Screens of three major autosomal arms of Drosophila have identified more than 15 loci that when mutated result in stronger pigmentation in a GMR-wIR background. One such locus, identified in the screen of the right arm of the second chromosome, was a homozygous viable complementation group consisting of 39 alleles. Noncomplementation was based on a strongly suppressed eye color phenotype in the presence of GMR-wIR. To genetically map the locus, Drosophila single nucleotide polymorphism (SNP) markers were used. Mapping placed the locus within a 568 kb interval of 54C, an interval that contains a Dicer gene. Drosophila contains two genes in the Dicer family, dicer-1 and dicer-2 (Lee, 2004).
The dcr-2 gene is located within the interval that contained the complementation group of suppressor mutations. The dcr-2 gene was sequenced in six independent mutants, and each mutant had base changes that significantly altered the predicted protein product. Two alleles contained premature stop codons that would produce truncated proteins, lacking the RNase III domains essential for dsRNA processing activity. These two mutations likely represent null alleles of dcr-2. To confirm that the mutant complementation group corresponded to dcr-2, transformation rescue was performed with a 7.2 kb genomic fragment that spans the dcr-2 transcription unit. This fragment completely rescued the phenotype associated with a homozygous dcr-2 null mutation (Lee, 2004).
It was next asked if the phenotype associated with dcr-2 mutants resulted from a defect in dsRNA processing. The levels of siRNAs generated from GMR-wIR were examined in the eyes of wild-type and dcr-2 mutants. The dcr-2 null mutants exhibit a large reduction in siRNA levels when compared to wild-type. This reduction did not result from instability or low-level expression of GMR-wIR dsRNA, since GMR-wIR precursor RNAs were present at levels comparable to wild-type. These data indicate that Dcr-2 plays a major role in dsRNA processing. Interestingly, substitution mutants in the Dcr-2 helicase domain were as impaired for siRNA production as null mutants. One of these, the dcr-2G31R mutant, changes one of the invariant GXGXXG residues in the ATP binding site of the helicase domain. Thus, Dcr-2 requires a functional helicase domain for dsRNA processing (Lee, 2004).
Flies homozygous for null dcr-2 alleles are viable and fertile, and are morphologically normal in external appearance. Since miRNAs are indispensable for growth and development in Drosophila, the dcr-2 phenotype suggests that Dcr-2 is not essential for pre-miRNA processing. To address this, levels of the miRNA let-7 were examined in dcr-2 null mutants. The dcr-2 mutants exhibited mature let-7 RNA levels comparable to those of wild-type controls. This confirms that Dcr-2 is not required for the processing of pre-miRNAs (Lee, 2004).
Previous work showed that RNAi is established in the Drosophila female germline (Kennerdell, 2002). To examine whether Dcr-2 is required for mRNA degradation in eggs, dcr-2 mutant eggs were injected with dsRNA corresponding to the bicoid gene, which is maternally expressed. Subsequently, bicoid mRNA levels were assayed by RT-PCR. Wild-type eggs displayed rapid reduction in bicoid transcript abundance after dsRNA injection. In contrast, dcr-2 null mutant eggs showed no significant reduction in bicoid transcript abundance, indicating that dcr-2 is required for effective RNAi in the female germline. A similar effect was observed in dcr-2 mutants bearing substitutions in the Dcr-2 helicase domain (Lee, 2004).
It was next asked whether the RNAi defect in dcr-2 eggs is simply due to defective siRNA production. To test this hypothesis, eggs were injected with a synthetic siRNA corresponding to the bicoid gene and subsequently bicoid transcript levels were assayed. Wild-type eggs exhibited loss of bicoid mRNA in response to siRNA injection. In contrast, dcr-2 null mutant eggs exhibited an impaired RNAi response to siRNA. Five-fold more bicoid mRNA was present in dcr-2 mutant eggs compared to wild-type eggs after siRNA treatment. This result indicates that Dcr-2 also functions downstream of siRNA production in the RNAi pathway. Interestingly, substitution mutants in the Dcr-2 helicase domain were unimpaired for siRNA-dependent RNAi, suggesting that a functional helicase activity is not required for Dcr-2 to mediate its downstream function (Lee, 2004).
These experiments established an important though not absolute role for Dcr-2 in Drosophila RNAi. Since another Dicer (Dcr-1) is present in Drosophila, it is possible that it has a redundant function with Dcr-2. The dcr-1 gene is located at 94C4 on the third chromosome, and a mutation was found that mapped by linkage and complementation analysis to that region. The coding sequence of dcr-1 in the mutant contained a premature stop codon such that the truncated product lacks the PAZ and RNase III domains. Moreover, dcr-1 mRNA is not detectable in the mutant as determined by RT-PCR and Northern blot analysis, suggesting that the transcript is unstable when truncated Dcr-1 protein is produced. This transcript null dcr-1Q1147X mutant exhibited an eye color phenotype when assayed in a GMR-wIR genetic background. The mutant appeared to partially suppress silencing by GMR-wIR, with patches of dark orange eye color. In addition, the eye was half its normal size, the organization of ommatidial facets was disrupted, and sensory bristles were missing over the eye surface. Other bristles, which flank the eye surface, were sometimes absent or exhibited hyperplasia (Lee, 2004).
Despite an effect on white gene silencing, the dcr-1 mutant had normal levels of wIR siRNAs. This observation is consistent with Dcr-2 processing the great majority of wIR dsRNA. It is further consistent with a central role for Dicer helicase activity in dsRNA processing, since Dcr-1 lacks a DExH-box helicase domain. If the dcr-1 mutant has normal dsRNA processing, why is it partially disrupted for gene silencing? To answer this, clones of homozygous dcr-1 mutant germ cells were generated in heterozygous females, and then dcr-1 mutant eggs were injected with either dsRNA or siRNA complementary to bicoid transcripts. Loss of bicoid mRNA was measured as a consequence. dcr-1 mutant eggs exhibit an impaired RNAi response to dsRNA and siRNA. Six-fold more bicoid mRNA was present in dcr-1 mutant eggs compared to wild-type eggs after either dsRNA or siRNA treatment. This result indicates that Dcr-1 acts downstream of siRNA production in the RNAi pathway. Dcr-1 plays an important though not absolute role in siRNA-dependent RNAi. Since Dcr-2 is also required downstream of siRNAs, these data suggest that Dcr-1 and Dcr-2 function might be partially redundant in some downstream activity (Lee, 2004).
