Effects of Mutation or Deletion

The Drosophila AGO2 protein is an essential factor for RNAi as a component of the RISC complex in cultured S2 cells (Hammond, 2001). However, the precise role that AGO2 plays in RNAi is not well understood. To gain an insight into the molecular functions of AGO2 in RNAi, fly strains that lack AGO2 were produced. To obtain fly strains bearing deletions in AGO2, a P{EP}element inserted in the first exon of the AGO2 gene on the EP(3)3417 chromosome was mobilized to produce several partial deletions of AGO2. One such mutation named AGO2⁴¹⁴ was selected for further characterization. Western blot analysis with anti-AGO2 antibody revealed that there is no AGO2 protein in homozygous AGO2⁴¹⁴ flies. The absence of AGO2 mRNA was also confirmed by Northern blot analysis in homozygous AGO2⁴¹⁴ flies. These results demonstrated that AGO2⁴¹⁴ are AGO2 null flies. Homozygous AGO2⁴¹⁴ flies proceed into adulthood, are fertile, and appear outwardly normal. Because it is not known whether AGO2 is required for RNAi in embryos, RNAi was tested in vivo by assaying the ability of long dsRNA corresponding to the fushi tarazu (ftz) gene to produce a ftz phenotype when injected into wild-type and AGO2 mutant embryos. AGO2 mutant adult females were used to make mutant eggs to remove the maternal contribution of AGO2 (simply called the 'AGO2 mutant embryo' or AGO2⁴¹⁴ embryos, hereafter). Wild-type embryos injected with ftz dsRNA exhibited segmentation defects in their cuticle. In contrast, AGO2 mutant embryos showed a complete absence of interference in response to ftz dsRNA, indicating that they were RNAi defective. This is consistent with previous findings made with cultured S2 cells (Hammond et al. 2001; Caudy, 2002; Ishizuka, 2002). To confirm that disruption of the AGO2 gene is directly responsible for the RNAi-defective phenotype, mutant flies were transformed with a P element containing wild-type AGO2 genomic sequences. The RNAi-defective phenotype was ameliorated by the introduction of this AGO2 minigene, demonstrating that the RNAi-defective phenotype is caused by the AGO2 mutation rather than a second site mutation elsewhere on the chromosome (Okamura, 2004).

To determine whether AGO2 is required for the formation of the siRNA duplex or for interference thereafter, synthetic ftz siRNA was injected into AGO2⁴¹⁴ embryos. siRNA produced a ftz phenotype in wild-type embryos. However, AGO2⁴¹⁴ embryos were not responsive to the ftz siRNA. Similarly, in vitro, AGO2⁴¹⁴ embryo lysate processed long ftz dsRNA into short ~21-nt fragments, similar to lysates from wild-type embryos. These findings suggest that AGO2 is necessary for RNAi after the formation of the siRNA duplex (Okamura, 2004).

Before target mRNA recognition, the siRNA duplex is unwound, and each RISC contains only one of the two strands of the siRNA duplex. Whether AGO2 activity is necessary for this unwinding process was tested. Unwound siRNA was detected in wild-type embryo lysate, whereas no such activity was detected in AGO2⁴¹⁴ embryo lysate, suggesting that AGO2 is required for the unwinding of the siRNA duplex. An alternative explanation of the results could be that the absence of AGO2 leaves the unwound single-stranded RNA exposed to nucleolytic degradation and therefore the single-stranded RNA could not be detected. However, this is unlikely because the amount of input siRNA duplex left in AGO2⁴¹⁴ embryo lysate was not significantly reduced even after 3 h incubation as opposed to that seen in wildtype embryo lysate, indicating that input siRNA duplex is indeed not unwound in AGO2 mutant embryo lysate. Using a recently developed RISC assembly assay, RISC formation was analyzed in AGO2 mutant embryo lysates. ds-siRNA was incubated with embryo lysates in a standard RNAi reaction, and then RISC complexes were resolved by electrophoresis through a native gel. Three major complexes (B, A, and RISC) were detected in wild-type embryo lysates. Complexes B and A are thought to be intermediate precursors to active RISC and contain ds-siRNA. Both complexes B and A were readily detected in AGO2 mutant embryo lysates. However, RISC formation was impaired in AGO2 mutant embryo lysates. Together, these results suggest that AGO2 is required in a step(s) in RISC assembly after binding of the siRNA duplex to RISC precursors (complexes B and A) (Okamura, 2004).

Both siRNAs and miRNAs have been shown to be associated with the same RISC and miRNAs can even direct cleavage of perfectly basepaired substrates in human cells. To see if this is also the case in Drosophila, whether the lack of AGO2 affects the miRNA-mediated cleavage of target RNAs was investigated. ³²P-radiolabeled target mRNA, containing a sequence fully complementary to an endogenous miRNA, miR-2b, as well as a sequence exactly complementary to one strand of a ftz siRNA duplex, was incubated with AGO2⁴¹⁴ embryo lysate in an in vitro RNAi reaction. Both siRNA-directed and miRNA-directed cleavage products are seen in the wild-type lysate. However, ftz siRNA did not direct cleavage in AGO2⁴¹⁴ embryo lysate; in contrast, miR-2b still directed cleavage of the target RNA in AGO2⁴¹⁴ embryo lysates. These results demonstrate that although AGO2 is an essential factor in siRNA-directed RNAi, AGO2 is dispensable for miRNA-directed RNA cleavage in RNAi (Okamura, 2004).

