In eukaryotic cells degradation of bulk mRNA in the 5' to 3' direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5' to 3' exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins, which are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional, processes P-body or essential pathway components were depleted from Drosophila cells and the effects of these depletions were analyzed on the expression of reporter constructs, allowing specific monitoring of NMD, RNAi, or miRNA function. The RNA-binding protein GW182 (Gawky) and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. This analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence in Drosophila (Rehwinkel, 2005).
In eukaryotic cells, bulk messenger RNA (mRNA) is degraded via two alternative pathways, each of which is initiated by the removal of the poly(A) tail by deadenylases. Following this first step, mRNAs can be degraded from their 3' ends by the exosome, a multimeric complex of 3' to 5' exonucleases. Alternatively, after deadenylation, the cap structure is removed by the DCP1:DCP2 decapping complex, and the mRNA is degraded by the major cytoplasmic 5' to 3' exonuclease XRN1 (Rehwinkel, 2005).
Proteins required for 5' to 3' mRNA degradation (e.g., DCP1, DCP2, and XRN1) colocalize in specialized cytoplasmic bodies or mRNA decay foci, also known as mRNA processing bodies (P-bodies) or GW-bodies, because of the accumulation of the RNA binding protein GW182 in these bodies. Additional components of P-bodies in yeast and/or human cells include the deadenylase Ccr4 (see Drosophila Twin), the cap binding protein eIF4E and its binding partner eIF4E-transporter (eIF4E-T), auxiliary decay factors such as the LSm1-7 complex, Pat1p/Mtr1p, and the putative RNA helicase Dhh1/rck/p54. Among these, human GW182, eIF4E-T, and Dhh1 are required for P-body formation, while the decapping enzymes and XRN1 are dispensable. In addition, mRNA decay intermediates, microRNA (miRNA) targets, and miRNAs have been localized to P-bodies, suggesting that these bodies are sites where translationally silenced mRNAs are stored before undergoing decay (Rehwinkel, 2005 and references therein).
Recently, proteins involved in other post-transcriptional processes have been localized to P-bodies in human cells. These include the proteins SMG5, SMG7, and UPF1 involved in the nonsense-mediated mRNA decay (NMD) pathway and the Argonaute (AGO) proteins that play essential roles in RNA silencing. Moreover, XRN1 is recruited by both the NMD and the RNA interference (RNAi) machineries to degrade targeted mRNAs, suggesting a possible link between NMD, RNAi, and P-bodies. NMD is an mRNA quality control (or surveillance) mechanism that degrades aberrant mRNAs having premature translation termination codons (PTCs), thereby preventing the synthesis of truncated and potentially harmful proteins. Core components of the NMD machinery include the proteins UPF1, UPF2, and UPF3, which form a complex whose function in NMD is conserved. The activity of UPF1 is regulated in multicellular organisms by additional proteins (i.e., SMG1, SMG5, SMG6, and SMG7) that are also required for NMD in all organisms in which orthologs have been characterized (Rehwinkel, 2005 and references therein).
In yeast and human cells, a major decay pathway for NMD substrates involves decapping and 5' to 3' degradation by XRN1. Although degradation of nonsense transcripts in Drosophila is initiated by endonucleolytic cleavage near the PTC, the resulting 3' decay intermediate is also degraded by XRN1. A molecular link between the NMD machinery and the decay enzymes localized in P-bodies is provided by SMG7 in human cells. Indeed, when overexpressed, human SMG7 localizes in P-bodies and recruits both UPF1 and SMG5 to these bodies, suggesting that NMD factors may reside at least transiently in P-bodies. RNA silencing pathways are evolutionarily conserved mechanisms that elicit decay or translational repression of mRNAs selected on the basis of complementarity with small interfering RNAs (siRNAs) or miRNAs, respectively. siRNAs are fully complementary to their targets and elicit mRNA degradation via the RNAi pathway. Animal miRNAs are only partially complementary to their targets and do not generally elicit decay, but repress translation instead (Rehwinkel, 2005 and references therein).
To perform their function, the siRNAs and miRNAs associate with the AGO proteins to form multimeric RNA-induced silencing complexes (RISC). Drosophila AGO1 mediates miRNA function, while AGO2 catalyzes the endonucleoytic cleavage of siRNA targets within the region complementary to the siRNA. Following this initial cleavage, the resulting 5' mRNA fragment is degraded by the exosome, while the 3' fragment is degraded by XRN1. The localization of AGO proteins in P-bodies in human cells provides a possible link between these bodies and silencing pathways (Rehwinkel, 2005 and references therein).
The NMD, the siRNA, and the miRNA pathways are therefore interlinked by the use of common decay enzymes and/or the coexistence of components of these pathways in P-bodies, suggesting a possible interdependence between these post-transcriptional mechanisms. Evidence for a link between NMD and RNAi has been reported in Caenorhabditis elegans where UPF1, SMG5, and SMG6 are required for persistence of RNAi, though not to initiate silencing. In contrast, UPF2, UPF3, and SMG1, which are also essential for NMD, are not required to maintain silencing, suggesting that UPF1, SMG5, and SMG6 may have evolved specialized functions in RNAi (Rehwinkel, 2005 and references therein).
This study investigates the interplay between NMD, RNAi, and the miRNA pathway using the Drosophila Schneider cell line 2 (S2 cells) expressing reporters allowing the monitoring of NMD, RNAi, or miRNA function. To this end, factors involved in NMD (UPF1, UPF2, UPF3, SMG1, SMG5, and SMG6), RNAi (AGO2), or the miRNA pathway (AGO1) were depleted and the effect on the expression of the reporters analyzed. These proteins showed a high degree of functional specificity. To determine the role of P-body components in these pathways the DCP1:DCP2 decapping complex, the decapping coactivators LSm1 and LSm3, the 5' to 3' exonuclease XRN1, GW182, and the Drosophila protein CG32016, which shares limited sequence homology with human eIF4E-T, were depleted. The results uncovered a crucial role for GW182 and the DCP1:DCP2 decapping complex in the miRNA pathway (Rehwinkel, 2005).
Components of the NMD, RNAi, and miRNA pathways exhibit functional specificity in Drosophila To investigate a potential role of components of RNA silencing pathways or of P-body components in NMD, use was made of cell lines expressing wild-type or PTC-containing reporter constructs in which the coding regions of the bacterial chloramphenicol acetyl transferase (CAT) or the Drosophila alcohol dehydrogenase (adh) genes were placed downstream of inducible or constitutive promoters. The PTCs were inserted at codon 72 and 83 of the CAT and adh open reading frames, respectively. P-body components and proteins involved in NMD, RNAi, or the miRNA pathway were depleted by treating the cells with double-stranded RNAs (dsRNAs) specific for the different factors. A dsRNA that targets green fluorescent protein (GFP) served as a control. The steady-state levels of the wild-type and PTC-containing mRNAs were analyzed by Northern blot and normalized to those of the endogenous rp49 mRNA (encoding ribosomal protein L32) (Rehwinkel, 2005).
Relative to the expression levels of the wild-type mRNAs, the levels of the corresponding PTC-containing transcripts are reduced because these transcripts are rapidly degraded via the NMD pathway. Depletion of UPF1 inhibits NMD, so the levels of the PTC-containing mRNAs are restored. Depletion of AGO1 or AGO2, both singly and in combination, does not interfere with the NMD pathway, although these depletions do inhibit siRNA- or miRNA-mediated gene silencing. The levels of the CAT wild-type transcript were not affected by the depletions. Similar results were obtained with the NMD reporter based on the adh gene. Together, these results indicate that inhibition of RNAi or of the miRNA pathway does not interfere with NMD. XRN1 is the only P-body component known to be required for degradation of decay intermediates arising from mRNAs undergoing NMD in Drosophila. Nevertheless, in cells depleted of XRN1 the NMD pathway is not inhibited, and only the 3' decay intermediate generated by endonucleolytic cleavage of the mRNA accumulates (Rehwinkel, 2005).