Dcr-1 is essential to generate mature miRNAs. This role was demonstrated by analyzing miRNA levels in dcr-1 mutant eggs. No mature miRNAs belonging to the miR-2 group were detected in dcr-1 mutant eggs. Thus, Dcr-1 is critical for miRNA production whereas Dcr-2 is required primarily for siRNA production (Lee, 2004).
Although Dcr-1 and Dcr-2 preferentially produce different types of small RNAs, both are required for efficient siRNA-dependent mRNA degradation. Does this dual requirement extend to the miRNA pathway as well? To test this possibility, a genetic assay for miRNA-dependent gene silencing was used in dcr-1 and dcr-2 mutants. Several classes of motifs are present in the 3′UTR regions of the E(spl) and Bearded genes (Lai, 1998; Lai, 1997). The 3′UTR motifs are complementary to a variety of miRNAs, and they mediate posttranscriptional repression of gene expression (Lai, 2002). A series of reporter transgenes was constructed that mimics this posttranscriptional repression (Lai, 1998; Lai, 1997). The reporter genes contain a constitutive promoter from armadillo, lacZ coding sequence, and the 3′UTR from the Bearded or E(spl)m8 gene. When the reporter contains a wild-type Bearded 3′UTR, its expression in the developing eye disc is very weak. It is somewhat more strongly expressed in the eye disc posterior to the morphogenetic furrow and is equally weak in the anterior eye disc and antennal disc. When the reporter contains a Bearded 3′UTR with its three B motifs mutated, expression is ubiquitously strong in the eye and antennal discs, confirming that the B motifs mediate a silencing effect on gene expression (Lee, 2004).
Expression of a wild-type reporter gene was examined in clones of mutant dcr-2 cells that were generated in the developing eye disc. Clones expressed the reporter at levels indistinguishable from wild-type tissue, indicating that Dcr-2 is not required for this gene silencing mechanism. In contrast, expression of a wild-type reporter gene in clones of mutant dcr-1 cells was much stronger than in wild-type tissue. The derepressive effect of the dcr-1 mutation requires intact B motifs in the Bearded 3′UTR, since mutant clones did not affect expression of a reporter gene with mutated B motifs. These results argue that dcr-1 but not dcr-2 is necessary for posttranscriptional gene silencing that is mediated by a miRNA mechanism. This conclusion is also validated by other mutant phenotypes associated with each gene. Loss of dcr-1 has profound effects on Drosophila development within both somatic- and germ-lineages, whereas loss of dcr-2 appears to have little or no effect on development (Lee, 2004).
The dcr-1 mutant clones exhibited an interesting pattern of reporter expression. Clones in the antennal disc and eye disc, anterior to the morphogenetic furrow, exhibited little or no derepression of the reporter gene. Typically, only a few mutant cells in each clone had high levels of reporter gene expression. No overt cell differentiation occurs in this region of the eye disc. In contrast, almost all eye disc clones posterior to the furrow, where cell differentiation actively occurs, exhibited extensive derepression. Many, if not most, mutant cells in a clone exhibited this behavior. A boundary of reporter gene expression within a clone could be detected if the clone was bisected by the furrow. This boundary coincided with the morphogenetic furrow. Three interpretations seem possible. (1) Different sets of miRNAs repress the reporter in different regions of the eye disc, one set of which requires Dcr-1 and one set of which does not. However, all of these miRNAs would have to act through the 3′UTR binding sites, since a mutated reporter is constitutively derepressed. Thus, this interpretation is not favored. (2) Dcr-1 might not be essential in anterior disc cells because Dcr-2 or another factor substitutes if Dcr-1 is missing. (3) Anterior disc cells may contain miRNAs that were originally generated in dcr-1+ progenitor cells, and may therefore not require dcr-1. Passage of the morphogenetic furrow may trigger miRNA turnover, resulting in renewed dependence on dcr-1 posterior to the furrow (Lee, 2004).
The RNAi pathway can be divided into discrete biochemical steps: dsRNA processing, maintenance of siRNA 5′ phosphate termini, siRNA loading into siRISC, and siRISC-based cleavage of target mRNA. It was confirmed that Dcr-2 but not Dcr-1 is required for dsRNA processing by incubating radiolabeled dsRNA substrate in lysates made from mutant embryos and monitoring siRNA formation. Labeled 21–23 nucleotide RNAs were readily detectable from reactions with wild-type and dcr-1 lysates, but were greatly reduced in reactions with dcr-2 lysate (Lee, 2004).
The genetic experiments suggested a role for Dcr-1 and Dcr-2 downstream of dsRNA processing. Therefore, lysates made from mutant embryos were assayed for steps downstream of siRNA production. siRNAs require 5′ phosphate termini for proper association with RISC, and the 5′ phosphates are maintained by a kinase that recognizes siRNAs (Nykanen, 2001). Both wild-type and dcr-2 mutant lysates efficiently converted synthetic siRNAs bearing 5′ hydroxyl groups into 5′-phosphorylated forms, indicating that the mutant lysates have normal end-maintenance activity (Lee, 2004).
Nykanen (2001) reported that siRNAs are incorporated into a 350 kDa complex when incubated with embryo lysate. The complex is converted to active siRISC using ATP hydrolysis. Radiolabeled siRNA was incubated with wild-type or dcr-2 mutant embryo lysates in the presence of ATP, and then the products were size-fractionated by gel filtration chromatography. siRNA was predominantly associated with a 350 kDa complex in wild-type lysate. However, most of the siRNA in the dcr-2 mutant lysate fractionated as if unbound by proteins, indicating that Dcr-2 is essential for siRNA entry into functional complexes. Thus, dcr-2 lysates should be defective for siRNA-directed mRNA cleavage. SiRNA complementary to a radiolabeled target mRNA was coincubated with embryo lysate. Incubation in wild-type lysate produced a truncated mRNA whose length was consistent with it being the 5′ cleavage product. Incubation in dcr-2 lysate generated 5- to 15-fold less cleavage product. This indicates that Dcr-2 is required for mRNA target cleavage by a siRNA, and is consistent with the in vivo observations (Lee, 2004).
