Innovative Viral Immunity initiated by Dicer

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1 Innovative Viral Immunity initiated by Dicer Yasuko Kitagishi, Mayumi Kobayashi and Satoru Matsuda Department of Food Science and Nutrition Nara Women's University, Japan

2 1 Introduction In eukaryotes, small RNA-mediated RNA silencing called RNA interference (RNAi) is able to suppress gene expression (Chang et al., 2011; Lilley et al., 2012). In mammalian cells, Dicer is a key enzyme of the RNAi pathway to cleave double-stranded RNA (dsrna) into small RNAs categorized as small interfering RNAs (sirnas) or micro-rnas (mirnas), which are largely involved in invasive nucleic acid defense and endogenous genes regulation, respectively (Katiyar-Agarwal & Jin, 2012; Ohrt et al., 2012) (Figure 1). The mirnas are short noncoding RNAs, which act on protein-encoding mrnas. Expressed from endogenous genes, mirna target the coding mrna for translational repression or degradation. The mirna pathway has been shown to be crucial in embryonic development, as shown by Dicer knockout analysis in mice (Bernstein et al., 2003). Specific patterns of mirnas have been reported to be expressed only in ES cells and in early phases of embryonic development. The mirnas have appeared as key regulators of stem cells (Calabrese et al., 2007). Similarly, Dicer in mammals is involved in a lot of tissue-developmental stages. Endogenous sirnas have been shown to be essential for oocyte maturation (Liu et al., 2010). Figure 1: Schematic representation and overview of Dicer signaling for RNA silencing. Several components of the RNAi cascade are important toward maturation of sirnas and mirnas. Examples of molecules known to act on the regulatory pathways are shown. Note that some critical pathways have been omitted for clarity.

3 The RNAi is an evolutionarily conserved gene silencing process triggered by dsrnas. Common to all cell types, is the production of about 21 nucleotides sirnas, which control the RNA-induced silencing complex (RISC) to identify and cleave target mrna sequences, participating in both the antiviral immune response and developmental regulation. For example, Drosophila harboring the Dicer mutant exhibited enhanced disease susceptibility to Drosophila C virus and cricket paralysis virus (van Rij et al., 2006). In addition, Caenorhabditis elegans harboring the Dicer mutant had developmental phenotype defects (Grishok et al., 2001; Ambros et al., 2003). The RNAi is a conserved eukaryotic gene silence mechanism that works at both the transcriptional and the posttranscriptional levels. Since its discovery in the end of 1990s by Fire and Mello (Fire et al., 1998), the RNAi has proven the most useful tool for scientists working in the fields of functional genomics, molecular biology, biotechnology, and medical therapeutic development. We fortuitously cloned and sequenced the human Dicer, (initially designated as HERNA) for the first time (Matsuda et al., 2000). Despite many advances, however, some of RNAi mechanisms are still under development. This review will mainly focus on the advances in understanding the intracellular mechanisms of the key components of the Dicer-initiated viral immunity. We also review studies on the features of small RNAs from virus-infected insects, plants, animals and discuss the properties of RNA-based antiviral immunity. 2 Structure and Characteristics of Dicer In mammalian cells, Dicer was identified as the ribonuclease that functions at the initiation stage of RNAi (Bernstein et al., 2001; Hutvágner et al., 2001). Dicer belongs to the RNase III family, a group of enzymes that exhibit specificity for dsrna, with ATP dependent RNA helicase, PAZ (Piwi/Argonaute/Zwille), dsrna binding, and RNase III domains (Figure 2). Dicer is responsible for cleaving long dsrnas or hairpin dsrna regions of single-stranded RNAs into sirnas or mirnas. The precursor mirnas are exported to the cytoplasm, and are further processed by Dicer to generate the mature duplex mirnas. The PAZ domain binds the single stranded 3 end of small RNA, and it might also function in protein-protein interaction (Yan et al., 2003; Lingel et al., 2003). All RNase III enzymes encode a homologous ribonuclease domain known as the RNase III domain. The dsrna cleavages by RNase III produce dsrna fragments with a structure consisting of a 5 -phosphate group and a twonucleotide overhang at the 3 -end (Ji, 2008). The RNase III usually acts as a homo-dimer (Zhang et al., 2004). Figure 2: Schematic representation of the predicted consensual domain structure for human Dicer protein.