In contrast to XRN1, none of the P-body components tested, including GW182 and the DCP1:DCP2 decapping complex, affected NMD or the accumulation of the 3' decay intermediate. The lack of a significant effect of the depletion of the DCP1:DCP2 complex was confirmed using the adh reporter. The decapping enzymes are certainly involved in NMD in yeast and human cells because the major decay pathway for NMD substrates is initiated by decapping in these organisms (for review, see Conti, 2005). Thus, it is possible that the requirement for P-body components and/or P-body integrity in NMD varies across species (Rehwinkel, 2005).
Two different approaches were used to investigate the RNAi pathway. In one approach, a cell line constitutively expressing the wild-type Drosophila adh gene was treated with a dsRNA complementary to a central region of ~300 nucleotides (nt) of adh mRNA (adh dsRNA). This dsRNA elicits decay of the adh mRNA via the RNAi pathway. Cells were treated with dsRNAs targeting various factors in the presence or absence of adh dsRNA. The steady-state levels of the adh mRNA were analyzed by Northern blot and normalized to those of the rp49 mRNA. In cells treated with GFP dsRNA, the normalized levels of the adh transcript were reduced to 4% after addition of adh dsRNA, relative to the levels detected in the absence of adh dsRNA. In cells depleted of AGO2, a sixfold increase of adh mRNA levels was observed despite the presence of adh dsRNA. In contrast, when AGO1 was depleted, adh dsRNA could still trigger a reduction of adh mRNA levels, though a slight increase in transcript levels was observed. Similarly, depletion of UPF1 did not prevent silencing of adh expression by adh dsRNA. These results indicate that UPF1 is not required for RNAi in Drosophila. Additional NMD components (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6) have been identified, but no SMG7 ortholog has been identified in Drosophila. No significant change was observed in the efficacy of RNAi under the conditions in which NMD was inhibited (Rehwinkel, 2005).
Similarly to the results reported for the NMD pathway, depletion of XRN1 leads to the accumulation of the 3' decay intermediate generated by endonucleolytic cleavage by RISC, while depletion of the DCP1:DCP2 decapping complex does not prevent RNAi or the degradation the 3' decay intermediate. In contrast, depletion of GW182 leads to a modest increase in the adh mRNA level in the presence of adh dsRNA, suggesting that this protein could influence the efficiency of RNAi (Rehwinkel, 2005).
In a second approach, RNAi was triggered by an siRNA instead of a long dsRNA, to uncouple RISC activity from processing of dsRNAs. To this end, S2 cells were transiently transfected with a plasmid expressing firefly luciferase (F-Luc) and an siRNA targeting the luciferase coding sequence (F-Luc siRNA) or a control siRNA. A plasmid encoding Renilla luciferase (RLuc) was included to normalize for transfection efficiencies. Cotransfection of the F-Luc reporter with the F-Luc siRNA led to a 50-fold inhibition of firefly luciferase activity relative to the activity measured when the control siRNA was cotransfected, indicating that F-Luc siRNA effectively silences firefly luciferase expression (Rehwinkel, 2005).
The results obtained with the luciferase reporter correlate well with those obtained with adh mRNA, in spite of differences between the methods used to detect changes in reporter levels (RNA levels vs. protein levels), and the nature of the RNA trigger (long dsRNA vs. siRNA). Indeed, depletion of AGO2 impaired silencing of firefly luciferase expression by the F-Luc siRNA, leading to an eightfold increase in firefly luciferase activity relative to the activity of the Renilla control. Depletion of AGO1 led to a twofold increase of firefly luciferase activity (Rehwinkel, 2005).
The observation that depletion of AGO2, but not AGO1, significantly inhibits RNAi is in agreement with previous reports showing that only AGO2-containing RISC is able to catalyze mRNA cleavage triggered by siRNAs. The results together with these observations indicate that Drosophila AGO1 and AGO2 are not redundant (Rehwinkel, 2005).
Depletion of GW182 or the DCP1:DCP2 complex led to a 1.5- to twofold increase of the firefly luciferase activity, although RNAi was not abolished. These results together with those obtained with the adh reporter suggest that GW182 and the DCP1:DCP2 complex are not absolutely required for RNAi but may modulate siRNA function (Rehwinkel, 2005).
Finally, depletion of core NMD components does not inhibit the silencing of firefly luciferase expression by F-Luc siRNA. The results are consistent with results from C. elegans showing that NMD per se is not required for the establishment of silencing (Rehwinkel, 2005).
To investigate the miRNA pathway firefly luciferase reporters were generated in which the coding region of firefly luciferase is flanked by the 3' UTRs of the Drosophila genes CG10011 or Vha68-1. These genes were identified as miRNA targets in a genome-wide analysis of mRNAs regulated by AGO1. The 3' UTR of CG10011 mRNA contains two binding sites for miR-12, while the 3í UTR of Vha68-1 has two binding sites for miR-9b. Expression of the firefly luciferase construct fused to the 3' UTR of CG10011 (F-Luc-CG10011) was strongly reduced by cotransfection of a plasmid expressing the primary (pri) miR-12 transcript, but not pri-miR-9. Conversely, expression of the firefly luciferase reporter fused to the 3' UTR of Vha68-1 (FLuc-Vha68-1) was inhibited by cotransfection of pri-miR-9b, but not of primiR-12 (Rehwinkel, 2005).
Silencing of luciferase expression by the cognate miRNAs was prevented in cells depleted of AGO1. Indeed, despite the presence of the transfected miRNAs, in cells depleted of AGO1 an 11-fold and a 16-fold increase of firefly luciferase expression was observed from the FLuc- CG10011 and F-Luc-Vha68-1 reporters, respectively. Notably, the firefly luciferase activity measured in AGO1-depleted cells in the presence of the transfected miRNAs was at least twofold higher than the activity measured in control cells in the absence of exogenously added miRNAs. Since endogenous miR-9b and miR-12 are expressed in S2 cells, these results suggest that depletion of AGO1 also suppresses silencing mediated by the endogenous miRNAs. Depletion of AGO2 does not suppress the effect of coexpressing the reporters with the cognate miRNAs. These results provide additional evidence supporting the conclusion that the siRNA and miRNA pathways are not interdependent (Rehwinkel, 2005).
miRNA-mediated silencing of firefly luciferase expression was not affected by depletion of UPF1 or by the additional NMD factors (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6). Thus, the individual NMD factors and NMD per se are not required for miRNA function. Unexpectedly, although the efficiency of NMD and RNAi was unaffected or only modestly affected in cells depleted of GW182 or the DCP1:DCP2 complex, miRNA-mediated silencing of firefly luciferase expression was effectively relieved in these cells. In the presence of cognate miRNAs, depletion of GW182 resulted in a sixfold increase of firefly luciferase expression. Therefore, despite the presence of transfected miRNAs, firefly luciferase activity in GW182-depleted cells was similar to that measured in controls cells in the absence of transfected miRNAs. Codepletion of DCP1 and DCP2 led to a fourfold increase of firefly luciferase expression. Finally, depletion of CG32016 resulted in a twofold increase of firefy luciferase activity, but only for the F-Luc-Vha68-1 reporter, suggesting that this effect may not be significant (Rehwinkel, 2005).
To investigate whether depletion of GW182 affects RISC activity directly, as opposed to interfering with miRNA processing, use was made of a tethering assay. This assay involves the expression of a lN-fusion of AGO1 that binds with high affinity to five BoxB sites (5-BoxB) in the 3í UTR of a firefly luciferase reporter mRNA. When AGO1 is tethered to this reporter transcript, luciferase expression is inhibited relative to the activity measured in cells expressing the lN-peptide alone. The inhibition was partially relieved in cells depleted of GW182 but not of AGO2. It is concluded that GW182 and the decapping DCP1: DCP2 complex play a critical role in the effector step of the miRNA pathway. These results are in agreement with the observation that Argonaute proteins localize to P-bodies and interact with DCP1 and DCP2 independently of RNA or of P-body integrity (Rehwinkel, 2005).