To examine siRISC formation in dcr-1 mutant lysate, native gel electrophoresis was used. Gel filtration chromatography requires large lysate volumes, which could not be obtained from dcr-1 embryos. Pham (2004) has developed a gel electrophoretic method to characterize siRNA complexes. The R1 gel complex corresponds to Dcr-2 and R2D2 proteins bound to labeled siRNA. R2 complex appears to be an intermediate that links R1 to a third complex, R3. The R3 complex corresponds to a siRISC that is competent to cleave cognate mRNA (Pham, 2004). To address the role of Dcr-1 in complex formation, complexes were sought in a dcr-1 mutant lysate. R1 complex was detected, but its mobility was slightly shifted and more heterogeneous. No complex with comparable mobility to R2 was detected. The data indicate that proper formation of the R2 intermediate from the R1 precursor complex is dependent upon Dcr-1 (Lee, 2004).
Bacterial RNase III is an antiparallel dimer containing a deep cleft within the catalytic domain. At each end of the cleft lies a symmetric cluster of acidic residues that are conserved among RNase III enzymes. Some of the residues at each end coordinate a divalent metal ion (Mg2+) that is essential for the nucleophilic attack on the RNA phosphodiester bonds at each active site. The metal ion makes a bidentate interaction (inner-sphere and outer-sphere) with an invariant glutamate residue. In one model of catalysis, the nucleophile is used twice, thereby cleaving both strands. In another model, based on a crystal structure, each active site uses two separate clusters of residues to cleave the two phosphodiesters of the RNA helix. One cluster involves the metal ion, and the other cluster acts independent of the metal ion (Lee, 2004 and references therein).
Dicers contain two catalytic domains, which based on the structure of bacterial RNase III, might fold into a pseudodimer structure or might associate within a dimeric holoenzyme. In either scenario, the catalytic repeats contain many of the invariant acidic residues implicated in RNase III catalysis. Moreover, like bacterial RNase III, Dicers cleave dsRNA to produce fragments with 3′ overhangs, and with 5′-phosphate and 3′-hydroxy termini in a reaction that requires a divalent metal ion. On this basis, the chemistry of phosphodiester hydrolysis is likely to be similar. Accordingly, certain invariant residues were mutated within Dcr-2 that ware predicted to specifically disrupt phosphodiester hydrolysis. E1371 and E1617 in the first and second RNase III repeats, respectively, are homologous to the E residue that extensively interacts with Mg2+ in the bacterial holoenzyme. An E→K mutant in E. coli RNase III fails to cleave dsRNA, but still binds the dsRNA substrate. To elucidate the functions of the homologous residues in Dcr-2, single and double E1371K and E1617K substitution mutants of dcr-2 were generated and the mutant genes were transformed into a Drosophila strain null for dcr-2. Their ability to silence white expression in a GMR-wIR background was tested. The single mutants gave barely detectable silencing activity, while the double mutant gave no detectable silencing. Thus, E1371 and E1617 are essential for Dcr-2 activity in vivo (Lee, 2004).
Two other conserved acidic residues in bacterial RNase III form an interdomain bridge near each metal binding site, but do not coordinate the metal. Interestingly, all known Dicers have acidic residues in homologous positions of the first domain repeat, but have nonacidic residues in the homologous positions of the second domain repeat. To test the functionality of the residues in the first domain, an E1210V or E1237A substitution was introduced into the dcr-2 gene. The same substitutions at the homologous positions of E. coli RNase III abolish activity. However, both dcr-2 point mutants fully rescued the dcr-2 null phenotype, indicating that E1210 and E1237 are not critical for Dcr-2 activity. These results are consistent with the notion that the nonacidic partner residues in the second repeat normally render these clusters nonfunctional (Lee, 2004).
These genetic and biochemical analyses support the idea that siRISC activity is dependent on Dcr-1 and Dcr-2. Pham (2004) has observed both Dcr-1 and Dcr-2 proteins in siRISC that is competent for target cleavage. At least three functions that Dicer could play in siRISC can be imagined. (1) Dicer may stably associate with siRISC after having passed an siRNA molecule to other RISC factors, but has no further role in siRISC activity. This is unlikely since siRNAs can UV-crosslink to Dcr-1 and Dcr-2 in assembled siRISC (Pham, 2004). (2) Dicer may use its dsRNA binding activity to retain double-stranded siRNA or a siRNA/mRNA duplex within siRISC. (3) The RNase III domain of Dicer may be responsible for RNA cleavage by siRISC. To test this latter possibility, the E1371K and E1617K variants of Dcr-2 were examined for siRISC activity (Lee, 2004).
Lysates were prepared from mutant embryos in which dcr-2E1371K, dcr-2E1617K, or dcr-2E1371K E1617K genes were expressed in place of the endogenous dcr-2 gene. Lysates were incubated with siRNA duplexes and a labeled mRNA substrate, and siRNA-directed cleavage of the substrate was monitored by 5′ product formation. All three mutants exhibited normal mRNA cleavage activity in vitro. To demonstrate that the mutant proteins are nevertheless defective for RNase III activity, they were tested for dsRNA processing. Neither dcr-2E1371K nor dcr-2E1617K lysates were able to support dsRNA cleavage to form siRNAs. These data indicate that siRISC activity is unaffected when Dcr-2 RNase III activity is specifically impaired (Lee, 2004).
Infection of Drosophila with Drosophila C virus triggers a transcriptional response that is dependent in part on the Jak kinase Hopscotch. Successful infection and killing of Drosophila with the insect nodavirus flock house virus is strictly dependent on expression of the viral protein B2, a potent inhibitor of processing of double-stranded RNA mediated by the essential RNA interference factor Dicer. Conversely, flies with a loss-of-function mutation in the gene encoding Dicer-2 (Dcr-2) show enhanced susceptibility to infection by flock house virus, Drosophila C virus and Sindbis virus, members of three different families of RNA viruses. These data demonstrate the importance of RNA interference for controlling virus replication in vivo and establish Dcr-2 as a host susceptibility locus for virus infections (Galiana-Arnoux, 2006).