In this experiment, however, the timings of miRNA loading and siRNA loading into RISC are different. miR- 2b is already incorporated into the RISC by the embryo before the lysate is made, whereas siRNA is added to the reaction mixture. Therefore synthetic Drosophila melanogaster let-7 precursor RNA was added to an in vitro RNAi reaction mixture containing the let-7 complementary target RNA. It should be noted that the sequence of the synthetic let-7 precursor used in this study is slightly longer than that of naturally processed pre-let-7, which contains short 3' overhangs. However, the synthetic let-7 precursor has been previously shown to be processed and produce mature let-7 with precisely the same 5' end as authentic let-7 in the Drosophila embryo lysates. In this experiment, the synthetic let-7 precursor RNA was also converted to mature let-7 in vitro, not only in the wild-type embryo lysate, but also in AGO2⁴¹⁴ embryo lysate. In the in vitro RNAi assay, target RNA was cleaved within the let-7 complementary sequence in AGO2⁴¹⁴ embryo lysate as efficiently as in wild type. Thus, miRNA that is not preloaded by the embryo is still capable of entering into the RNAi pathway without AGO2 activity. These results demonstrate that AGO2 is not required for miRNA production or the loading of miRNAs into the RISC and suggest that AGO2 has a specific role for siRNA activity, likely as a specific unwinding and loading factor of siRNA into RISC, and possibly as a necessary component of an siRNA-initiated RISC (Okamura, 2004).

Whether siRNA- and miRNA-directed RNA cleavage pathways have differential requirements for AGO1 was tested. Mutations in AGO1 result in late embryonic/ early larval lethality with developmental defects (Kataoka, 2001). Although AGO1 has been shown necessary for efficient RNAi in embryos (Williams, 2002), AGO1 appears to have little or no effect on the efficiency of RNAi in cultured S2 cells (see also Caudy, 2002). AGO1^k08121 is a strong allele (Kataoka 2001). AGO1^k08121 was balanced over a CyO chromosome carrying a kruppel-Gal4 (Kr-Gal4) and a UAS-GFP transgene. Homozygous embryos were separated from heterozygous and CyO;Kr-Gal4;UAS-GFP homozygous embryos on the basis of GFP expression. Because there is strong reduction of AGO1 transcripts in AGO1^k08121 embryos (Kataoka, 2001) and AGO1 protein was not detected in lysate from 14- to 16-h staged AGO1^k08121 embryos, 14-16-h staged AGO1^k08121 embryo lysate was tested for its ability to direct cleavage of siRNA or miRNA complementary RNA targets. ftz siRNA directed its cleavage in AGO1^k08121 embryo lysate as effectively as in wild type. AGO1^k08121 embryo lysate also processed long ftz dsRNA into short ~21-nt fragments. Furthermore, RISC formation directed by siRNA duplex was not impaired in AGO1^k08121 embryo lysate as judged by the native gel shift assay. These findings suggest that AGO1 is not necessary for the production of the siRNA duplex, siRNA-initiated RISC formation, or siRNA-directed cleavage. However, cleavage of the target RNA directed by miR-2b was suppressed in AGO1^k08121 embryo lysate by a factor of fivefold compared with that in wild type, suggesting that AGO1 is necessary for efficient target RNA cleavage mediated by miRNAs. Residual miRNA activity seen in AGO1^k08121 could be due to some degree of functional complementation among Argonaute family proteins expressed in embryos (Williams, 2002). The in vitro experiments with AGO1^k08121 mutant embryo lysates argue against AGO1 being required for siRNA-mediated RNAi in embryos when injected with either long dsRNA or siRNA (Williams, 2002). Although the discrepancy is currently not understood, these findings are at least consistent with the findings (Caudy, 2002) using cultured S2 cells (Okamura, 2004).

It was hypothesized that AGO1 might be involved in the maturation and/or function of miRNAs. To directly determine the contribution of AGO1 and AGO2 to miRNA production in Drosophila cells, each was depleted from S2 cells by dsRNA soaking and Northern blot analyses were perfomed to monitor the abundance of bantam miRNA (miR-ban). A marked reduction of mature miR-ban was observed in AGO1-depleted S2 cells but not in AGO2-depleted S2 cells. It was also confirmed that the miRNA level was significantly reduced in the AGO1^k08121 embryos. The accumulation of pre-miR-ban was not observed in both cases. Similar results were obtained when the same RNA preparations were probed for the expression of another miRNA, miR-2b, which is also known to be expressed both in S2 cells and embryos. These results suggest that AGO1 is involved in the maturation and/or stability of miRNAs (Okamura, 2004).

Production of both siRNAs and miRNAs require Dicers, which interact with RISC components, suggesting that Dicer action is coupled to loading small RNAs onto the RISC. Two Dicers, Dicer-1 and Dicer-2, have been identified in Drosophila. siRNA production is associated with Dicer-2, but not Dicer-1. Dicer-2, together with the dsRNA-binding protein R2D2, facilitates siRNA loading onto RISC. It was found that the targeted destruction of only one of the two Dicers, Dicer-1, leads to an accumulation of the miR-ban precursor in S2 cells, consistent with the recent genetic studies showing that Dicer-1 is required for miRNA processing. The amount of pre-miR-ban was further increased after prolonged Dicer-1 knockdown for 8 d. In contrast, prolonged suppression of AGO1 expression did not result in the accumulation of pre-miR-ban. These findings suggest that mature miRNAs are processed from premiRNAs by Dicer-1 and are stabilized by AGO1. It was next asked whether AGO1 is physically associated with Dicer-1 by purifying AGO1-associated complexes from S2 cells using a TAP method. It was found that AGO1 was associated with Dicer-1. This interaction of AGO1 with Dicer-1 seems to be quite specific for AGO1 because Dicer-1 was not detected in AGO2- and in dFMR1-associated complexes under the conditions used for the purification. RNA preparations from AGO1-, AGO2-, and dFMR1-associated complexes were then probed for the presence of miR-ban. AGO2- and dFMR1-associated complexes contained the mature form of miR-ban. Only the AGO1-associated complex contained the pre-miR-ban and mature forms. The interaction of AGO1 with Dicer-1 and pre-miRNA further suggests that AGO1 is involved in miRNA biogenesis (Okamura, 2004).

Involvement of microRNA in AU-rich element-mediated mRNA instability: i>Ago1 and Ago2 are required for ARE-mediated RNA degradation

AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation (Jing, 2005).