Thus, despite convergence in P-bodies, NMD, RNAi, and the miRNA pathway are not interdependent in Drosophila. This conclusion is based on the observation that the inhibition of one pathway by depleting key effectors may slightly interfere with, but does not significantly inhibit, the functioning of the other pathways. The lack of interdependence between RNAi and the miRNA pathway is further supported by the observation that knockouts of AGO1 or AGO2 in Drosophila have different phenotypes. Nevertheless, cross-talk between the RNAi and the miRNA pathways may still occur at the initiation step, since Dicer-1 plays a role in RISC assembly (Rehwinkel, 2005).
Biochemical and genetic approaches in several organisms have led to the identification of essential components of the miRNA pathway. These include AGO1 and the enzymes required for miRNA processing, such as Drosha and Dicer-1 and their respective cofactors, Pasha and Loqs. However, the mechanisms by which miRNAs inhibit protein expression without affecting mRNA levels are not completely understood. Recent evidence suggests that translation initiation is inhibited and that the targeted mRNAs are stored in P-bodies, where they are maintained in a silenced state either by associating with proteins that prevent translation or possibly by removal of the cap structure. This study identified the P-body components GW182 and the DCP1:DCP2 decapping complex as proteins required for the miRNA pathway. The precise molecular mechanism by which these proteins participate in this pathway remains to be established. These proteins may have an indirect role in the miRNA pathway by affecting P-body integrity. Alternatively, these proteins may play a direct role in this pathway by escorting miRNA targets to P-bodies or facilitating mRNP remodeling steps required for the silencing of these targets. Consistent with a direct role for the DCP1:DCP2 decapping complex, and thus for the cap structure, in miRNA function is the observation that mRNAs translated via a cap-independent mechanism are not subject to miRNA-mediated silencing. In conclusion, the results uncover an important role for the P-body components, GW182 and the DCP1:DCP2 complex, in miRNA-mediated gene silencing (Rehwinkel, 2005).
MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the GW182 protein, which interacts with the AGOs and is required for miRNA function. Depletion of GW182 (Gawky) leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1, NOT1, or the decapping DCP1:DCP2 complex. The N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. These findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets (Behm-Ansmant, 2006).
To accomplish their regulatory function miRNAs associate with the Argonaute proteins to form RNA-induced silencing complexes (RISCs), which elicit decay or translational repression of complementary mRNA targets. In plants, miRNAs are often fully complementary to their targets, and elicit mRNA decay. In contrast, animal miRNAs are only partially complementary to their targets, and silence gene expression by mechanisms that remain elusive. Recent studies have shown that miRNAs silence gene expression by inhibiting translation initiation at an early stage involving the cap structure; mRNAs translated via cap-independent mechanisms escape miRNA-mediated silencing. Other studies have suggested that translation inhibition occurs after initiation, based on the observation that miRNAs and some targets remain associated with polysomes. In addition, animal miRNAs can also induce significant degradation of mRNA targets despite imperfect mRNA-miRNA base-pairing (Behm-Ansmant, 2006 and references therein).
The existence of a link between the miRNA pathway and mRNA decay is supported by the observation that mammalian Argonaute proteins (AGO1-AGO4), miRNAs, and miRNA targets colocalize to cytoplasmic foci known as P-bodies. These mRNA processing bodies are discrete cytoplasmic domains where proteins required for bulk mRNA degradation in the 5'-to-3' direction accumulate (e.g., the decapping DCP1:DCP2 complex and the 5'-to-3' exonuclease XRN1). Additional components of P-bodies in yeast and/or human cells include the CCR4:NOT deadenylase complex, auxiliary decapping factors (e.g., the LSm1-7 complex and Pat1p/Mtr1p), the cap-binding protein eIF4E, and the RNA helicase Dhh1/Me31B involved in translational repression. In metazoa, P-bodies are also marked by the presence of GW182, a protein with glycine-tryptophan repeats (GW repeats) required for P-body integrity (Behm-Ansmant, 2006 and references therein).
The presence of Argonaute proteins, miRNAs, and miRNA targets in P-bodies has led to a model in which translationally silenced mRNAs are sequestered to these bodies, where they may undergo decay. At present, it is unclear whether the localization in P-bodies is the cause or consequence of the translational repression, though several lines of evidence point to a direct role for P-body components in miRNA-mediated gene silencing. First, DCP1, GW182, and its paralog TNRC6B associate with AGO1 and AGO2 in human cells. Second, depletion of GW182 in human cells impairs both miRNA function and mRNA decay triggered by complementary short interfering RNAs (siRNAs). Similarly, miRNA function is impaired in Drosophila Schneider cells (S2 cells) depleted of GW182 or the decapping DCP1:DCP2 complex (Rehwinkel, 2005). Finally, the Caenorhabditis elegans protein AIN-1, which is related to GW182, is required for gene regulation by at least a subset of miRNAs (Behm-Ansmant, 2006 and references therein).
In Drosophila, siRNA-guided endonucleolytic cleavage of mRNAs (RNA interference [RNAi]) is mediated by AGO2, while gene silencing by miRNAs is mediated by AGO1. That siRNAs and miRNAs enter separate pathways in Drosophila is further supported by the observation that depletion of GW182 inhibits miRNA-mediated, but not siRNA-mediated gene silencing (Rehwinkel, 2005). The precise role of GW182 in the miRNA pathway is unknown. GW182 could have an indirect role by affecting P-body integrity. Alternatively, it could be more directly involved, localizing miRNA targets to P-bodies or facilitating the mRNP remodeling steps required for the silencing and/or decay of these targets (Behm-Ansmant, 2006 and references therein).
This study investigates the role of Drosophila GW182 in the miRNA pathway. Depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of AGO1, indicating that GW182 is a genuine component of the miRNA pathway. In cells in which miRNA-mediated gene silencing is suppressed by depletion of AGO1, GW182 can still silence the expression of bound mRNAs, suggesting that GW182 acts downstream of AGO1. It is further shown that GW182 triggers silencing of bound transcripts by inhibiting protein expression and promoting mRNA decay via a deadenylation and decapping mechanism. Finally, evidence is provided that mRNA degradation by miRNAs requires GW182, the CCR4:NOT deadenylase, and the DCP1:DCP2 decapping complexes. Together with the observation that GW182 interacts with AGO1, these results indicate that binding of GW182 to miRNA targets induces silencing and can trigger mRNA degradation, providing an explanation for the observed changes in mRNA levels, at least for a subset of animal miRNA targets (Behm-Ansmant, 2006).
These results indicate that GW182 is a genuine component of RNA silencing pathways, associating with the Argonaute proteins and with components of the mRNA decay machinery and, providing a molecular link between RNA silencing and mRNA degradation. Depletion of GW182 or AGO1 from Drosophila cells leads to correlated changes in mRNA expression profiles, indicating that these proteins act in the same pathway. Transcripts commonly up-regulated by AGO1 and GW182 are enriched in predicted and validated miRNA targets. These results, together with the observation that GW182 associates with AGO1, identify GW182 as a component of the miRNA pathway (Behm-Ansmant, 2006).
GW182 belongs to a protein family with GW repeats, a central UBA domain, and a C-terminal RRM. Multiple sequence alignment of all proteins possessing these domains revealed that there are three paralogs (TNRC6A/GW182, TNRC6B, and TNRC6C) in vertebrates, a single ortholog in insects, and no orthologs in worms or fungi. At present, it is unclear whether the vertebrate paralogs have redundant functions, but both GW182 and TNRC6B have been shown to associate with human AGO1 and AGO2 (Behm-Ansmant, 2006).