4 Most vertebrates are reported to have only one Dicer-1 protein, which generates both mirnas and sirnas (Birchler & Kavi, 2008). While insects, fungi, and plants have more than one Dicer or Dicer-like proteins, Dicer enzymes in Drosophila melanogaster are classified into Dicer-1 and Dicer-2 in terms of their specialized functional activities (Ye & Liu, 2008). Dicer-1 can process loop pre-mirna to mature mirna, while Dicer-2 can process dsrna precursors into sirnas molecules. The Dicer-2 of Drosophila melanogaster participated in antiviral responses by processing viral RNAs into anti-viral small RNAs (Wang et al., 2006). The Dicer-1 of Caenorhabditis elegans participates in fragmenting chromosomal DNA during apoptosis, and undergoes a protease-mediated conversion from a ribonuclease to a deoxyribonuclease in addition to the processing of small RNAs (Nakagawa et al, 2010). Both sirnas and mirnas are Dicer products processed from dsrna and hairpin dsrna regions of single-stranded RNA precursors (Figure 1). Small RNAs includes PIWI-associated RNAs (pirnas), short single stranded RNAs arising from a Dicer-independent pathway, which are found in germ cells and associate with the PIWI subfamily of Argonaute proteins (Saito et al., 2009). Many zebrafish pirnas are derived from repetitive sequences (Thomson & Lin, 2009). Mutations in the Piwi homologue protein result either in loss of germ cells or in defects in meiosis and chromosome segregation in Drosophila eggs (Blumenstiel et al., 2008). These small RNAs are found in effector complexes such as RISC that contain an Argonaute protein as an essential component (Gagnon & Corey, 2012; Naqvi et al., 2012). Upon viral infection, insects and plants produce virus-derived sirnas to direct antiviral immunity (van Mierlo et al., 2011; Pantaleo, 2011). By contrast, infection of mammals with certain nucleus replicating DNA viruses induces production of virus-derived mirnas capable of silencing mrnas of the viruses (Aliyari & Ding, 2009; Parameswaran et al., 2010; Perez et al., 2010). Genetic studies showed a critical requirement for Dicer in vivo. However, recent studies have demonstrated that Dicer can function in an RNAi pathway independent manner (Wang et al., 2006; Aoki et al., 2007). Ablation of Dicer in the mouse germ-line produces a lethal phenotype, and conditional deletion of Dicer in various hematopoietic lineages has been shown an impaired cell differentiation, proliferation, and survival (Guo et al., 2010; Mizoguchi et al., 2010). Disruption of Dicer has also a major impact on the overall immune system. Similarly, Dicer deletion in developing B cells, NK cells (Zhou et al., 2009) and thymocytes result in increased cell death, suggesting that the Dicer pathway plays an important role in controlling immune cell survival. The functionally important sites are shown. Note that the sizes of each domain are modified for clarity. Helicase: N-terminal helicase domains DUF283: unknown function PAZ: Pinwheel-Argonaute-Zwille domain, which binds to sirnas RNase III: bidentate ribonuclease III domains dsrbd: double strand RNA binding domain 3 Innate immunity and Dicer The innate immune response provides primary defense against infection by external pathogens, which represents an early defense mechanism conserved in various multicellular creatures (Faure & Rabourdin- Combem, 2011). The immune receptors play a key role in innate immunity against pathogens. These

5 receptors are referred to as pattern recognition receptors, as they recognize conserved molecular patterns associated with microbes. The Toll-like receptors, the NOD-like receptors and the RIG-I-like DExD/H box RNA helicases are among the best characterized molecules of pattern recognition receptors (Opitz et al., 2010; Lech et al., 2010). These receptors interact with microbial signatures shared by major classes of microbes and activate the downstream signaling events, leading to transcription of effector genes with anti-microbial activities. Both of the sirnas and mirnas are potent activators of the innate immune system (Han et al., 2011; Taganov et al., 2006). Because Dicer was found to be involved in the forming these small RNAs, it had predicted that Dicer might play a role in the initiation of the RNA silencingbased antiviral immunity, when virus-specific small RNAs were detected after viral infection. In eukaryotic RNA-based antiviral immunity, viral double-stranded RNA is recognized as a pathogenassociated molecular pattern and processed into the sirnas by the host Dicer. In other words, Dicer recognizes a viral RNA like some pattern recognition receptors to initiate protective immunity against the viruses. Dicer then processes the viral RNA into virus-derived small RNAs. By the way, recent studies indicate that prokaryotes also produce virus-derived small RNAs to guide antiviral defense (Brouns et al., 2008). Prokaryotes make use of small RNAs encoded by CRISPR (clustered regularly interspaced short palindromic repeat) to deliver immunity against bacteriophage (Mulepati et al., 2012). Dicer also generates functional mirnas, which are also known to influence the fate of immune cells as well as to regulate adaptive immune responses (Schlauder et al., 2009). Those RNA silencing provide protection against various RNA viruses (Figure 3). Figure 3: Small RNAs-dependent functions of Dicer. Schematic illustrations of the tentative model for immunological and developmental functions of Dicer are shown. Note that implication of sirna and mirna in immunity against viruses is depicted for easy to understand. Natural antiviral defense mechanism exhibits features of both innate and adaptive immunity. The innate immune response induces cytokines production, which impacts downstream activation of adaptive