The ARE motif (AUUUA) is the most studied cis-acting element responsible for rapid turnover of unstable mRNAs in mammalian cells. In the quest for a genetic system that allows a comprehensive search for components involved in ARE-mediated decay of mRNA, Drosophila S2 cells were examined and it was found that the decay of ARE-containing RNA in S2 cells is regulated in a manner similar to that in mammalian cells. Inhibition of gene expression by RNAi is much easier and more cost effectively conducted in Drosophila S2 cells compared to mammalian cells: this allowed for an investigation of a large number of genes for their involvement in ARE-mediated RNA decay. Surprisingly, knockdown of Drosophila Dicer1 gene expression leads to stabilizing an ARE-RNA reporter. Further studies revealed that Drosophila Ago1 and Ago2 are required for ARE-mediated RNA degradation, suggesting involvement of the miRNA system. It was then confirmed that human Dicer is required in ARE-RNA degradation in HeLa cells, which implies that this underlying mechanism is conserved in the mammalian cells. Given the involvement of Dicer in HeLa cells, it was reasoned that miRNA(s) are involved in ARE-mediated RNA decay and a search was conducted for miRNAs that possess a complementary sequence to the canonical AUUUA sequence of ARE. miR16 is a potential candidate due to the presence of the sequence UAAAUAUU, and it was shown that downmodulation and overexpression of miR16 increases or decreases, respectively, the stability of a RNA reporter containing ARE of TNF or Cox2, but not uPAR. Furthermore, it was determined that the regulation of ARE^TNF-RNA decay by miR16 is sequence specific. Just as with Dicer, a function of Ago family members in ARE^TNF-RNA degradation is likely to be the processing of miR16. However, the interaction with the ARE binding protein TTP indicates that Ago/eiF2C family members also play a crucial role in the targeting of miR16 to ARE. These data demonstrate the involvement of miR16 in controlling ARE-RNA turnover and suggest that cooperation of miRNA and ARE binding proteins is essential in the recognition of ARE and in triggering mRNA degradation (Jing, 2005).

Studies have shown that the ability of miRNA to target mRNA is directed by the pairing of miRNA to mRNA. The ARE-complementary sequence in miR16 is indeed required for miR16 function in destabilizing ARE-RNA. However, pairing with no more than an eight-base ARE-sequence may not be sufficient for miR16 to target ARE-RNA. In addition, the pairing of miR16 to ARE is not in the 5′ region of miRNA, which is believed to be more critical in causing gene repression than the 3′ region. It is speculated, then, that TTP is a factor that assists miR16 targeting to ARE sequences due to its ability to interact with the ARE and RISC complex. This explains why miR16-mediated ARE-RNA instability requires TTP. In addition, the requirement of miR16 in TTP-mediated destabilization of ARE-RNA suggests that targeting of miR16 to ARE is a necessary step for RNA degradation (Jing, 2005).

ARE sequences from different mRNA can vary dramatically, with some containing multiple AU-rich elements that allow for simultaneous interaction with more than one miRNA. This could influence the ability of miRNA to promote RNA degradation because of the potential synergistic effect of miR16 to bind to multiple sites. This synergism has been demonstrated in a study that shows the addition of multiple binding sites of CXCR4 siRNA into 3′UTR of a reporter results in more translation inhibition than expected when summing up the individual effects of each binding site. The number of pairs that miR16 can form with different ARE sequences varies from five to eight, and the strength of interaction between miR16 and different AREs in a given mRNA may also vary. The number of miRNAs targeted to an mRNA and the strength of the interaction may both contribute to the quantitative control of mRNA turnover or translation. Perhaps since no more than six pairs can form between miR16 and ARE of uPAR and since uPAR has only one AUUUA motif in the 3′UTR, miR16 does not have a significant effect on the stability of mRNA containing uPAR 3′UTR (Jing, 2005).

miR16 is conserved in mammals. Although a homolog of miR16 has not been found in Drosophila, miR289 contains UAAAUAUUUA, and four other known Drosophila miRNAs contain a UAAAU sequence. Among them, at least miR277, miR289, and miR304 are expressed in S2 cells. 2′-O-methyl oligonucleotides were used to test for Drosophila miRNA that could be involved in ARE-RNA degradation in S2 cells. The anti-miR289 oligo significantly stabilizes mRNA containing TNF-α ARE, while the other four oligos have no or very modest effects on the stability of ARE^TNF-RNA. miR289 has a similar effect on the stability of ARE^IL-6-RNA and ARE^IL-8-RNA. Sequence comparisons showed that miR289 partially complements with ARE, but not the other regions of these 3′UTRs. Thus, miR289 is likely to be a miRNA that has a role in regulating ARE-RNA in S2 cells (Jing, 2005).

Though the association of miR16 with ARE-RNA in the presence of TTP and S-100 in vitro has been demonstrated, the exact mechanism of miRNA targeting of ARE and regulation of RNA degradation remains undetermined. Because of the similarity between siRNA and miRNA in regulating gene expression, miR16-mediated ARE-RNA degradation could be similar to siRNA-mediated mRNA decay. It is theoretically possible that the targeting of ARE with miRNA leads to mRNA cleavage at the targeting site since RISC has been shown to be an RNA endonuclease in vitro. However, translational suppression caused by miRNA or imperfect pairing of siRNA suggests that endonuclease activity is not always associated with RISC. Since ARE-RNA degradation is believed to be initiated by deadenylation and subsequent targeting by the exosome pathway, and since endocleaved ARE-RNA was not detected in the experimental system that was used, it is believed that the RISC involved in ARE-RNA decay is not associated with endonuclease activity. At the present, it is not clear if RISC can execute an exonuclease function, although an exonuclease, Tudor-SN, has been found in the RISC complex. TTP has been shown to bind to extended ARE sequences by virtue of its zinc finger and associates with components of exosomes; this study shows that TTP is also associated with eiF2C/Ago family members. A recent study reported that an exosome associated DexH box helicase facilitates ARE-RNA deadenylation and decay in mammalian cells. Interestingly, a C. elegens homolog of this DexH box protein has been shown to interact with a protein complex containing Dicer, RDE-1, and RDE-4. It appears that ARE binding proteins, miRNA, deadenylase, and exosomes cooperate with each other in regulating mRNA degradation. A model is favored in which TTP binds to an ARE and transiently interacts with the RISCs that scan mRNA. When a RISC containing miR16 encounters TTP, it stays with ARE and TTP due to base complementarity between miR16 and ARE. It is conceivable that RISC, in conjunction with TTP, serves to recruit proteins for deadenylation and/or exosomes for mRNA degradation (Jing, 2005).