In Drosophila, GW182 interacts with AGO1 in vivo and in vitro. No stable interaction with AGO2 was detected under the same conditions, suggesting that AGO2 may act independently of GW182. This is consistent with the observation that depletion of GW182 does not affect siRNA-guided mRNA cleavage or RNAi, which is mediated exclusively by AGO2 in Drosophila. Nevertheless, since AGO2 also regulates the expression levels of a subset of miRNA targets (Rehwinkel, 2006), the lack of interaction with GW182 raises the question of whether this regulation occurs by a similar or different mechanism from that mediated by AGO1. Further studies are needed to elucidate the mechanism by which Drosophila AGO2 regulates the expression of a subset of miRNA targets (Behm-Ansmant, 2006).
The N-terminal GW repeat region of GW182 encompasses two highly conserved motifs (I and II) and is expanded in vertebrates. This region is shorter in insects and bears similarity to the GW-like regions in the C. elegans protein AIN-1, involved in the miRNA pathway. However, AIN-1 does not contain UBA, Q-rich, or RRM domains. This lack of common domain architecture suggests that AIN-1 represents a functional analog. Nevertheless, the observation that C. elegans AIN-1 also localizes to P-bodies and interacts with AGO1 (i.e., worm ALG-1), and the finding that the N-terminal GW repeats of Drosophila GW182 interact with the PIWI domain of AGO1, suggest a conserved role for these repeats in mediating the interaction with Argonaute proteins. It would be of interest to determine the molecular basis of the specific interaction between the N-terminal GW repeats of GW182 and the PIWI domain of AGOs, and whether this interaction affects the catalytical activity of the domain (Behm-Ansmant, 2006).
Apart from the interaction with AGO1, the N-terminal repeats and the UBA and Q-rich domains contribute to the localization of GW182 in P-bodies, which is in turn required for P-body integrity. This suggests that GW182 may act as a molecular scaffold bringing together AGO1-containing RISCs and mRNA decay enzymes, possibly nucleating the assembly of P-bodies. Understanding the precise role of the various GW182 domains in the interaction with mRNA decay enzymes and AGO1 as well as in P-body integrity awaits further biochemical characterization (Behm-Ansmant, 2006).
Tethering GW182 to a reporter transcript silences its expression, bypassing the requirement for AGO1. Silencing by GW182 occurs by two distinct mechanisms: repression of protein expression, and mRNA degradation. It remains to be elucidated how GW182 represses translation. mRNA degradation by GW182 is inhibited in cells depleted of CAF1, NOT1, or the DCP1:DCP2 complex, indicating that GW182 promotes mRNA deadenylation and decapping. Thus, binding of GW182 appears to be a point of no return, which marks transcripts as targets for degradation (Behm-Ansmant, 2006).
More studies are needed to determine whether decapping triggered by GW182 requires prior deadenylation or whether these two events occur independently. The observation that mRNA levels are fully restored in cells depleted of DCP1:DCP2, suggests that deadenylation followed by 3'-to-5' exonucleolytic degradation is unlikely to represent a major pathway by which these mRNAs are degraded. Future studies should also reveal the identity of the nuclease(s) acting downstream of the decapping enzymes (Behm-Ansmant, 2006).
Previous studies indicate that miRNAs can reduce the levels of the targeted transcripts, and not just the expression of the translated protein. Consistently, transcripts up-regulated in cells depleted of AGO1 or GW182 are enriched in predicted and validated miRNA targets. In this paper further evidence is provided indicating that miRNAs silence gene expression by two mechanisms: one mechanism involving translational silencing, and one involving mRNA degradation. The contribution of these mechanisms to miRNA-mediated gene silencing appears to differ for each miRNA:target pair. Indeed, of the three reporters analyzed, Nerfin is silenced mainly at the translational level, silencing of the CG10011 reporter can be attributed to mRNA degradation, while Vha68-1 is regulated both at the translational and mRNA levels. Regardless of the extent of the contribution of these two mechanisms to silencing, both require AGO1 and GW182, because the levels of the mRNA reporter and luciferase activity are restored in cells depleted of any of these two proteins (Behm-Ansmant, 2006).
In contrast, although the levels of the mRNA reporter are restored in cells depleted of CAF1 or NOT1, translational repression is not fully relieved, indicating that deadenylation is required for mRNA decay, but not for translational silencing by miRNAs. In agreement with this, two reports published while this manuscript was in preparation have shown that miRNAs trigger accelerated deadenylation of their targets (Giraldez, 2006; Wu, 2006). This study extends these observations further by demonstrating: 1) deadenylation is mediated by the CCR4:NOT complex; 2) decapping is also required for miRNA target degradation; and 3) both deadenylation and decapping triggered by miRNAs requires GW182 (Behm-Ansmant, 2006).
Based on the results presented in this study and the observations that GW182 associates with AGO1 and is required for miRNA-mediated gene silencing, the following model is proposed: AGO1-containing RISCs binds to mRNA targets by means of base-pairing interactions with miRNAs; AGO1 may then recruit GW182, which marks the transcripts as targets for decay via a deadenylation and decapping mechanism (Behm-Ansmant, 2006).
A question that remains open is whether miRNA-mediated translational repression is the cause of mRNA degradation or whether these represent two independent mechanism by which miRNAs silence gene expression as proposed by Wu (2006). Indeed, changes in mRNA levels are not observed for all miRNA targets (Rehwinkel, 2006), suggesting that inhibition of translation is not always followed by mRNA decay. Conversely, depletion of CAF1 or NOT1 prevents mRNA decay but does not relieve translational silencing, suggesting that these two processes are independent (Behm-Ansmant, 2006).
An important finding is that miRNAs elicit degradation to different extents. One possible explanation is that the extent of degradation depends on the stability of the miRNA:mRNA duplexes. Also, the extent of degradation might depend on the particular set of proteins associated with a given target. For instance, some targets may assemble with a set of proteins that antagonize degradation. Finally, GW182 might interact only with a subset of AGO1-containing RISCs, as suggested for AIN-1. A major challenge will be to identify the specific features of miRNA targets and/or RISC complexes that lead to regulation of gene expression at the level of translation or at the level of mRNA stability (Behm-Ansmant, 2006).
Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage ('Slicer') activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown to possess the Slicer activity. Here it is shown that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. Upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a new model in which AGO2 is directly involved in RISC formation as 'Slicer' of the passenger strand of the siRNA duplex (Miyoshi, 2005).
Drosophila Polycomb group (PcG) proteins silence homeotic genes through binding to Polycomb group response elements (PREs). Fab-7 is a PRE-containing regulatory element from the homeotic gene Abdominal-B. When present in multiple copies in the genome, Fab-7 can induce long-distance gene contacts that enhance PcG-dependent silencing. Components of the RNA interference (RNAi) machinery are involved in PcG-mediated silencing at Fab-7 and in the production of small RNAs at transgenic Fab-7 copies. In general, these mutations do not affect the recruitment of PcG components, but they are specifically required for the maintenance of long-range contacts between Fab-7 copies. Dicer-2, PIWI, and Argonaute1, three RNAi components, frequently colocalize with PcG bodies, and their mutation significantly reduces the frequency of PcG-dependent chromosomal associations of endogenous homeotic genes. This suggests a novel role for the RNAi machinery in regulating the nuclear organization of PcG chromatin targets (Grimaud, 2006).
The RNAi machinery has been implicated in a wide variety of biological processes. One of these processes is the formation of heterochromatin. In S. pombe, this involves bidirectional transcription of RNA molecules from repetitive sequences and their cleavage into short interfering RNAs (siRNAs) of 21–23 nt by an RNase III enzyme called Dicer-1. siRNAs guide the RNA-induced initiation of transcriptional gene silencing (RITS) complex to homologous sequences in the nucleus (Noma, 2004; Verdel, 2004). Clr4, the homolog of the histone methyltransferase Su(Var) 3-9, is recruited along with the RITS complex to chromatin, where it methylates lysine 9 of histone H3 (H3K9). This epigenetic mark promotes the formation of heterochromatin by recruiting the heterochromatin protein Swi6, the homolog of HP1, via its chromodomain (Grewal, 2004). Consistent with these data, a redistribution of H3K9 methylation has been observed in Drosophila chromosomes in flies mutant for components of the RNAi machinery (Pal-Bhadra, 2004; Grimaud, 2006).