6 immunity. Synthetic sirna in delivery vehicles can also induce inflammatory cytokines and interferons after systemic administration (Judge & MacLachlan, 2008). This activation is predominantly mediated by immune cells, normally via a Toll-like receptor pathway (Schlee et al., 2006). The sirna sequence dependency of these pathways varies with the type and location of the TLR involved. Alternatively, nonimmune cell activation may also occur. The virus-derived sirnas conduct specific antiviral immunity through RNA interference and related RNA silencing effector mechanisms. New developments in this field implicate RNAi in the innate immune response to infection in insects, plants, animals, and so forth. However, the induction of innate immunity by sirna is dependent on sirna structure and sequence, method of delivery, and cell type. 4 Dicer functions involved in antiviral machinery 4.1 Insect Insect RNA viruses induce production of virus-derived small RNAs of predominantly 21 nucleotides in length, which are sirnas processed by Dicer-2 (Dcr-2) (Kim et al., 2006). Thus, insect RNA based antiviral immunity restricts infection of diverse RNA viruses, which induce production of viral sirnas. For example, Drosophila mounts an innate immune response to insect viruses that requires the RNAi machinery. Double stranded RNA intermediates of RNA viruses are recognized by Dcr-2 and further processed into virus-derived sirnas to guide silencing of the cognate viral RNAs by Argonaute-2 in Drosophila. Mosquito RNAi is also the major innate immune pathway controlling arbovirus infection and transmission (Blair, 2011). This innate response is distinct from known bacterial and fungal defense systems provided by the Toll and immune deficiency pathways. In fruit flies and nematodes, dsrna replicative intermediates of viral RNA genomes are also recognized and processed into virus-derived sirnas by Dicer (Wu et al., 2010). In Drosophila melanogaster, Dcr-1 and Dcr-2 initiate the mirna pathway and the sirna pathway, respectively (Okamura et al., 2011). The pirna pathway is Dicer independent. Drosophila cells that express both Ago2 and PIWI proteins produce virus derived pirnas of 24 to 30 nucleotides in addition to viral sirnas. Similar viral pirnas are detected in the mosquito cells that are Dcr-2 deficient. Caenorhabditis elegans secondary sirnas are also Dicer independent (Pak & Fire, 2007). Therefore, it is not perfect what features define the population of viral sirnas in an insect host in which potent antiviral silencing occurs. 4.2 Plants Pathogen-responsive endogenous small RNAs also seem to regulate gene expression and adjustment of plant immunity. Evidences suggest that endogenous small RNAs and host RNA-silencing machinery represent a fundamental control in plant immune responses. Production of viral sirnas in plants involves multiple Dicer-like proteins (DCLs). Four DCLs of Arabidopsis thaliana plants have specific roles in the biogenesis of distinct classes of endogenous small RNAs (Blevins et al., 2006). mirnas are predominantly made by DCL1. DCL1 and DCL2 are required for the production of natural antisensederived sirnas. The 22 nucleotides viral sirnas produced by DCL2 in the presence of DCL4 often constitute viral small RNA population. DCL3 produces 24 nucleotides repeat-associated sirnas that target transposons and repetitive DNAs (Figure 3). The DCL4 dependent transacting sirnas lead RNA silencing of endogenous targets. Viral sirnas detected in Arabidopsis thaliana infected with positive-