Hundreds of miRNAs have been identified, but the targets of most miRNAs are unknown. Since perfectly or nearly perfectly paired sequences can only be found for a few miRNAs, computational as well as experimental approaches have been developed to identify potential miRNA targets that do not contain perfect complementary sequences. Although these approaches have been shown to be very useful, ARE was not identified as the target of miR16 through currently available computer programs. The current data suggest that additional factors, such as sequence-specific RNA binding proteins, needs to be considered in studying the function of miRNA. As in the case of miR16, many miRNAs may require specific proteins in binding to their mRNA targets. The role of many miRNAs may need to be studied, not only in the context of miRNA-mRNA interaction, but also the interaction of miRNA complexes with other proteins (Jing, 2005).

Drosophila argonaute-2 is required early in embryogenesis for the assembly of centric/centromeric heterochromatin, nuclear division, nuclear migration, and germ-cell formation

The RNA-induced silencing complex (RISC) or the RISC complex mediates RNAi and is comprised of proteins belonging to the dicer and Argonaute family proteins. Argonaute-2 is required for proper nuclear migration, pole cell formation, and cellularization during the early stages of embryonic development in Drosophila. These defects have been traced back to the nuclear division cycles. Unlike wild type, nuclear division is asynchronous in ago-2 embryos and there are defects in chromosome condensation, nuclear kinesis, and assembly of spindle apparatus. The aberrations in the nuclear division cycle are correlated with defects in the formation of centric/centromeric heterochromatin and point to a failure in the assembly of functional centromeres (Deshpande, 2005).

Since the RNAi machinery has been implicated in the formation of centric heterochromatin and mitosis in unicellular organisms, it was anticipated that a loss of ago-2 activity might influence the rapid nuclear divisions in early fly embryos. To investigate this possibility, wild-type and ago-2⁴¹⁴ mutant embryos stained with the DNA dye Hoechst were compared. Several abnormalities were evident in ago-2 embryos. The spacing between nuclei in wild type is relatively constant during the early cleavage cycles, and the nuclei are distributed uniformly through the central region of the embryo. In contrast, in ago-2⁴¹⁴ embryos, the distance from one nucleus to the next is quite variable, and nuclei are found clustered in some regions of the embryo while they are absent in other regions. When the nuclei migrate out to the cortex in wild type during cycles 8-9, they are spaced at regular intervals around the entire surface of the embryo. This is not the case in ago-2⁴¹⁴ embryos. Many ago-2⁴¹⁴ nuclei fail to migrate to the surface, while others appear to reach the surface but then fall back into the interior of the embryo. Consequently, the nuclear distribution at the surface is quite irregular. In some regions there is a high concentration of nuclei, often linked together in 'strings' of two, three, or four incompletely separated nuclei, while in other regions there are very few. There are also some very brightly stained nuclei that appear to have more than the normal complement of DNA, while other nuclei are smaller than normal (some are just small dots) and seem to be missing DNA. Defects in chromosome segregation are also evident prior to nuclear migration. Consistent with a requirement for ago-2 in the proper execution of the mitotic cycle, anomalous anaphase figures with incompletely condensed and irregularly positioned chromosomes are observed in the ago-2⁴¹⁴ mutant (Deshpande, 2005).

Similar defects in nuclear division, migration, and spacing were observed for a second ago-2^51b allele and for trans-combinations between the two ago-2 mutations. In all three cases, the frequency of embryos showing at least some defect in nuclear division and/or nuclear migration was between 50% and 60%. These abnormalities prompted a re-examination of the effects of ago-2 mutations on viability. 10%-15% of ago-2⁴¹⁴ embryos failed to hatch, while nearly 40% of ago-2^51B did not hatch. The viability of the trans-combination was intermediate between the homozygous mutants (Deshpande, 2005).

To further characterize the progression of the nuclear division cycles in ago-2, Histone H3 phosphorylation on Ser 10 was examined. In wild type, H3-Ser 10 phosphorylation can be detected nearly simultaneously in all nuclei when chromosome condensation commences at the onset of mitosis. High levels of H3 phospho-Ser 10 persist through mitosis and then disappear as the chromosomes start to decondense before the beginning of the next round of DNA synthesis. A variety of anomalies are evident in ago-2⁴¹⁴ embryos. In the early cleavage stage embryo, these include fragmented nuclei and nuclei that appear to have incompletely separated but are still entering mitosis. In the older embryo, the most striking defect is the asynchrony in the nuclear division cycles. Asynchrony can also sometimes be seen in incompletely separated nuclei (Deshpande, 2005).

The nuclear division defects suggested that ago-2 may be important for the assembly or functioning of the mitotic spindle apparatus. In the wild-type embryo, the chromosomes in each mitotic figure are aligned on the metaphase plate. Emanating from the chromosomes in opposite directions are microtubule arrays that converge at each centrosome. Nuclei that appear to be undergoing a normal mitosis are seen in ago-2⁴¹⁴ embryos. However, a variety of defects are also evident in more than half of the ago-2⁴¹⁴ embryos. Among the more striking are 'orphan' centrosome pairs. While the duplicated centrosomes occasionally remain in close proximity, most appear to migrate to 'opposite poles'. However, these orphan centrosome pairs are not associated with a mitotic spindle apparatus or with mitotic chromosomes, nor does there appear to be any nearby interphase nucleus. While other centrosome pairs appear to properly nucleate the mitotic spindle apparatus, the spindles are abnormally short and do not extend to the chromosomes or make connections with the centromeres. In these mitotic figures the chromosomes often appear to be displaced from their normal position in the center of the metaphase plate. In other cases there are DNA bridges that extend between two adjacent mitotic figures. There are also 'mitotic spindle' bridges that connect two different mitotic figures. Similar centrosomal and mitotic spindle defects are seen in ago-2^51b embryos (Deshpande, 2005).