The RNAi machinery is also required for cosuppression, a phenomenon whereby the introduction of multiple transgenic copies of a gene phenocopies its loss of function instead of increasing its expression. In Drosophila, cosuppression can act at either the transcriptional or posttranscriptional level and involves PcG proteins as well as the RNAi machinery (Pal-Bhadra, 1997, Pal-Bhadra, 1999 and Pal-Bhadra, 2002; Grimaud, 2006 and references therein).
The Drosophila RNAi machinery includes two Dicer proteins encoded by the dicer-1 (dcr-1) and dicer-2 (dcr-2) genes. Dcr-2 is specifically required to process double-stranded RNAs into siRNAs and mediates the assembly of siRNAs into the RNA-induced silencing complex (RISC). Dcr-1 is involved in the metabolism of siRNAs as well as the processing of pre-microRNAs into microRNAs. RNA silencing also involves several highly conserved genes coding for PAZ-domain proteins. Argonaute1 (AGO1) and Argonaute2 (AGO2) are involved in microRNA biogenesis and RNA interference (RNAi). piwi is involved in cosuppression, silencing of retrotransposons, and heterochromatin formation. aubergine (aub) was first isolated based on its role in germline development but is also responsible for maintaining the silenced state of an X-linked male fertility gene locus (Stellate) via RNAi. The Aub protein is required for RNAi and RISC assembly in ovaries. In addition, homeless/spindle-E (hls) is involved in silencing of Stellate and in heterochromatin formation. This study tested whether RNAi components are involved in the PcG pathway. The results show that the RNAi machinery affects the PcG response via a novel regulatory function in nuclear organization (Grimaud, 2006).
The role of a variety of RNAi components in a specific transgenic line called Fab-X was tested. This line contains a construct carrying a 3.6 kb fragment from the Fab-7 region, cloned upstream of a mini-white reporter and inserted into the X chromosome. In the Fab-X line, the presence of the Fab-7 sequence is sufficient to induce PcG-dependent silencing, both of the mini-white eye-color reporter gene and of the endogenous scalloped (sd) gene, which is required for wing-blade morphogenesis and is located 18.4 kb downstream of Fab-7. These two repressed phenotypes are abolished in the presence of mutations in PcG genes and are not present in heterozygous females and hemizygous males, indicating that both mini-white and sd expression are subject to PSS (Grimaud, 2006).
The eye-color and wing phenotypes were used as a basis to analyze the effect of the RNAi machinery on PcG-dependent repression. Mutations in RNAi components were introduced into the Fab-X line and placed over a balancer chromosome containing a GFP marker. As the AGO1 mutant alleles involve P element insertions containing the mini-white reporter gene, they could not be tested using the eye phenotype. A null mutation in dcr-2 (dcr-2L811fsX) decreased silencing of the mini-white reporter gene relative to the Fab-X line when in the homozygous state. Likewise, two different mutant alleles of piwi (piwi1 and piwi2) decreased mini-white silencing, with the effect being more pronounced in piwi2 mutant flies. This effect was not restricted to the Fab-X line since it was also observed when piwi2 was recombined into another Fab-7-containing line. In contrast to the effects seen for dcr-2 and piwi alleles, Fab-X females homozygous mutant for hlsE1 or that carried the heteroallelic hlsE1/hlsE616 combination silenced mini-white like wt Fab-X females (Grimaud, 2006).
The sd phenotype was then analyzed in all mutant backgrounds at 28.5°C, a temperature inducing a strong wing phenotype in Fab-X. A preselection of non-GFP female larvae was carried out in order to selectively analyze homozygous or trans-heterozygous mutant adults. This analysis revealed that mutating any of the components of the RNAi machinery, except for hls and the heterozygous dcr-1 mutation, leads to a strong decrease in the sd phenotype. These data show that the RNAi machinery can affect PcG-mediated silencing. The fact that hls mutants had no effect suggests that this process might be mechanistically distinct from the role of RNAi components in heterochromatin formation (Grimaud, 2006).
While most RNAi components are not required for binding of PcG proteins to PREs, they are required to mediate long-range contacts between multiple copies of the Fab-7 element. Moreover, Dcr-2, PIWI, and AGO1 colocalize with PH in the cell nucleus, and their effect correlates with the presence of small RNAs homologous to Fab-7 sequences. Finally, in addition to their effects on transgenic Fab-7 copies, mutations in these genes also reduce the frequency of long-distance contacts between endogenous PcG target genes. Taken together, these results reveal a novel and unexpected role for the RNAi machinery in the regulation of euchromatic genes in the nuclear space (Grimaud, 2006).
The effects caused by mutations in different RNAi components suggest the existence of distinct molecular roles for these proteins in the regulation of PcG function. First, the hls gene does not seem to play a major role in silencing at the Fab-7 PRE or maintaining long-distance Fab-7 contacts. Since Hls has been shown to play a central role in heterochromatin formation (Pal-Bhadra, 2004), there may be different subtypes of nuclear RNAi machineries for heterochromatin formation and for regulating PcG function. No effect was found when a mutation in the dcr-1 gene was analyzed at the heterozygous state, but the elucidation of the function of dcr-1 in PcG-mediated silencing awaits further analysis in a homozygous mutant background. A second class of RNAi components that participate in PcG-mediated repression contains dcr-2, AGO1, and the aub gene. Loss of any of these RNAi gene products affects PcG-dependent silencing at Fab-7, although it does not impact the binding of PcG proteins to Fab-7. Only mutations in piwi affected the binding of PcG proteins to Fab-7, at least in polytene chromosomes, but even PIWI did not affect recruitment of PcG factors at endogenous genes. In S. pombe, both RNAi components as well as DNA binding proteins are involved in recruiting heterochromatin proteins to the mating-type region (Jia, 2004). At PREs, multiple DNA binding factors and chromatin-associated proteins are known to contribute to PcG protein recruitment, such as PHO, the GAGA factor, DSP1, and the CtBP proteins. Their combinatorial action might play a key role in the robust and specific chromatin tethering of PcG proteins, while, in contrast to the situation in S. pombe, the RNAi machinery might play a relatively minor role (Grimaud, 2006).
It is interesting to note that piwi mutations affected recruitment of E(Z) and PC to Fab-7 in polytene chromosomes but had no effects on PH, another PRC1 component. In the current model for recruitment of PcG proteins to PREs, histone H3 methylation by the E(Z) protein recruits PRC1 via the chromodomain of PC. The current results indicate that multiple mechanisms might be used to anchor different PRC1 components to PREs and that the loss of PC does not necessarily lead to the disintegration of the entire PRC1 complex at PREs (Grimaud, 2006).
To date, all transcriptional gene-silencing phenomena that depend on the RNAi machinery involve the production of small-RNA molecules. RNAi components were also shown to affect telomere clustering in S. pombe, although binding of Swi6 and H3K9me to individual telomeres is not affected. The production of siRNAs is believed to be essential for the nuclear clustering of telomeres since cells carrying a catalytically dead RNA-dependent RNA polymerase (which abolishes siRNA production) are defective in telomere clustering. Consistent with a role for small RNAs in mediating gene contacts, sense and antisense transcription of Fab-7 as well as small-RNA species were found in Fab-7 transgenic lines. Moreover, a mutant allele of dcr-2 producing a truncated polypeptide lacking the RNase III domain, which is required for dsRNA processing, is defective in long-range interactions of PcG target sequences as well as in accumulation of Fab-7 small RNAs. These data suggest that small-RNA species could be involved in these gene contacts. However, no small Fab-7 RNA species was detected in the wt situation, although RNAi mutants affect the contact of the endogenous Fab-7 locus with the Antp gene. This might indicate that other RNA species produced in the endogenous Hox genes could contribute to gene clustering. However, the possibility remains that RNA-independent functions of RNAi proteins contribute to the maintenance of gene contacts, in particular in the case of endogenous PcG target genes (Grimaud, 2006).