These data demonstrate that Dicer-2 is part of a potent effector mechanism in vivo for controlling virus infection in Drosophila. Three published studies have indicated involvement of RNAi in antiviral silencing in the nematode Caenorhabditis elegans. Those studies have shown that worms with mutations in rde-1 (which encodes a member of the Argonaute family) or rde-4 (which encodes a dsRNA-binding protein facilitating the loading of siRNA onto the RISC) contain higher viral loads after infection with FHV or the rhabdovirus vesicular stomatitis virus. However, any potential benefit of RNAi for infected worms was not addressed in those studies (Galiana-Arnoux, 2006).
By using insect flock house virus (FHV) and Drosophila as a model system, it was shown that point mutations inactivating the viral protein B2 or the host protein Dicer-2 have substantial effects on viral replication and on the resistance of flies to infection. Although B2 has been shown to bind tightly to dsRNA and to prevent its cleavage by Dicer in vitro, the data have demonstrated critical involvement of B2 in countering the Dicer-2-dependent viral RNA silencing mechanism in vivo. Unfortunately, for technical reasons, an initial attempt to express both the RNA1DeltaB2 and RNA2 transgenes on a Dicer-2 mutant background, to demonstrate restoration of the accumulation of viral RNAs and a similar effect on virulence, was unsuccessful. Nevertheless, the importance of Dicer-2 in preventing accumulation of FHV RNA in vivo was formally demonstrated by showing substantial accumulation of FHV RNA1 and RNA3 in the absence of B2 in Dicer-2 mutant flies (Galiana-Arnoux, 2006).
The potent antiviral activity of the Dicer-2-dependent RNAi mechanism was further confirmed with two other insect RNA viruses, DCV and SINV, indicating that Dicer-2 has broad antiviral functions in Drosophila. In particular, the data using SINV have shown that the outcome of the infection (death versus recovery) depends on the presence of a functional Dcr-2 gene. Although focus was placed on drosophila, the findings may be relevant to other insects, including disease vectors that transmit viruses to mammals, including humans. Indeed, increased viral loads have been reported in Anopheles gambiae mosquitoes with silencing of the gene encoding the Argonaute protein AGO2, which functions together with Dicer-2 in the RNAi pathway (Galiana-Arnoux, 2006).
The results, in conjunction with the information now available on RNAi, particularly in plants lead to a proposal that in flies Dicer-2 detects and cleaves newly synthesized viral dsRNA, generating siRNA that then specifically recognizes viral RNA and 'guides' the RISC to degrade the viral RNA. That proposed mechanism is supported more strongly by data obtained using plants, in which a nucleic acid–based antiviral defense was first described. In particular, A. thaliana plants deficient in the Dicer factor DCL2 have increased susceptibility to infection by the RNA virus turnip crinkle virus, with higher viral titers and a more deleterious disease phenotype than that of wild-type plants. However, DCL2-mutant A. thaliana are as susceptible as wild-type plants to infection by two other RNA viruses, turnip mosaic virus and cucumber mosaic virus, indicating that DCL2 does not have a general function in antiviral defenses against RNA viruses (Galiana-Arnoux, 2006).
An opposite result was demonstrated for Drosophila, for which all cases tested so far have indicated that Dicer is a global antivirus defense mechanism. The difference in results obtained with plants and Drosophila probably reflects the fact that A. thaliana expresses four DCL factors, which may have partially overlapping functions. In contrast, Drosophila has two Dicer genes: Dcr-1, which controls the production of 'micro RNA' and accomplishes important developmental functions; and Dcr-2, which controls the production of siRNA and participates in the control of viral infection. In contrast, there are no indications thus far that the sole Dicer protein found in mammals, which is essential for development, participates in the control of viral infections. Instead, mammals seem to have a diverse set of cytosolic receptors (RIG-I and MDA5) and transmembrane receptors (Toll-like receptors 3, 7, 8 and 9) that recognize viral RNA or DNA and trigger antiviral responses (Galiana-Arnoux, 2006).
Despite its importance, as demonstrated here, RNAi is certainly not the sole effector mechanism controlling virus infection in flies. The modest increase in viral RNA in FHV-infected Dicer-2 mutant flies, in contrast to the considerable effect on survival, was unexpected. Further experiments will be needed to determine whether the small differences in viral RNA concentrations in whole flies reflect tissue-specific requirements for Dcr-2 and can explain the enhanced death of infected flies or if they indicate that Dicer-2 exerts an additional function beneficial to the host other than 'dicing' viral RNA (Galiana-Arnoux, 2006).
In addition to RNAi, other antiviral effector mechanisms in plants and metazoans have been characterized, including programmed cell death, which has been reported to participate in the control of viral infections and can be blocked by specific viral inhibitors such as the baculovirus caspase inhibitor p35. Furthermore infection of Drosophila with DCV triggers induction of some 150 genes by a factor of two or more. At least some of those genes encode proteins that participate in controlling the infection, an hypothesis supported by the fact that the genes are not induced after virus infection in Jak-deficient flies, mutant flies that have higher viral loads than wild-type control flies and are generally more susceptible to infection (Galiana-Arnoux, 2006).
Strictly speaking, there is at present evidence for two types of responses to virus infection in Drosophila: degradation of viral RNA by the RNAi machinery and cytokine-mediated induction of many genes (via hopscotch-encoded Jak activated by the gp130-related cytokine receptor Domeless), some of which may counter viral infection. The coexistence of those two types of response may reflect an important difference in RNAi in plants versus Drosophila: whereas RNAi is cell autonomous in Drosophila, in plants the RNAi response triggered in infected cells spreads systemically to the plant to induce protective RNAi at distant sites. That cell-to-cell transfer of the silencing signal is essential for the host to counter viral infection, as the presence of dsRNA is in most cases detected after viral replication at a stage at which the cells may not succeed in blocking or destroying the virus. It is proposed that in Drosophila, RNAi functions to limit viral replication in infected cells and is coupled to other defense mechanisms triggered by cytokine signaling in uninfected cells. A principal challenge for future work will be to elucidate how the integration of these responses allows Drosophila to resist viruses (Galiana-Arnoux, 2006).