The mitotic defects in S. pombe and T. brucei lacking Ago activity have been attributed to a disruption in centromere function because of a failure to properly assemble centromeric heterochromatin and the flanking centric heterochromatin. It therefore seemed possible that the abnormal nuclear division and the defects in the spindle apparatus evident in ago-2 embryos might also arise from a failure in the formation of heterochromatin across the centromeric repeats. Drosophila centromeric heterochromatin contains a centromere-specific histone H3 variant, called centromeric identifier (CID), that is critical for centromere function and assembly of the kinetochore. To examine centromeric heterochromatin, ago-2⁴¹⁴ embryos were probed with antibody against CID and Centrosomin. During anaphase the chromosomes on the metaphase plate begin moving toward the two centrosomes. At this point in the mitotic cycle, the centromeres are located in close proximity to the centrosomes, while the chromosomal arms extend back toward the metaphase plate. The centromeres can be visualized in wild-type chromosomes as several prominent dots of CID staining located near the base of the spindle apparatus in close proximity to each centrosome. While prominent CID dots can be seen on chromosomes throughout mitosis in wild-type embryos, this is not true in ago-2⁴¹⁴ embryos (Deshpande, 2005).

Flanking the centromere is the centric heterochromatin, which is marked by elevated levels of histone H3 methylated on Lys 9 (H3 meK9) and by the Swi6 homolog, Heterochromatin protein 1 (HP-1). Association of HP-1 with centric heterochromatin is abnormal in ago-2⁴¹⁴ precellular blastoderm embryos. There also appear to be defects in K9 methylation (Deshpande, 2005).

If ago-2 is required for the formation of centromeric/centric heterochromatin then ago-2 mutations should suppress silencing of mini-white transgenes inserted into pericentric heterochromatin. Males carrying mini-white transgenes inserted into pericentric regions of the second, third, or fourth chromosomes were mated to ago-2⁴¹⁴ females, and the eye color phenotype of ago-2⁴¹⁴/+ adults was examined. For inserts on all three chromosomes, it was found that mini-white expression is up-regulated in ~7% of the ago-2⁴¹⁴/+ flies. Significantly, the pattern of suppression points to a very early role for ago-2 in the establishment of silenced heterochromatin domains. Thus, while the eye color varies from one fly to the next, in the flies showing suppression, both eyes typically have very similar pigmentation. Moreover, instead of a highly variegated phenotype, the pigmentation is (with the exception of a few ommatidia) usually quite uniform across each eye. These results suggest that maternally derived ago-2 activity is required for establishing silenced heterochromatin domains in the paternal chromosomes at a point prior to nuclear migration (Deshpande, 2005).

The occurrence of 'orphaned' centrosomes prompted an examination of the cytoskeleton in ago-2 embryos with antibodies against Anillin, a component of the actin-myosin contractile ring, and Peanut, a septin. During interphase in wild-type Drosophila nuclear cycle 13-14 blastoderm embryos, cortical actin forms a cap on the apical surface of the embryo above the nucleus. Anillin is localized just underneath the actin cap in a ring that surrounds the nucleus, and in surface views it gives a quite regular mesh-like appearance. In ~50% of the ago-2⁴¹⁴ embryos this regular lattice is replaced by a broken network with irregularly shaped contractile rings that vary in thickness from one part of the ring to the next. Some of the anillin rings appear to contain multiple nuclei, while other rings have neither nuclei nor DNA. Similar irregularities are also observed in the Peanut lattice/network (Deshpande, 2005).

The anillin lattice is not present in wild-type embryos during earlier nuclear cycles, and instead there is a donut-like structure around each nucleus. While the anillin 'donut' is also seen around many nuclei in ago-2 embryos of a similar age, it is often absent altogether. In other cases, the DNA seems to surround the 'donut-hole', instead of being the 'donut-hole' as it is in wild type. Anillin can also be seen concentrated inside the nucleus. Interestingly, when defects are observed in the anillin donuts, they are typically associated with abnormalities in nuclear morphology or with fragmented or otherwise anomalous chromosomes. This finding would suggest that there is a link between the assembly and functioning of the mitotic apparatus and the proper organization of actin-myosin cytoskeleton. For example, the assembly of the anillin donuts/contractile rings may depend on the presence of a functional mitotic spindle and/or the reformation of the nuclear envelope at the end of mitosis. When the mitotic spindle is absent or disrupted, or if the nuclear envelope does not reform because of chromosome fragmentation, this could prevent the assembly of the regular anillin donuts/contractile rings (Deshpande, 2005).

Since proper nuclear migration is necessary for pole cell formation, the number of pole cells were counted at the syncytial blastoderm stage (nuclear cycles 12-13) in ago-2⁴¹⁴ embryos stained with Vasa antibody and counterstained with the DNA dye Hoechst. The total number of pole cells in ago-2⁴¹⁴ (eight per embryo; n = 20) is also much less than wild type (20 per embryo; n = 15). Many ago-2⁴¹⁴ embryos had only two or three pole cells, and the pole cells were often not positioned correctly at the very posterior or were separated from each other by somatic cells. There was also a great deal of variability in the number of pole cells in the ago-2 mutant that is not evident in wild type. While some ago-2⁴¹⁴ embryos had only two or three pole cells, there were a few that had near wild-type numbers. A similar, though not quite as dramatic reduction in pole cell number was observed for a second allele, ago-2^51B (12 per embryo). The reduction in the number of pole cells can be traced back to nuclear cycle 8-9 when migrating nuclei first enter the posterior pole plasm (Deshpande, 2005).