Interestingly, none of the RNAi mutants tested are defective in the establishment of long-distance chromosomal interactions. Fab-7 contacts are correctly established during embryogenesis but decay during later stages of development. This suggests that the RNAi machinery is not required to initiate contacts but rather to maintain them via the stabilization of gene clustering at specific nuclear bodies. This clustering could be important in cosuppression, where transgene silencing can occur at the transcriptional and posttranscriptional levels, both requiring the RNAi machinery (Pal-Bhadra, 2002). It is difficult, however, to understand how a relatively modest increase in transcript levels caused by an increase in the copy number of a gene could trigger a robust silencing of all copies. This is particularly puzzling considering that the transcript levels of endogenous single-copy genes can vary, e.g., during normal physiological gene regulatory processes, without triggering gene silencing. One explanation might be that cosuppressed genes are clustered in the cell nucleus. Indeed, clustering of multiple gene copies has been reported in plant cells (Grimaud, 2006).
It is proposed that the RNAi machinery, perhaps in conjunction with PcG proteins, might stabilize this gene-clustering phenomenon. Specifically, the colocalization of multiple gene copies with components of the RNAi machinery might increase the local concentration of RNA species. Once this concentration overcomes a critical threshold, double-stranded RNAs might assemble and be cleaved in situ by the enzymatic activity of the RNAi machinery. RNA molecules might contribute to hold together loci containing PcG proteins that produce noncoding transcripts encompassing PREs. This gene clustering might involve contacts with components of the RNAi machinery as well as PcG proteins assembled in the same nuclear compartments (Grimaud, 2006).
One important question is, what is the role of the RNAi machinery in the regulation of endogenous PcG target genes? The data indicate that RNAi components affect only a subset of these genes since the colocalization of PcG bodies with RNAi bodies is limited. Hox loci are characterized by extensive noncoding RNA transcription, and, recently, other PcG target genes have been shown to be associated to intergenic transcription. RNAi components might be targeted to this subset of PcG target genes, while other PcG target genes that are characterized by the absence of noncoding transcripts might be independent on RNAi factors (Grimaud, 2006).
The fact that no homeotic phenotypes are visible in RNAi mutant backgrounds suggests that the function of RNAi components can be rescued by other chromatin factors. Indeed, the decrease in the level of nuclear interaction between the homeotic complexes was incomplete in RNAi mutant backgrounds. The data suggest that, while the RNAi machinery does not act in the establishment of PcG-dependent gene silencing, RNAi factors might help stabilize silencing during development by clustering PcG target genes at RNAi nuclear bodies. Thus, in addition to its role in defending the genome against viruses, transposons, and gene duplications, the RNAi machinery might participate in fine tuning the expression of PcG target genes through the regulation of nuclear organization. Finally, it must be noted that the developmental expression profile of the components of the RNAi machinery is highly specific. The function of specific RNAi components is therefore likely to be highly variable in different cell types and as a function of time. It will be of great interest to explore this issue in the developmental context of the whole organism, in Drosophila as well as in other species (Grimaud, 2006).
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide distinct classes of RNA-induced silencing complexes (RISCs) to repress mRNA expression in biological processes ranging from development to antiviral defense. In Drosophila, separate but conceptually similar endonucleolytic pathways produce siRNAs and miRNAs. Despite their distinct biogenesis, double-stranded miRNAs and siRNAs participate in a common sorting step that partitions them into Ago1- or Ago2-containing effector complexes. These distinct complexes silence their target RNAs by different mechanisms. miRNA-loaded Ago2-RISC mediates RNAi, but only Ago1 is able to repress an mRNA with central mismatches in its miRNA-binding sites. Conversely, Ago1 cannot mediate RNAi, because it is an inefficient nuclease whose catalytic rate is limited by the dissociation of its reaction products. Thus, the two members of the Drosophila Ago subclade of Argonaute proteins are functionally specialized, but specific small RNA classes are not restricted to associate with Ago1 or Ago2 (Forstemann, 2007).
Animal miRNAs are produced by the sequential action of two distinct RNase III endonucleases. Drosha converts primary miRNAs, most of which are full-length RNA polymerase II transcripts, into pre-miRNAs, 70 nt RNAs that fold into a stem-loop or hairpin structure. Dicer then excises the mature miRNA, bound to its miRNA* strand, from the pre-miRNA. In Drosophila, distinct Dicer enzymes produce siRNA and miRNA. Dicer-1 (Dcr-1) acts with a double-stranded RNA (dsRNA)-binding protein partner, Loquacious (Loqs), to convert pre-miRNA to a miRNA/miRNA* duplex, whereas Dicer-2 (Dcr-2) produces siRNA duplexes by cleaving long dsRNA. Dcr-2 also acts with its dsRNA-binding partner protein, R2D2, to load an siRNA duplex into Ago2, a function that is separable from its role in siRNA production (Forstemann, 2007).
Both siRNAs and miRNAs act as components of RNA-induced silencing complexes (RISCs); the core protein component of all RISCs is a member of the Argonaute family of small RNA-guided RNA-binding proteins. The Drosophila genome encodes five Argonaute proteins, which form two subclades. The Ago subclade comprises Ago1 and Ago2, which have been reported to bind miRNAs and siRNAs, respectively. Piwi, Aub, and Ago3 form the Piwi subclade of Argonaute proteins and bind repeat-associated siRNAs (rasiRNAs; also called piRNAs), which direct silencing of selfish genetic elements such as transposons (Forstemann, 2007).
In lysates from Drosophila embryos, in cultured Drosophila S2 cells, and in adult flies, miRNA can be loaded into both Ago1 and Ago2. The data suggest that sorting miRNAs into Ago1- and Ago2-RISC generates silencing complexes with distinct functional capacities: Ago1-RISC represses expression of targets with which its guide miRNA matches only partially, whereas Ago2 silences fully matched target RNAs. These differences result, in part, from the surprisingly different catalytic efficiencies of Ago1 and Ago2: only Ago2 catalyzes robust, multiple-turnover target cleavage (Forstemann, 2007).
In mammals, only Ago2 retains the ability to catalyze guide RNA-directed endonucleolytic cleavage of RNA; the three other mammalian Argonaute proteins, Ago1, Ago3, and Ago4, lack a functional active site that is presumed to have been present in the evolutionarily ancestral Argonaute protein. Why then has Drosophila Ago1 retained any endonuclease activity at all, if it is so inefficient at target cleavage that it cannot measurably contribute to small RNA-directed RNAi? One potential explanation is that the primary role of the Ago1 endonuclease activity is to facilitate loading of Ago1-RISC. That is, the predominant substrate for the Ago1 endonuclease is not target RNA but, rather, miRNA* strands and perhaps the occasional siRNA passenger strand. Because miRNA* strand cleavage would occur only in cis and only once per loaded Ago1-RISC, efficient, multiple-turnover cleavage of target RNA would not be required (Forstemann, 2007).
These data reveal an important biochemical difference between Ago2 and Ago1, but they do not explain the molecular basis for the inefficiency of Ago1-directed cleavage of target RNA. Two explanations can be envisioned for the more than 40-fold lower kcat of Ago1 compared to Ago2. First, the active site of Ago1 might be less well suited to catalyzing phosphodiester bond cleavage. Alternatively, Ago1 might be slow to assume a catalytically active conformation. In this second model, the rate of a conformational rearrangement would limit the speed of target RNA cleavage by Ago1 (Forstemann, 2007).
The genome of Drosophila contains no mRNA with complete complementarity to miR-277. Why then do flies load miR-277 into Ago2-RISC? Perhaps there are as yet unknown iral RNAs targeted by Ago2-loaded miR-277. Such an innate immune response function has previously been proposed for miRNAs in mammals. Regardless of the biological purpose for loading miR-277 into Ago2, miR-277 provides an important in vivo test of the controversial proposal that the production of small RNA duplexes by Dicer is uncoupled from the loading of Argonaute proteins. That Dcr-2 and R2D2 act in vivo to load Ago2 with miR-277, a miRNA produced by Dcr-1 and Loqs, confirms previous in vitro data suggesting that both ends of a small RNA duplex are available for examination by the Ago2 loading machinery. The results suggest that the miR-277/miR-277* duplex dissociates from Dcr-1 after the dicing of pre-miR-277 and is then bound by the Dcr-2/R2D2 heterodimer, which loads it into Ago2 (Forstemann, 2007).