Innate immunity against bacterial and fungal pathogens is mediated by Toll and immune deficiency (Imd) pathways, but little is known about the antiviral response in Drosophila. This study demonstrates that an RNA interference pathway protects adult flies from infection by two evolutionarily diverse viruses. The work also describes a molecular framework for the viral immunity, in which viral double-stranded RNA produced during infection acts as the pathogen trigger whereas Drosophila Dicer-2 and Argonaute-2 act as host sensor and effector, respectively. These findings establish a Drosophila model for studying the innate immunity against viruses in animals (Wang, 2006).
RNA interference (RNAi) silences gene expression through small interfering RNAs (siRNAs) and microRNAs (miRNAs). In Drosophila, Dicer-2 (Dcr-2) produces siRNAs, whereas Dicer-1 (Dcr-1) recognizes precursors of miRNAs. The small RNAs are assembled with an Argonaute (Ago) protein into related effector complexes, such as RNA-induced silencing complex (RISC), to guide specific RNA silencing (Wang, 2006).
RNA silencing provides an antiviral mechanism in plants and animals. Plant viruses have evolved diverse strategies for evading the RNA silencing immunity, and expression of viral suppressors of RNAi (VSRs) is essential for infection and virulence. However, it is unknown whether antiviral silencing in plants is controlled by a specific small RNA pathway targeted by plant VSRs. Bacterial and fungal infections of Drosophila induce Toll and immune deficiency (Imd) pathways, leading to transcriptional induction of antimicrobial peptide effectors via NF-KappaB)–like signaling processes. However, it has been unclear whether either pathway plays a role in Drosophila innate immunity against viruses. Previous work in cell culture has indicated that RNAi might mediate viral immunity in Drosophila. This study investigated whether RNAi indeed provides protection against virus infection in Drosophila embryos and adults (Wang, 2006).
Flock house virus (FHV) contains an RNA genome divided among two plus-strand molecules, RNAs 1 and 2. RNA2 (R2) encodes the single virion structural protein, whereas RNA1 (R1) encodes protein A, the viral RNA–dependent RNA polymerase (RdRP), and B2, a VSR expressed after RNA1 replication from its own mRNA, RNA3. In the absence of R2, R1 replicates autonomously, accumulates to high levels, and produces abundant RNA3 in wild-type (WT) Drosophila embryos 30 hours after injection with R1 transcripts synthesized in vitro. No FHV RNAs accumulate in WT embryos injected with R1fs transcripts that contain a frameshift mutation in the RdRP open reading frame (ORF). FHV RNAs are also not readily detected in WT embryos injected with a second mutant of R1, R1DeltaB2, which does not express the VSR. However, abundant accumulation of R1DeltaB2 but not FR1fs occurs in mutant Drosophila embryos that carry a homozygous null mutation in ago-2 (ago-2414), which is essential for RNAi in Drosophila. These data indicated that viral RNA replication in Drosophila embryos triggers an RNAi-mediated virus clearance in an Ago-2–dependent manner and that effective RNAi suppression by B2 is necessary to achieve normal accumulation of FHV RNAs (Wang, 2006).
In Drosophila, Ago-2 acts downstream of Dicer-2 (Dcr-2) to recruit siRNAs, the products of Dcr-2 activity, into the siRNA-dependent RISC (siRISC). Thus, a genetic requirement for ago-2 in FHV RNA clearance implicates Dcr-2 in the RNAi antiviral effector mechanism. To test this hypothesis, R1, R1fs, and R1DeltaB2 transcripts were injected into embryos carrying a homozygous dcr-2 null mutation, dcr-2L811fsX. Northern blot hybridizations showed that, although FHV RNAs remained undetectable in dcr-2L811fsX embryos injected with R1fs, viral RNA accumulation is rescued in the dcr-2L811fsX embryos injected with R1DeltaB2 transcripts. This result shows that Dcr-2 is required to initiate RNAi-mediated clearance of FHV RNAs in Drosophila embryos (Wang, 2006).
To investigate whether the RNAi pathway protects Drosophila from virus infection, adult flies of either WT or dcr-2L811fsX genotype were injected with purified FHV virions. The FHV isolate was of low virulence in WT flies, because about 50% of infected flies survived 15 days postinoculation (dpi) despite a detectable virus load. Inoculation with the same dose of FHV resulted in 60% mortality by 6 dpi and 95% by 15 dpi in dcr-2L811fsX flies. Mock inoculation with buffer had little effect on either WT or dcr-2L811fsX flies for as long as observations were made. Both Northern and Western blot analyses revealed that the virus accumulated more rapidly and to much greater levels in dcr-2L811fsX than WT flies. Thus, dcr-2 mutants exhibit enhanced disease susceptibility to FHV in comparison with WT flies, demonstrating that Dcr-2 is also required to mount an immune response that protects adult Drosophila against FHV infection (Wang, 2006).
R2D2 contains tandem double-stranded RNA (dsRNA)–binding domains and forms a heterodimer with Dcr-2 in vivo that is required for siRNA loading into RISC. Flies homozygous for a loss-of-function mutation in r2d2 exhibit a phenotype of enhanced disease susceptibility to FHV infection similar to that of dcr-2L811fsX. Thus, R2D2 also participates in the innate immunity pathway that protects adult flies from FHV infection. Notably, although FHV accumulates to extremely high levels in both dcr-2 and r2d2 mutant flies, abundant viral siRNAs were detected only in r2d2 mutant flies, and viral siRNAs were below the level of detection in dcr-2L811fsX flies. Thus, FHV infection is detected by Dcr-2, leading to production of FHV siRNAs. However, R2D2 is not required for the production but is essential for the function of viral siRNAs, which is consistent with the genetic requirements for processing the artificially introduced dsRNA (Wang, 2006).