Two questions are posed by the findings. The first is the relationship between the phenotypes seen in early embryos and ago-2 function. Although the possibility that ago-2 participates in several distinct pathways, it is suspected that many of the phenotypes are attributable either directly or indirectly to a requirement for ago-2 in the proper execution of the nuclear division cycles. In principle, ago-2 could impact the division cycles by promoting the degradation and/or translational repression of mRNAs encoding factors that somehow impede the cleavage cycles. However, since the RNAi machinery has been implicated in the establishment of heterochromatin domains, an alternative possibility is that ago-2 is important for generating the appropriate chromatin organization of cleavage-stage chromosomes. In S. pombe, deletion of Ago-1 (the only Ago gene) disrupts the formation of transcriptionally silenced heterochromatin domains near the centromeres, and this impairs the assembly/functioning of the centromeres. Most of the abnormalities in the division cycles and mitotic apparatus evident in ago-2 embryos could also be explained by perturbations in the assembly/functioning of the centromeres. Probably the most compelling evidence in favor of this hypothesis is the defects in CID association with ago-2 chromosomes (Deshpande, 2005).

Dividing nuclei were observed in which a subset of chromosomes or in some cases all of the chromosomes have little or no detectable CID. Strikingly, in these anaphase figures the chromosomes that have little CID typically remain on the metaphase plate, while the chromosomes that have CID are seen to migrate toward the centrosomes. The effects of ago-2 are not limited to centromeric heterochromatin. It was found that the silencing of paternally inherited mini-white transgenes inserted into centric heterochromatin can be suppressed by reducing ago-2 activity in the mother. Significantly, the pattern of suppression -- a nearly uniform level of pigmentation that is the same in both eyes -- argues that ago-2 activity is required very early in development during the initial 'establishment' of silenced heterochromatin. Consistent with an early role in heterochromatin assembly, abnormalities were also found in HP1 localization. While these results do not provide insights into how Ago-2 might actually function in this process, the idea that the RNAi machinery plays some special role in the de novo assembly of centromeric/centric heterochromatin during the embryonic nuclear division cycles is supported by the finding that siRNAs are highly enriched in early embryos but not at later stages of development (Deshpande, 2005).

The second question is why do the ago-2 mutants show incomplete penetrance and expressivity. One reason may be the plasticity of early embryos that enables cells in regions of the embryo that are normal to compensate for cells in regions that have major defects. Consistent with this suggestion, mutations have been described in three genes, l(3)malignant brain tumor, shakleton, and out of sync, that have a range of nuclear division and migration phenotypes similar to those seen in the two ago-2 alleles. In spite of the defects observed in these mutants, most of the embryos hatch and go on to form fertile adults. Another reason is that the two mutants examined may not be complete nulls. Consistent with this possibility, a low level of Ago-2 protein was detected in mutant embryos. Finally, there could be other partially redundant mechanisms that ensure the formation and/or maintenance of centromeric/centric heterochromatin and functional centromeres. This idea is supported by the studies that found that like ago-2, mutations in two other ago-2-like proteins, piwi and aubergine, suppress heterochromatic silencing of mini-white transgenes and disrupt the localization of heterochromatin proteins HP1 and HP2. Thus, it is possible that either of these two genes or perhaps other components of the RNAi machinery are able to substitute for ago-2 and promote the establishment and/or maintenance of heterochromatin domains and functional centromeres in early embryos (Deshpande, 2005).

RNA interference directs innate immunity against viruses in adult Drosophila

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-2^L811fsX. Northern blot hybridizations showed that, although FHV RNAs remained undetectable in dcr-2^L811fsX embryos injected with R1fs, viral RNA accumulation is rescued in the dcr-2^L811fsX 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-2^L811fsX 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-2^L811fsX flies. Mock inoculation with buffer had little effect on either WT or dcr-2^L811fsX 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-2^L811fsX 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-2^L811fsX. 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-2^L811fsX 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-2^L811fsX, 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-2^L811fsX, 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-2^L811fsX 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-2^L811fsX 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-2^L811fsX 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).

Overlapping functions of Argonaute proteins in patterning and morphogenesis of Drosophila embryos

Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs) of unknown function. This study shows that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, it is further demonstrated that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. These findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning (Meyer, 2006).

This study characterizes the maternal-effect mutation drop out (dop), which causes specific developmental defects at the midblastula transition. The mutant embryos show a transient block in membrane growth and fail to undergo a developmental switch in the microtubule-based polarized transport of lipid droplets. Surprisingly, dop mutations represent special alleles of ago2. Two independently generated dop alleles reduce the copy number of the GRRs, providing the first evidence of a functional role of this domain. These mutations render Ago2 only partially defective in siRNA responses. However, these alleles interact genetically with Ago1, suggesting the possibility of crosstalk between Ago1- and Ago2-mediated pathways. This conclusion is further supported by double-mutant analysis using loss-of-function alleles of ago2 and ago1; it was demonstrated that the two gene products function redundantly in embryonic patterning. The results reveal novel functions of Argonaute proteins in early embryogenesis and suggest a regulatory role for the GRR domain of Ago2 (Meyer, 2006).

In Drosophila, two major molecular pathways of RNA silencing have been defined: miRNA-induced silencing and siRNA-induced RNAi. At the level of Argonaute family members, Ago1 has been implicated in miRNA function while Ago2 was shown to be essential for siRNA function. This analysis provides genetic and biochemical evidence that Ago1 and Ago2 have overlapping functions both in siRNA-triggered RNAi and during early embryogenesis (Meyer, 2006).

In addition to the PAZ and PIWI domains conserved in all family members, insect orthologs of Ago2 contain an amino-terminal GRR domain. The ago2^dop alleles allowed the function of this domain to be probed. Even the subtle alterations in these alleles have striking organismal phenotypes, but the absence of Ago2 (in the reported null alleles) does not. While the mutant Ago2 proteins still support siRNA function to some extent, they also interfere with Ago1-dependent processes (Meyer, 2006).