7 strand RNA viruses are predominantly 21 nucleotides in length made by DCL4, which also produces endogenous transacting sirnas and sirnas targeting transgenes. Accordingly, elimination of both DCL4 and DCL2 leads to dramatically increase disease susceptibility to distinct RNA viruses (Mlotshwa et al., 2008). Plant transacting sirnas resemble secondary sirnas because production of transacting sirnas requires AGO1 (Argonaute1) -mediated cleavages of transcripts targeted by mirnas (Baumberger & Baulcombe, 2005). It is assumed that degradation of viral RNA by Dicer proteins alone is not sufficient for virus resistance. 4.3 Animals Infection of mammalian cells with DNA viruses also induces production of virus-derived mirnas. Viral transcripts are recognized by the RNA silencing machinary in mammals, which includes processing of primary mirnas by Drosha in the nucleus, then maturation of mirnas by Dicer in the cytoplasm (Jaskiewicz & Filipowicz, 2008). Viral mirnas function to target and regulate the expression of host genes in mammals (Pegtel et al., 2011; Plaisance-Bonstaff & Renne, 2011). Some of the known mammalian viral mirnas exhibit features of viral sirnas, suggesting that they act to silence viral sequences as part of mammalian immune responses to infection. Host-encoded mirnas have also been implicated in the regulation of innate and adaptive immune responses in mammals. For example, influenza virus infection of human respiratory cells induces primary mirna expression (Buggele et al., 2012). In response to host RNAi-dependent immunity, however, viruses have evolved several countermeasures. Both plant and animal viruses encode protein suppressors of RNA silencing (SRSs). Examples of mammalian SRSs are the human immunodeficiency virus type 1 Tat, influenza A virus NS1 and vaccinia virus E3L (Li et al., 2004). Human disease caused by ebola virus is marked by early immunosuppression of innate immune signaling events, involving silencing and sequestration of dsrna by the viral protein VP35 (Haasnoot et al., 2007; Fabozzi et al., 2011). The VP35 functions as an SRS. The VP35 could mask key recognition sites of Dicer to block the entry of viral dsrnas into the RNA silencing pathways. However, the molecular mechanism by which VP35 interacts with the RNAi pathway has not yet been well elucidated. 5 Perspective As studies have shed light on the RNA silencing pathway, remarkable progress has been made in understanding of the mechanism of RNAi, which plays a role in the defense against viral infection. In conclusion, Dicer may be critical in defining the actual modes of substrate recognition in precise processing of RNAs. Whether RNAi mechanisms, aside from its function in counteracting viruses, are also used to fight bacterial diseases remains unknown. In Arabidopsis, the bacterial component, flagellin, induces the expression of a specific mirna, which in turn leads to the down-regulation of the signaling pathways (Navarro et al., 2006). This down-regulation then increases the resistance to the infection. RNAi is also becoming a technique of choice both for analysing gene function and for drug target validation. In this process, sequence specific gene inhibition is initiated by sirnas. The possibility that exogenously delivered sirnas or endogenously expressed hairpin sirnas can cause the destruction of specific target mrna has been demonstrated. However, the key challenges for the development of sirnas as novel therapeutics are largely dependent on the development of suitable delivery agents. The

8 function of Dicer is not limited to mirna biogenesis, but also required for the processing of sirnas derived from endogenous dsrna transcripts and exogenous dsrnas of viruses. One of the obstacles of making sirnas as a therapeutic agent includes off-target effects and competition with endogenous mirnas for cellular mirna-processing machinery (Sioud, 2010). As immune activation by sirnabased drugs represents an undesirable side effect due to the considerable toxicities associated with excessive cytokine release in humans. Abrogating the activity will be critical for the development of safe and effective therapeutics. Furthermore, the translation of RNAi technology into the clinic depends on the development of new strategies to understand the competition between endogenous and exogenous dsrnas. Acknowledgments This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology in Japan. In addition, this work was supported in part by the grant from SHIN-EI Pharmaceutical Co., Ltd. The authors declare that they have no competing financial interests. Abbreviations RNAi: RNA interference RISC: RNA-induced silencing complex dsrna: double-stranded RNA mirnas: micro-rnas sirnas: small interfering RNAs pirnas: PIWI-associated RNAs, or PIWI-interacting RNAs SRSs: suppressors of RNA silencing Dcr-1: Dicer-1 Dcr-2: Dicer-2 DCLs : Dicer-like proteins Ago1: Argonaute1 PAZ: Pinwheel-Argonaute-Zwille domain, which binds to sirnas dsrbd: double strand RNA binding domain References Aliyari, R. & Ding, S. W. (2009). RNA-based viral immunity initiated by the Dicer family of host immune receptors. Immunol Rev, 227, Ambros, V., Lee, R. C., Lavanway, A., Williams, P. T., & Jewell, D. (2003). MicroRNAs and other tiny endogenous RNAs in C. elegans. Curr Biol, 13, Aoki, K., Moriguchi, H., Yoshioka, T., Okawa, K., & Tabara, H. (2007). In vitro analyses of the production and activity of secondary small interfering RNAs in C. elegans. EMBO J, 26,

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