To investigate whether the RNAi pathway in Drosophila is specific against nodaviruses and not other classes of RNA viruses, the susceptibility of WT, dcr-2L811fsX, and r2d2 mutant flies to cricket paralysis virus (CrPV) was assessed. CrPV contains a nonsegmented plus-strand RNA genome but belongs to a group of picorna-like viruses. CrPV is substantially more virulent than FHV in Drosophila; injection of CrPV at much lower titers resulted in mortality of 70% of WT flies by 15 dpi. CrPV was also found to induce enhanced disease susceptibility in both dcr-2 and r2d2 mutant flies. About 60% of the infected mutant flies were dead by 6 dpi, and more than 95% were dead by 15 dpi. In addition, Northern blots indicated that the virus accumulated more rapidly and to greater levels in the mutant flies. Thus, both dcr-2 and r2d2 are required for protection of Drosophila against CrPV (Wang, 2006).
CrPV infection of cultured S2 cells induced antiviral silencing, illustrated by detection of CrPV-specific siRNAs. Antiviral silencing against FHV in S2 cells induced by FR1gfp is suppressed by CrPV superinfection, leading to derepression of green fluorescent protein (GFP). Two ORFs are encoded by the CrPV RNA genome. No suppression of antiviral silencing was observed in S2 cells cotransfected with a plasmid expressing either the entire downstream ORF of CrPV or the individual mature virion proteins processed from the polyprotein. In contrast, RNAi suppression was detected after cotransfection with a plasmid expressing either the entire upstream ORF of CrPV or its N-terminal 140 codons. However, the suppressor activity was not detected after a frameshift mutation was introduced into pA, thus identifying the N-terminal fragment of 140 amino acids of the CrPV nonstructural polyprotein as a VSR (Wang, 2006).
In Drosophila, Imd signaling is stimulated by Gram negative (Gram–) bacterial infection, whereas Toll signaling is triggered by Gram positive (Gram+) bacterial infection. To determine whether loss of the RNAi pathway initiated by Dcr-2 has an impact on the Toll and Imd signaling processes, WT, dcr-2L811fsX, and r2d2 mutant flies were subjected to immune challenge by inoculation with Escherichia coli (Gram–) or Micrococcus luteus (Gram+). Northern blot hybridizations detected substantial transcriptional induction of the antimicrobial peptide gene Diptericin A 6 hours postimmune challenge (hpi) with either E. coli or M. luteus, which declined at 24 hpi as described. Similar induction patterns for Diptericin A were observed in dcr-2L811fsX and r2d2 mutant flies inoculated with Gram+ and Gram– bacteria. Furthermore, it was found that induction of either Attacin A or Drosomycin by Gram+ and Gram– bacteria was also not altered in dcr-2L811fsX and r2d2 mutant flies as compared to WT flies. These results indicate that induction of antimicrobial peptide genes via Toll and Imd signaling pathways is not compromised in dcr-2L811fsX and r2d2 mutant flies (Wang, 2006).
Nodaviruses and the polio-like CrPV belong to two different superfamilies of animal RNA viruses. The same set of RNAi pathway genes is required for Drosophila defense against FHV and CrPV and both viruses encode a potent VSR. These results collectively show that RNAi pathway functions as a common viral immunity mechanism in Drosophila and that RNAi suppression represents a general counterdefensive strategy used by insect viruses. Furthermore, a genetic requirement for Dcr-2, R2D2, and Ago-2 in antiviral silencing establishes a molecular framework for the innate immunity against viruses in Drosophila. None of Dcr-2, R2D2, and Ago-2 plays a detectable role in either the production or function of miRNAs in Drosophila. Thus, this work identifies the dsRNA-siRNA pathway of RNAi as providing the innate immunity against virus infection in Drosophila and establishes that dsRNA produced during virus replication acts as the pathogen trigger whereas Dcr-2 and Ago-2 act as host sensor and effector of the immunity, respectively. These results support and extend the previous findings on antiviral silencing in C. elegans (Wang, 2006).
Although NF-KappaB-like signaling in the Toll and Imd pathways do not appear to play a role in the RNAi-directed viral immunity mechanism in Drosophila, the fly mutant defective in the Janus kinase (JAK) Hopscotch exhibit a modest increase in susceptibility to infection with Drosophila C virus, suggesting an antiviral role for JAK–signal transducer and activator of transcription (STAT) signaling. Nonetheless, it is believed that RNAi-based immunity is independent of JAK-STAT signaling, because virus infection is not known to induce the RNAi pathway in Drosophila and FHV induction of the JAK-STAT responsive gene vir-1 is unaltered in the dcr-2 and r2d2 mutants. Because the Toll and Imd pathways are highly conserved in vertebrates, the Drosophila model established for RNAi may also be useful for the analyses of the innate antiviral immunity in vertebrates (Wang, 2006).
P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. This study shows that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity (Eulalio, 2007).
The first proteins found in P bodies are those functioning in the degradation of bulk mRNA. In eukaryotes, this process is initiated by removal of the poly(A) tail by deadenylases. There are several deadenylase complexes in eukaryotes: the PARN2-PARN3 complex is thought to initiate deadenylation, which is then continued by the CAF1-CCR4-NOT complex. Following deadenylation, mRNAs are exonucleolytically digested from their 3' end by the exosome, a multimeric complex with 3'-to-5' exonuclease activity. Alternatively, the cap structure is removed by the decapping enzyme DCP2 after deadenylation, rendering the mRNA susceptible to 5'-to-3' degradation by the major cytoplasmic exonuclease XRN1 (Eulalio, 2007).
Decapping requires the activity of several proteins generically termed decapping coactivators, though they may stimulate decapping by different mechanisms. In the yeast Saccharomyces cerevisiae, these include DCP1, which forms a complex with DCP2 and is required for decapping in vivo, the enhancer of decapping-3 (EDC3 or LSm16), the heptameric LSm1-7 complex, the DExH/D-box RNA helicase 1 (Dhh1, also known as RCK/p54 in mammals), and Pat1, a protein of unknown function that interacts with the LSm1-7 complex, Dhh1, and XRN1. In human cells, DCP1 and DCP2 are part of a multimeric protein complex that includes RCK/p54, EDC3, and Ge-1 (also known as RCD-8 or Hedls), a protein that is absent in S. cerevisiae (Eulalio, 2007).