In other proteins, glutamine-rich domains have been implicated in protein aggregation, such as in certain neurodegenerative diseases that involve the formation of long-lived protein aggregates (e.g., the PolyQ domain of mutant Huntingtin). Extension of the glutamine-rich region promotes aggregation, and the length of the polyglutamine extension correlates with the severity of the disease. Glutamine-rich domains are also involved in the mechanism by which yeast prions switch between soluble and aggregated states. For the translation factor Sup35, e.g., increases in the copy number of GRRs in the prion domain favor the aggregated, inactive state; decreases in the copy number favor the soluble, active state. Genetic and molecular analyses of the ago2^dop alleles thus raise the tantalizing possibility that the GRRs regulate Ago2 by modulating its aggregation state. Unlike in the polyglutamine diseases, however, it is the reduction, rather than the expansion, of the GRR region that leads to an aberrant Ago2 protein. Drosophila Ago2 may therefore provide a unique inroad for dissecting the normal organismal function of glutamine-rich or PolyQ domains (Meyer, 2006).

Since Ago2 is an essential component of protein complexes, such as the RISC, control of its aggregation state is conceivably important for its function. Mammalian Argonaute proteins are localized to GW bodies, cytoplasmic compartments analogous to yeast P-bodies, which are centers of mRNA degradation. Central components of GW bodies, like GW182 and decapping enzymes DCP1:DCP2, have been shown to also be involved in miRNA-mediated gene silencing in Drosophila cultured cells. The presence of both Ago1 and Ago2 in GW bodies is consistent with the biochemical studies. An important next step for unraveling the molecular function of the Ago2 GRR domain will be to determine whether the ago2^dop alleles alter the recruitment of Ago2 to particular cytoplasmic mRNA degradation complexes. Such recruitment via glutamine-rich domains need not necessarily inactivate the protein: in the translation factor CPEB from Aplysia, a glutamine-rich prion-like amino-terminal domain promotes protein aggregation, and it is the aggregated form that has the greatest capacity to stimulate translation (Meyer, 2006).

Previous analyses have suggested a simple model of division of labor between Argonaute proteins in Drosophila, with Ago1 specific for miRNA-directed silencing and Ago2 involved in siRNA-triggered RNAi. However, the genetic data add to emerging evidence that these proteins play much broader roles. Ago2, for example, appears to have functions beyond siRNA-induced RNAi. It has been proposed that in larval neurons Ago2 is recruited via the dFMR1 protein to certain RNP complexes, including those containing the PPK1 mRNA. This recruitment is functionally important since in the ago2^51B allele PPK1 mRNA levels are not properly downregulated; thus, Ago2 may play a role in the turnover of specific transcripts (Meyer, 2006).

For Ago1, in contrast, it is well established that it has a function in miRNA-directed RNA silencing. But while in biochemical assays Ago1 is not essential for siRNA function, ago1 mutations impair the response of siRNA-triggered RNAi in vivo. The data provide further evidence for overlapping functions of Ago2 and Ago1 in siRNA-directed RNAi. It is possible that although Ago2 is in principle sufficient to promote siRNA-directed RNA decay, in vivo the two proteins act in concert to make this process more efficient (Meyer, 2006).

It is unlikely that the morphogenesis phenotypes of ago2^dop mutant embryos are simply caused by disturbing the function of Ago2 in RNAi. Unlike ago2^dop1 mutants, ago2 alleles that completely abolish experimental siRNA-induced responses do not cause these gross morphological defects and exhibit problems with nuclear migration only during syncytial stages; these phenotypes occur with a moderate penetrance such that animals homozygous for these alleles can be kept as a stock. Rather, genetic data suggest that ago2^dop mutations compromise the function of both Ago2 and Ago1 in controlling specific aspects of the MBT. A genome-wide analysis of mRNA targets regulated by Argonaute proteins has recently shown that Ago1 and Ago2 are required for the regulation of a common set of miRNA targets, despite the fact that only Ago1 is essential for miRNA function in vitro. In S2 cells, both Ago1 and Ago2 coprecipitate with specific miRNAs, suggesting that not only Ago1, but also Ago2, is able to bind miRNAs. Based on the results, it is conceivable that the interaction of miRNAs with Ago2 is indirect, namely that Ago2 coprecipitates those miRNAs that are bound to Ago1. While the exact mechanisms need to be resolved, the available data provide ample support for the conclusion that Ago1 and Ago2 act in a partially redundant fashion during early embryogenesis (Meyer, 2006).

It is conceivable that the ago2^dop mutations not only interfere with Ago1 and Ago2 function but might affect a common factor that is essential for both Ago1 and Ago2 or for Argonaute protein function in general. Preliminary observations suggest that mutations in other Argonaute family members, piwi or aubergine, might also interact genetically with ago2^dop alleles. The model is favored that disrupting both Ago1 and Ago2 function is sufficient to cause the observed defects at the MBT because ago2^dop1 mutants can be rescued by zygotic expression of either ago1 or ago2. A test of this notion will be to determine the phenotypic consequences for embryos when both the maternal and zygotic expression of ago1 and ago2 has been eliminated. In addition, the interactions of ago2^dop alleles with other components of RNA silencing pathways should be examined to further understand the genetic and molecular basis for the altered activity of Ago2^dop proteins during the MBT (Meyer, 2006).

Mutations in ago1 were originally discovered in a genetic screen for modifiers of the Wg pathway. Overexpression of ago1 rescues a defect in Wg signaling induced by depletion of cytoplasmic Arm in the wing imaginal disc. However, because embryos homozygous for a loss-of-function mutation in ago1 did not exhibit defects in segment polarity, the relevance of Ago1 for normal Wg signaling remained unclear. The data presented in this paper now provide an explanation for this result. By combining loss-of-function mutations in both ago1 and ago2, it is demonstrated that the two Argonaute genes have partially overlapping functions and together are required for establishing segment polarity (Meyer, 2006).

The requirement of Ago1 and Ago2 for the initial expression of Wg protein is striking. No other genes have been identified that are similarly essential for the general expression of Wg. Two possible explanations are proposed for this result. Ago1 and Ago2 might act to eliminate a general repressor of wg transcription or translation. In this case, it is conceivable that specific miRNAs exist that modulate wg expression by negatively regulating a repressive mechanism. Alternatively, Ago1 and Ago2 might be part of RNPs that contain wg mRNA, and the reduction in Argonaute function might interfere with the microtubule motor-driven localization of the transcripts. It is well established that compromising the apical localization of wg mRNA strongly affects the intracellular distribution and the signaling activity of the protein. A detailed analysis of the expression and the localization of wg transcripts will be required to discriminate between these possibilities (Meyer, 2006).

