficient to disrupt let-7-mediated repression of a reporter transcript. These effects are likely due to a defect in miRNA biogenesis as Ars2-depleted cells had decreased levels of a subset of miRNAs including let-7 and miR-21. In contrast, miR-30a, which is implicated in the regulation of hepatobiliary development, and miR-16, which has been implicated in the regulation of apoptosis and proliferation, were not affected by depletion of Ars2 (Gruber, 2009).

Several lines of evidence indicate that Ars2 functions at the level of the primary miRNA transcript to promote miRNA maturation. The decreased levels of primary miRNA transcripts in the absence of Ars2 are different from the effect of Drosha or DGCR8 depletion, which causes stabilization of primary transcripts. This suggests that Ars2 and CBC contribute to the stability of pri-miRNAs in proliferating cells. As the CBC binds and protects the 5' cap structure of PolII transcripts, depletion of Ars2 or CBC may lead to increased decapping and transcript decay (Gruber, 2009).

As demonstrated in the accompanying manuscript (Sabin, 2009), Ars2 and the CBC are also required for miRNA function in Drosophila cells and interact with Drosophila homologs of the evolutionarily conserved Microprocessor complex. In addition, Sabin demonstrated in Drosophila that Ars2 and CBP20 are required for miRNA processing at the level of the pri-miRNA and that pri-miRNA levels decline in the absence of Ars2. Because the nuclear CBC is cotranscriptionally recruited to nascent transcripts it is ideally positioned to promote recruitment of the Microprocessor complex to pri-miRNA transcripts. However, the observed alteration in pri-miRNA processing in Ars2-depleted extracts argues for a more direct role for Ars2 in modulating the activity of the Microprocessor complex. Consistent with these findings, recent work in plants has shown that the Ars2 homolog SERRATE increases the processing efficiency and accuracy of DCL1 in vitro (Dong, 2008). Sabin also report decreased processing activity of Dcr-2 in extracts depleted of Drosophila Ars2. Taken together, these observations suggest that Ars2 homologs may contribute to the regulation of RNase III enzyme complexes in several species (Gruber, 2009).

The data from Sabin suggest that Ars2 also is a required component of small RNA-mediated viral suppression. While depletion of Ars2 from adult flies had no effect on organismal viability under baseline conditions, a requirement for Ars2 emerged during viral infection. Adult flies do not depend on cell proliferation for organismal viability or viral immunity. Thus, Ars2 may play an inducible role in small RNA regulation in nonproliferating cells when infected. Based on the data, it appears that Ars2 is not essential for miRNA processing during cell quiescence but is required to maintain the efficiency of miRNA-mediated silencing as cells commit to engage in cell division or are subjected to viral infection. Recent studies have shown that translational repression of target mRNAs by miRNAs is relieved upon exit from the cell cycle. Induction of Ars2 expression upon entry to cell proliferation may regulate the repression of genes specific to states of cellular quiescence. Alternatively, as cells engage in increased and more complex gene expression associated with cell division, compensatory upregulation of the efficiency or specificity of miRNA production may be required to meet the complex demands of exponential growth (Gruber, 2009).

ARS2 is a conserved eukaryotic gene essential for early mammalian development

ARS2 is an evolutionarily conserved gene that confers arsenite resistance on arsenite-sensitive Chinese hamster ovary cells. Little is known regarding the function of ARS2 in mammals. This study reports that ARS2 is transcribed throughout embryonic development and is expressed ubiquitously in mouse and human tissues. The mouse ARS2 protein is predominantly localized to the nucleus, and this nuclear localization is ablated in ARS2-null embryos, which in turn die around the time of implantation. After 24 h of culture, ARS2-null blastocysts contained a significantly greater number of apoptotic cells than wild-type or heterozygous blastocysts. By 48 h of in vitro culture, null blastocysts invariably collapsed and failed to proliferate. These data indicate ARS2 is essential for early mammalian development and is likely involved in an essential cellular process. The analysis of data from several independent protein-protein interaction studies in mammals, combined with functional studies of its Arabidopsis ortholog, SERRATE, suggests that this essential process is related to RNA metabolism (Wilson, 2008).

Search PubMed for articles about Drosophila Ars2

Bezerra, I. C., et al. (2004). Lesions in the mRNA cap-binding gene ABA HYPERSENSITIVE 1 suppress FRIGIDA-mediated delayed flowering in Arabidopsis, Plant J. 40: 112-119. PubMed ID: 15361145

Dong, Z., Han, M. H. and Fedoroff, N. (2008). The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc. Natl. Acad. Sci. 105: 9970-9975. PubMed ID: 18632569

Galiana-Arnoux, D., et al. (2006). Essential function in vivo for Dicer-2 in host defense against RNA viruses in Drosophila. Nat. Immunol. 7: 590-597. PubMed ID: 16554838

Gregory, B. D., et al. (2008). A link between RNA metabolism and silencing affecting Arabidopsis development. Dev. Cell 14: 854-866. PubMed ID: 18486559

Grigg, S. P., Canales, C., Hay, A. and Tsiantis, M. (2005). SERRATE coordinates shoot meristem function and leaf axial patterning in Arabidopsis, Nature 437: 1022-1026. PubMed ID: 16222298

Gruber, J. J., et al. (2009). Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation. Cell 138: 328-339. PubMed ID: 19632182

Laubinger, S., et al. (2008). Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc. Natl. Acad. Sci. 105: 8795-8800. PubMed ID: 18550839

Lobbes, D., et al. (2006). SERRATE: a new player on the plant microRNA scene. EMBO Rep. 7: 1052-1058. PubMed ID: 16977334

Rossman, T. G. and Wang, Z. (1999). Expression cloning for arsenite-resistance resulted in isolation of tumor-suppressor fau cDNA: possible involvement of the ubiquitin system in arsenic carcinogenesis. Carcinogenesis 20: 311-316. PubMed ID: 10069470

Sabin, L. R., et al. (2009). Ars2 regulates both miRNA- and siRNA- dependent silencing and suppresses RNA Virus infection in Drosophila. Cell 138: 340-351. PubMed ID: 19632183

van Rij, R. P., et al. (2006). The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster. Genes Dev. 20: 2985-2995. PubMed ID: 17079687

Wang, X. H., et al. (2006). RNA interference directs innate immunity against viruses in adult Drosophila. Science 312: 452-454. PubMed ID: 16556799

Wilson, M. D., et al. (2008). ARS2 Is a conserved eukaryotic gene essential for early mammalian development. Mol. Cell. Biol. 28: 1503-1514. PubMed ID: 18086880

Yang, L., et al. (2006). SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis. Plant J. 47: 841-850. PubMed ID: 16889646

Zhou, R., et al. (2008). Comparative analysis of argonaute-dependent small RNA pathways in Drosophila. Mol. Cell 32: 592-599. PubMed ID: 19026789

date revised: 22 March 2022

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