The decapping enzymes, decapping coactivators, and XRN1 colocalize in P bodies. Additional P-body components in multicellular organisms include the protein RAP55 (also known as LSm14; Drosophila homolog - Trailer hitch), which has a putative role in translation regulation, and GW182, which plays a role in the microRNA (miRNA) pathway (Eulalio, 2007).
The P-body marker GW182 localizes to cytoplasmic foci in Drosophila S2 cells together with the decapping enzyme DCP2 and the decapping coactivator DCP1, suggesting that these foci represent P bodies. To characterize D. melanogaster P bodies further, antibodies were raised to the Drosophla orthologs of two proteins found in human-cell P bodies. These correspond to Ge-1 and Tral (LSm15), which is closely related to human RAP55 (or LSm14) (see Tanaka, 2006). Both antibodies stained the cytoplasm diffusely and also stained discrete cytoplasmic foci with a diameter ranging from 100 nm to 300 nm. The antibody signals are specific, as they are lost in cells in which the cognate proteins were depleted. The foci are present in about 95% of the cell population and are readily detectable because the concentration of Tral or Ge-1 in these foci is significantly higher than that in the surrounding cytoplasm (Eulalio, 2007).
The distribution of green fluorescent protein (GFP)-tagged versions of proteins found in P bodies was examined in yeast and/or human cells. These include DCP1, DCP2, GW182, Me31B (the D. melanogaster ortholog of S. cerevisiae Dhh1 and vertebrate RCK/p54), CG5208 (the D. melanogaster homolog of S. cerevisiae Pat1, referred to as HPat hereafter), and EDC3 (also known as LSm16). All of these proteins formed cytoplasmic foci that costained with the anti-Tral or anti-Ge-1 antibodies. Importantly, the expression of the GFP-tagged proteins did not significantly alter the number and size of endogenous P bodies. Together, these results indicate that the localization of decapping enzymes and decapping coactivators into P bodies is evolutionarily conserved. The localization of GW182 in Drosophila P bodies is in agreement with the proposal that GW-bodies and P bodies overlap, as reported for mammalian cells (Eulalio, 2007).
The localization of proteins implicated in translational regulation was examined in Drosophila oocytes whose corresponding transcripts are detectable in S2 cells, in particular, Smaug and the dsRNA binding protein Staufen. Smaug is a translational repressor that also promotes deadenylation of bound mRNAs by recruiting the CAF1-CCR4-NOT1 complex (Zaessinger, 2006). Both proteins localized to P bodies with endogenous Tral. Strikingly, P bodies increased in size in cells expressing Staufen at high levels but not in cells overexpressing GFP fusions of Smaug, suggesting that Staufen promotes P-body formation. Drosophila Staufen, Tral, DCP1, DCP2, XRN1, and Me31B have also been detected in RNP granules in neuronal cells and/or in oocytes, indicating that P bodies and other RNP granules observed in neuronal cells or during development share common components (Eulalio, 2007).
P-body formation requires nontranslating mRNPs and/or mRNPs undergoing decapping. A conserved feature of P bodies in human and yeast cells is that their formation depends on RNA and is enhanced in cells in which the concentration of nontranslating mRNAs or of mRNAs undergoing decapping increases. These observations indicate that mRNAs must exit the translation cycle to localize to P bodies. In agreement with this, it was observed that Drosophila P bodies decline when cells are treated with RNase A or with cycloheximide (which inhibits translation elongation and stabilizes mRNAs into polysomes). In contrast, P-body sizes increase in cells treated with puromycin, which causes premature polypeptide chain termination and polysome disassembly. Both puromycin and cycloheximide inhibit protein synthesis in S2 cells, as judged by the reduction of F-Luc and R-Luc activities after the treatment of cells transiently expressing these proteins with these drugs (Eulalio, 2007).
The size of Drosophila P bodies also depends on the fraction of mRNAs undergoing decapping, in agreement with the results reported for yeast and human cells. Indeed, blocking mRNA decay at an early stage, for instance, by preventing deadenylation in cells in which NOT1 (a component of the CAF1-CCR4-NOT deadenylase complex) is depleted, leads to the dispersion of P bodies, whereas P bodies are on average more prominent in cells from which DCP2 or XRN1 is depleted (in which decapping and subsequent 5'-to-3' mRNA decay are inhibited) (Eulalio, 2007).
Several lines of evidence show that P bodies do not serve as storage sites for the effectors of posttranscriptional process but are sites where mRNA degradation and silencing can take place. For instance, P-body formation is RNA dependent, and decay intermediates, siRNAs, and miRNAs and their targets are detected in P bodies. Moreover, the size and number of P bodies depends on the fraction of mRNAs undergoing decapping. However, the question of whether mRNA decay and silencing require the environment of microscopic, wild-type P bodies to occur or whether these processes can also occur outside of P bodies in soluble protein complexes remains open. This study shows that formation of large P bodies visible in the light microscope as observed in wild-type cells is not required for several processes associated with P-body components, including NMD, mRNA decay, and RNA-mediated gene silencing (Eulalio, 2007).
The question addressed in this study was whether the environment of macroscopic P bodies is required for posttranscriptional regulation. P bodies are defined as the large cytoplasmic foci visible by light microscopy in wild-type cells. These foci are on average 100 to 300 nm in diameter and are readily detected as bright cytoplasmic dots because the concentration of proteins in these foci is significantly higher than in the surrounding cytoplasm. Nevertheless, most P-body components are also detected diffusely throughout the cytoplasm. For a limited number of examples that have been analyzed, it has been shown that P-body components are not confined to these structures but dynamically exchange with the cytoplasmic pool. Quantitative information regarding the fractionation of P-body components between P bodies and the cytoplasm is still lacking, but given the volume of P bodies relative to that of the cytoplasm, it is likely that the diffuse cytoplasmic fraction is significantly larger. This suggests that posttranscriptional processes are likely to occur and may even be initiated in the diffuse cytoplasm or in soluble protein complexes that aggregate to form P bodies. Whether these processes take place in submicroscopic aggregates or soluble protein complexes in the absence of detectable microscopic P bodies remains to be solved. However, it is considered that aggregates or large multiprotein assemblies that are not detectable by light microscopy cannot be defined as bodies (Eulalio, 2007).