Although no direct evidence was found that any of the ago2 alleles interfere with miRNA function in vivo or in vitro, it is interesting to note that ago1;Dcr-1 double mutants exhibit the same segment polarity phenotypes as ago1, ago2 double mutants. This result further strengthens the notion that in the embryo Ago1 and Ago2 might both be important for miRNA function. An eye reporter assay was employed to test if ago2^dop alleles interfere with the function of the bantam miRNA, no interactions were detected. This result might be due to the observed redundancy of Ago2 with Ago1 function; such a redundancy was recently described for S2 cells. Future studies to identify the miRNAs involved and their targets might yield novel insight into the regulation of Wg expression (Meyer, 2006).

An alternative explanation is that this analysis has uncovered a novel function of Argonaute protein family members. Intriguingly, ectopically expressed Ago1 constructs can suppress Wg pathway defects even if they lack a functional PIWI domain. This result may suggest that Ago1 function in Wg signaling does not involve its PIWI domain, hinting at an uncharacterized biochemical property of Ago1. Although too little is known at this point to speculate what such a new function might entail, it is interesting to note that there are intriguing connections between microtubules and the RNA silencing machinery: Armitage, a putative helicase required to assemble Ago2-containing RISC, is associated with microtubules in developing oocytes; the dop alleles of Ago2 interfere with microtubule-based processes at the MBT; and it is conceivable that Ago1 and Ago2 control the microtubule-dependent localization of wg mRNA. Whether or not these phenomena are explained by a shared molecular mechanism remains to be established (Meyer, 2006).

In summary, the genetic interactions described in this paper are not easily reconciled with the model that different pathways in gene silencing are strictly separated. Rather, the data suggest that in the living organism these pathways, or at least crucial components of these pathways, might act in concert. Observation that ago1 and ago2 cooperate in Wg signaling provides a powerful new tool to resolve some of these issues since now the function of these Argonaute proteins can be assessed using a clearly defined phenotype of a well-characterized signaling pathway (Meyer, 2006).

Freshly laid Drosophila embryos contain large amounts of maternally supplied mRNAs that encode proteins essential for the earliest stages of embryogenesis. As development proceeds, these maternally supplied transcripts need to be replaced by transcripts synthesized by the zygote. This process is a hallmark of the MBT. Maternal transcripts are degraded via two pathways: a maternal pathway switched on at egg activation, and a zygotic pathway activated at the MBT. Genetic analysis has shown that although ago2^dop alleles represent maternal-effect mutations, they specifically perturb processes shortly after the onset of zygotic transcription at the MBT. It is therefore proposed that Ago1 and Ago2 are key mediators of the zygotic pathway of maternal transcript degradation. Precedence for such a scenario has recently been provided by the identification of the miR-430 miRNA family in zebrafish. miR-430 expression is strongly upregulated at the MBT and is required to specifically downregulate a set of maternal mRNAs. Conversely, embryos deficient for Dicer activity display defects shortly after the MBT. It remains to be determined whether miRNAs are also required for maternal transcript degradation in Drosophila (Meyer, 2006).

The known functions and structural features of Argonaute proteins suggest a model for the underlying molecular mechanisms. It is well established that Argonaute proteins can act as ribonucleases and provide slicer activity in RISC. During early development, Ago2 and Ago1 might act as ribonucleases that cleave maternal transcripts at the MBT. Abnormal persistence of maternal mRNAs could then interfere with the morphogenetic events usually triggered by zygotic transcription, such as membrane growth during cellularization and correct directionality of lipid-droplet transport. Alternatively, Argonaute proteins might regulate the translation of such maternal or zygotic transcripts. Since no significant changes in the expression pattern of known regulators of membrane growth and droplet transport (Halo, Slam, Klar) were detected, the relevant targets are likely novel components of these regulatory pathways. Identifying them should not only give insight into the regulation of these fundamental cell-biological processes but will also shed light on the mechanisms by which the Argonaute proteins Ago1 and Ago2 work together to control developmental events (Meyer, 2006).

RNA interference machinery influences the nuclear organization of a chromatin insulator

RNA interference (RNAi) is a conserved silencing mechanism that can act through alteration of chromatin structure. Chromatin insulators promote higher-order nuclear organization, thereby establishing DNA domains subject to distinct transcriptional controls. Evidence is presented for a functional relationship between RNAi and the gypsy insulator of Drosophila. Insulator activity is decreased when Argonaute genes required for RNAi are mutated, and insulator function is improved when the levels of the Rm62 helicase, involved in double-stranded RNA (dsRNA)-mediated silencing and heterochromatin formation, are reduced. Rm62 interacts physically with the DNA-binding insulator protein CP190 in an RNA-dependent manner. Finally, reduction of Rm62 levels results in marked nuclear reorganization of a compromised insulator. These results suggest that the RNAi machinery acts as a modulator of nuclear architecture capable of effecting global changes in gene expression (Lei, 2006).

These results suggest the existence of an RNA species required for the formation or integrity of insulator bodies, perhaps a product of processing by Argonautes and the other RNAi machinery. The putative RNA helicase Rm62 may be recruited to insulator complexes through physical interaction with CP190 and RNA. Although it is unknown at what mechanistic step Rm62 acts in RNAi, Rm62 may act downstream of Argonautes to unwind or remodel RNA-insulator protein complexes, thereby disrupting gypsy insulator activity and nuclear organization. Proper insulator body localization requires an intact nuclear matrix, and early observations identified RNA as an important component of this nuclear scaffold. Future studies should determine the identity of putative gypsy insulator associated RNAs. These results suggest a previously unknown function of the RNAi machinery in the control of nuclear architecture to effect changes in gene expression (Lei, 2006).

P-body formation is a consequence, not the cause, of RNA-mediated gene silencing; AGO2 is required for P-body integrity

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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