It was reasoned that Ago1 loading is also uncoupled from dicing. In all animals, some miRNAs are found on the 5' and others on the 3' arm of their pre-miRNA stem loops. In contrast, the geometry of Dcr-1 with respect to the two arms of the pre-miRNA stem is essentially the same for all miRNAs: Dcr-1 always makes staggered cuts that separate the pre-miRNA loop from the miRNA/miRNA* duplex. If Dcr-1 were to load miRNAs directly into Ago1, without first releasing the miRNA/miRNA* duplex, it would expected that all miRNAs would reside on the same arm of the pre-miRNA stem. The simplest explanation, and one most consistent with the partitioning of miR-277 into both Ago1- and Ago2-RISCs, is that miRNA/miRNA* duplexes are released from Dicer immediately after their production, then rebound by the Ago1- and Ago2-loading machineries. Such a model allows both the terminal thermodynamics of the miRNA/miRNA* duplex to determine the mature miRNA strand (rather than its position within the pre-miRNA) and the pattern of mismatches within the duplex to determine how the miRNA partitions between Ago1 and Ago2 (Forstemann, 2007).
In mammals, siRNAs produce off-target effects largely by acting like miRNAs. In flies, siRNAs loaded into Ago2 are believed to defend against viral infection. Virus-derived siRNAs might therefore trigger widespread, off-target silencing of host genes as flies mount an antiviral RNAi response. The partitioning of siRNAs into Ago2-RISC appears to circumvent this problem, because silencing by Drosophila Ago2 requires greater complementarity between the siRNA and its target than silencing by Ago1. It is tempting to speculate that a similar functional specialization among Argonaute proteins has gone undetected in mammals (Forstemann, 2007).
In Drosophila, small interfering RNAs (siRNAs), which direct RNA interference through the Argonaute protein Ago2, are produced by a biogenesis pathway distinct from microRNAs (miRNAs), which regulate endogenous mRNA expression as guides for Ago1. siRNAs and miRNAs are sorted into Ago1 and Ago2 by pathways independent from the processes that produce these two classes of small RNAs. Such small-RNA sorting reflects the structure of the double-stranded assembly intermediates the miRNA/miRNA* and siRNA duplexes from which Argonaute proteins are loaded. The Dcr-2/R2D2 heterodimer acts as a gatekeeper for the assembly of Ago2 complexes, promoting the incorporation of siRNAs and disfavoring miRNAs as loading substrates for Drosophila Ago2. A separate mechanism acts in parallel to favor miRNA/miRNA* duplexes and exclude siRNAs from assembly into Ago1 complexes. Thus, in flies small-RNA duplexes are actively sorted into Argonaute-containing complexes according to their intrinsic structures (Tomari, 2007).
In Drosophila the structure of a small-RNA duplex determines its partitioning between Ago1- and Ago2-RISC. These data suggest a simple model for this partitioning, with a central unpaired region serving as both an antideterminant for the Ago2-loading pathway and a preferred binding substrate for the Ago1 pathway. Supporting this view, miRNAs that contain central mismatches, such as let-7 and bantam, assemble primarily into Ago1-RISC. miR-277, whose central region is base paired, partitions between Ago1 and Ago2 in vivo (Tomari, 2007).
A model for small silencing RNA sorting in Drosophila. Dcr-2/R2D2 bind well to highly paired small-RNA duplexes but poorly to duplexes bearing central mismatches; such duplexes are therefore disfavored for loading into Ago2. Ago1 favors small RNAs with central mismatches, but no Ago1-loading proteins have yet been identified. Ago1- and Ago2-loading compete each other, increasing the selectivity of small-RNA sorting. The partitioning of a small-RNA duplex between the Ago1 and Ago2 pathways reflects its structure. A typical miRNA/miRNA* duplex, such as let-7 or bantam, loads mainly Ago1, whereas a standard siRNA duplex loads mostly Ago2. Some miRNA/miRNA* duplexes containing extensively paired central regions, such as miR-277/miR-277*, partition between Ago1 and Ago2. Sorting of small-RNA duplexes into Ago1 and Ago2 produces pre-RISC, in which the duplex is bound to the Argonaute protein. Subsequently, mature RISC, which contains only the siRNA guide or miRNA strand of the original duplex, is formed. The separation of the miRNA and miRNA* or the siRNA guide and passenger strands also reflects the structure of the small-RNA duplex. For Ago1, it is hypothesized that mismatches between the miRNA and the miRNA* or siRNA guide and passenger strands in the seed sequence are required for the efficient conversion of pre-RISC to mature RISC. For Ago2, such seed sequence mismatches are not needed because Ago2 can efficiently cleave the passenger or miRNA* strand, liberating the guide or miRNA from the duplex (Tomari, 2007).
Both the Ago2- and Ago1-loading pathways are selective. For Ago2, the affinity of the Dcr-2/R2D2 heterodimer for a small-RNA duplex provides the primary source of small-RNA selectivity. In the absence of either the Ago2-loading machinery or Ago2 itself, Ago1 is nonetheless preferentially loaded with a miRNA/miRNA* duplex; an siRNA duplex still loads poorly into Ago1. Thus, the Ago1-loading pathway is also inherently selective and not a default pathway that assembles small RNAs rejected by the Ago 2 pathway. It is not yet know if this selectivity is a direct property of Ago1, of an Ago1-loading machinery that remains to be identified, or both (Tomari, 2007).
Previous bioinformatic analyses noted that a central region of thermodynamic instability was a common feature of miRNA/miRNA* duplexes. The current data ascribe a function in flies to this common miRNA/miRNA* structural feature: directing the miRNA into Ago1 and away from Ago2. Mammalian miRNA/miRNA* duplexes also typically contain a central unpaired region, but it is not yet known if they are preferentially loaded into one of the four mammalian Ago-subclade Argonaute proteins (Tomari, 2007).
What is the biological significance in flies of sorting miRNAs into Ago1 and siRNAs into Ago2? One idea is that Ago1 and Ago2 are functionally distinct, with only Ago2 silencing targets that possess extensive complementarity to the small-RNA guide and only Ago1 directing repression of targets that contain multiple but only partially complementary miRNA-binding sites. Sorting small RNAs between Ago1 and Ago2 may also prevent miRNAs from saturating the Ago2 machinery, which might compromise Ago2-mediated antiviral defense. Conversely, excluding from Ago1 siRNAs produced in response to viral infection may minimize competition between such antiviral siRNAs and endogenous miRNAs, protecting flies from misregulation of gene expression during a viral infection. Restricting a robust RNAi (i.e., target cleavage) response to siRNAs loaded into Ago2 may also minimize undesirable, miRNA-like regulation of cellular genes by virally derived siRNAs. Thus, small-RNA sorting ensures that miRNAs are largely restricted to Ago1, whose relaxed requirement for complementarity between a miRNA and a regulated mRNA target allows each miRNA to control many different mRNAs, and that siRNAs are restricted to Ago2, whose silencing activity requires more extensive complementarity between the target and the siRNA guide. Nonetheless, a final question remains unanswered: why do some iconoclastic miRNA/miRNA* duplexes contain features that favor their loading into Ago2 (Tomari, 2007)?