Translation factors or ribosomes are generally not present in P bodies (with the exception of cap binding protein eIF4E), indicating that mRNAs leave the translation cycle prior to entering P bodies. Consistently, releasing mRNAs from polysomes leads to increases in P-body sizes and numbers, whereas the stabilization of mRNAs into polysomes disrupts P bodies. These observations suggest that a critical step in P-body formation is the release of mRNPs from a translationally active state associated with polysomes to a translationally inactive state. This paper has shown that releasing mRNAs from polysomes by puromycin treatment is not sufficient to elicit P-body formation and that functional silencing pathways or proteins generically termed decapping coactivators are required for P-body assembly. These proteins include Me31B (Dhh1 in yeast), HPat (Pat1 in yeast), Ge-1, and the LSm1-7 complex (Eulalio, 2007).
What could be the role of these proteins in P-body formation? Me31B is an RNA helicase which could facilitate rearrangements in mRNP composition upon release from polysomes. The role of HPat is unclear, but the yeast ortholog interacts with Dhh1, XRN1, and the heptameric LSm1-7 complex. Coimmunoprecipitation assays indicate that the interaction between Dhh1 and Pat1 orthologs (i.e., Me31B and HPat) is conserved in Drosophila. Finally, the LSm1-7 complex associates with deadenylated mRNAs and stimulates decapping. Clearly, many details regarding the precise molecular function of these proteins remain to be discovered, but their requirement for P-body assembly indicates that mRNAs that are not actively translated do not enter into P bodies by default: the activity of a defined set of proteins is required. Alternatively, nontranslating mRNAs may enter silencing pathways, and this would also lead to changes in mRNP composition due to the recruitment of Argonaute proteins and binding partners, which include P-body components such as GW182, decapping enzymes, and RCK/p54 (Eulalio, 2007).
Once P-body components are bound to an RNP, P-body formation may then be triggered by protein-protein interactions. Indeed, proteins required for P-body assembly are known to interact to form multimeric protein complexes. Consistently, in addition to the interactions mentioned above, DCP1, DCP2, Ge-1, RCK/p54, and EDC3 form a multimeric protein complex in human cells. The absolute requirement of RNA for P-body formation could be explained if affinities between these proteins increased upon RNA binding. Additionally, proteins like GW182 and Ge-1 are multidomain proteins that could bind more than one RNP simultaneously, bringing into close proximity several components and thus nucleating the formation of P bodies (Eulalio, 2007).
RNAs targeted by silencing pathways nucleate P bodies. In this study, it is shown that both the RNAi and miRNA pathways contribute to the generation of a pool of nontranslating mRNPs and/or of mRNPs committed to decay which are required for P-body formation. Nevertheless, silencing can occur in the absence of microscopic P bodies. The results provide support to previous models proposing that silencing is initiated in the cytoplasm and that the localization of the silencing machinery into P bodies is a consequence, rather than the cause, of silencing (Eulalio, 2007).
An unexpected observation from these studies is that AGO2 and Dicer-2, which function in siRNA-mediated gene silencing in Drosophila, are required for P-body integrity. The role of these proteins in P-body assembly is unlikely to be structural, because P bodies are restored upon puromycin treatment in cells from which AGO2 or Dicer-2 is depleted. The most likely explanation for the requirement of these proteins is, therefore, that silencing by siRNAs also generates RNPs that elicit P-body formation. The requirement for AGO2 could be at least partially explained by the observation that the expression levels of a small subset of endogenous miRNA targets are affected in AGO2-depleted cells, suggesting that some miRNAs may be loaded into AGO2-containing RNA-induced silencing complexes. Furthermore, the AGO1 and AGO2 genes interact, although it is unclear how this interaction affects the activities of these proteins (Eulalio, 2007).
The requirement for Dicer-2 in P-body assembly, however, suggests that endogenous siRNA targets also contribute to P-body formation. Because the levels of dsRNA synthesis from endogenous loci that could provide precursors for the production of endogenous siRNAs are currently unknown, the fraction and origin of transcripts regulated by endogenous siRNAs cannot be estimated. Nonetheless, a possible source of endogenous dsRNAs is the bidirectional transcription of pseudogenes and transposable elements, in agreement with the role of the RNAi pathway as a defense mechanism against RNA viruses and mobile genetic elements (Eulalio, 2007).
The essential role of silencing pathways in P-body formation in Drosophila, and presumably in human cells, raises the question of how P bodies are assembled in S. cerevisiae, which lacks silencing pathways. One possibility is that other posttranscriptional processes generate nontranslating mRNPs required to nucleate P bodies. For instance, the NMD pathway contributes to P-body assembly in yeast cells, because depletion of Upf2 or Upf3 leads to increases in P-body size and number in a Upf1-dependent manner, whereas similar experiments with Drosophila cells do not affect P bodies (Eulalio, 2007).
With the exception of the proteins involved in silencing, the composition of P bodies and the effects of drugs such as cycloheximide and puromycin on P-body size and number are strikingly similar in yeast, Drosophila, and human cells, raising the question of what the role of these structures accounting for their conservation in eukaryotic cells could be. The results show that the environment of microscopic P bodies is not essential for mRNA decay or silencing but do not exclude that the formation of P bodies confers a kinetic advantage. Moreover, the results do not rule out a role for large P bodies in sequestering a specific set of nontranslating mRNPs and reinforcing their repression by shielding them from the translation machinery (Eulalio, 2007).
Finally, the conservation of P bodies may reflect a role for these structures in other cellular processes that is not yet fully appreciated. A role in some steps of retroviral or retrotransposon life cycles is suggested by the localizations of the antiretroviral proteins APOBEC3G and APOBEC3F in human cell P bodies and of the protein and RNA components of the retrovirus-like element Ty3 in yeast P bodies. A link between P bodies and the regulation of retrotransposition would be consistent with the role of RNAi pathways in silencing the expression of transposable elements. Because all known essential P-body components play roles in decapping and/or silencing and proteins playing an exclusively structural role in P-body assembly have not yet been identified, it is currently not possible to evaluate the role of P bodies for cell, tissue, or organism survival (Eulalio, 2007).
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date revised: 10 March 2008
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