Several lines of evidence suggest that some members of the AGO1 superfamily may bind RNA or may be a component of a protein-RNA complex. For example, C. elegans rde-1 is required for RNA interference (Tabara, 1999). Because soluble recombinant AGO1 proteins were not available, an alternative approach was taken to investigate whether AGO1 has an RNA binding activity. Extracts of adult flies were prepared and incubated with poly(A)- or poly(U)-conjugated Sepharose 4B beads. AGO1 protein co-precipitates with the RNA-conjugated beads, but not with unconjugated control ones, indicating that AGO1 binds RNA either directly or indirectly. Although a unit volume of beads was conjugated with a similar amount of, and a similar range of lengths of, either poly(U) or poly(A), AGO1 was recovered more efficiently with poly(U)-beads than with poly(A)-ones. To determine the region required for the association with the RNA-beads, extracts were made of transgenic lines that produced DeltaN or DeltaC. Both forms of AGO1 precipitate with poly(U)-Sepharose, indicating that a deletion of either region alone does not abolish the association of AGO1 with the beads (Kataoka, 2001).
microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Drosophila Dicer-1 interacts with Loquacious, a double-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments revealed that along with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the specific pre-miRNA processing activity. Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqsf00791 mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis. These results support a model in which Loquacious mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs (Forstemann, 2005; Saito, 2005).
Argonaute protein AGO1 is required for stable production of mature miRNAs and associates with Dicer-1. Thus, attempts were made to ascertain if Loqs is also present in an AGO1-associated complex, and if so, if the AGO1 complex is capable of processing pre-miRNA in vitro. Flag-Loqs and AGO1 tagged with TAP were simultaneously expressed in S2 cells, and the AGO1-TAP complex was purified through immunoglobulin G (IgG) bead-binding. The IgG bound was then subjected to Western blot analysis using anti-Dicer-1, anti-AGO1, or anti-Flag (for Loqs detection) antibodies. Not only Dicer-1 but also Loqs was detected in the AGO1 complex. These results indicate that all three proteins are present in the same complex, although they cannot exclude the possibility that there is one complex that contains AGO1 and Dicer-1 but not Loqs, and another complex that contains AGO1 and Loqs but not Dicer-1. The pre-miRNA processing activity of the AGO1 complex was then examined. Pre-miR-ban was utilized as a substrate. The AGO1 complex is able to efficiently process pre-miR-ban into the mature form. In contrast, another Argonaute protein AGO2-associated complex shows no such activity, which is consistent with the finding that the AGO2-associated complex does not contain Dicer-1. Considered together, these results showed that Dicer-1 and Loqs form a functional complex that mediates the genesis of mature miRNAs from pre-miRNAs, and suggested that the resultant mature miRNAs are loaded onto an AGO1-associated complex, which probably is miRNA-associated RISC, through specific interaction of AGO1 with Dicer-1 and Loqs (Saito, 2005).
AGO1 is a cytoplasmic protein that is present in many tissues. RNA in situ hybridization of embryos showed that AGO1 mRNA is provided maternally and that zygotic expression is initiated ubiquitously at around stage 9 and continues during subsequent embryonic stages. At stage 16, mRNA localization is prominent in the primordia of imaginal discs and the central nervous system. In imaginal discs of third-instar larva, the mRNA seems to be widely present at a low level (Kataoka, 2001).
To study the subcellular localization of AGO1 protein, antibodies were prepared against AGO1. A protein of the predicted size, 106 kDa, was detected in extracts of wild-type flies and its level is elevated in extracts of transgenic flies in which AGO1 cDNA expression is induced under a heat-shock promoter. This confirms that the 106 ka protein is AGO1. Although it was difficult to visualize endogenous molecules, the cytoplasmic distribution of overproduced AGO1 molecules was observed in cells of peripordial membranes of imaginal discs and in other cell types. This appears to be consistent with the localization of rat protein GERp95 of the AGO1 subfamily to the Golgi complex or the endoplasmic reticulum (Cikaluk, 1999), in contrast to the nuclear distribution of Piwi (Kataoka, 2001).
Attempts were made to verify the genetic interaction of AGO1 with the Wg signaling pathway. Shotgun over-expression sequesters Armadillo (Arm) and causes the wg-like phenotype. This phenotype is rescued when Arm is co-overproduced with Shotgun. Co-over-expression of AGO1 with Shotgun is also able to suppress the phenotype, almost as efficiently as does overexpression of arm with Shotgun. This result suggests that AGO1 overproduction overcomes an interruption of the Wg signaling pathway at the level of Arm, implying a role of AGO1 as a positive regulator (Kataoka, 2001).
The AGO1 protein was dissected to narrow down the region responsible for the rescue activity. Production of the DeltaN form, which lacks most of the N region, does not rescue but rather enhances the phenotype. This result supports the idea that the N region is necessary for the rescue and that DeltaN performs as a dominant-negative form. Unexpectedly, a deletion mutant without the AGO1 box (the DeltaC form), can rescue the phenotype, although less effectively than the normal protein; this finding shows that the rescue activity does not absolutely require the highly conserved box (Kataoka, 2001).
To address whether AGO1 plays a role in the Wg pathway in normal development or not, AGO1 mutations were examined to see if they cause any segment polarity defects in embryonic cuticle patterning like those seen in wg and arm mutants. Homozygotes of l(2)k08121 are embryonic lethal, but gave no detectable phenotypes in denticles. Germ-line clones were made to remove the maternal contribution of AGO1. Maternal and zygotic AGO1 mutant embryos showed no obvious abnormality in segment polarity pattern; instead, a decrease in the number of denticle-forming epidermal cells (a reduction which was severe in the most anterior row) was detected. This pattern defect is indeed due to loss of AGO1 function, since the expression of a AGO1 transgene in the embryo rescues this phenotype. Apparently normal engrailed and arm stripe expression confirmed the formation of segment polarity in the mutant. Furthermore, the cuticle pattern was not affected -- not in the wild-type background by overproducing the normal AGO1 protein, the DeltaN form, nor the DeltaC form. These results indicate that AGO1 is not involved in the segment polarity formation in Wg signal transduction. Whether AGO1 over-expression is able to form a double-axis in a Xenopus embryo, a standard assay for examining the role of a protein in the Wnt pathway was performed. To overproduce either the normal or the deletion forms, DNA constructs or mRNA were synthesized in vitro and injected into the ventral side of the embryos; however, no sign of body malformation was observed, except for slightly smaller eyes compared with the normal controls (Kataoka, 2001).
Phenotypes were examined in the maternal and zygotic mutant embryos; severe deformation was found in the central and peripheral nervous systems. At stages 14-16, bundles of longitudinal and commissural axons in the CNS are disrupted, and pan-neuronal labelling of the PNS exhibits a reduction in the number of neurons. One class of peripheral neurons decreased in number by more than 80%. These neuronal phenotypes were seen in every maternal and zygotic mutant. In contrast, the nervous systems looked normal in zygotic AGO1-/- mutants and in zygotic +/- mutants without maternal contribution; animals of the latter genotype survived and grew up to adult flies, indicating that either maternal or zygotic expression of AGO1 is sufficient for neural development (Kataoka, 2001).
To clarify the primary effects of a loss of AGO1 function on neural development, the maternal and zygotic mutant (simply called the 'mutant' below) were examined with markers for subsets of neurons or glia. Both neurons and glial cells were decreased in number, which excluded the possibility that neurons were transformed into glia or vice versa. It seems unlikely that the specification of neuroblasts and glioblasts is blocked, since no major differences are found in the numbers of these precursor cells between the wild-type and the mutant. This finding suggests defects subsequent to the specification of neural precursor cells in the mutant (Kataoka, 2001).
A sublineage of the longitudinal glioblast was followed to examine if loss of AGO1 arrests cell cycles. In the wild-type at stage 11/12, two cells were doubly positive for Repo and Prospero in the abdominal hemisegment. Each cell divided once, and then two additional cells of unknown origin appeared nearby, generating a cluster of six cells in total at stage 13/14. In every mutant hemisegment, the two cells did not always appear in a synchronous fashion; the number of the double-positive cells subsequently increased but ranged between two and four as opposed to six in the wild-type at stage 14. This phenotype could be interpreted as indicating that a loss of AGO1 function arrests cell divisions stochastically. However, the decreases in cell numbers may not be solely due to the cell-division arrest, but also due to cell death, as suggested by the fact that more Tunnel-positive cells were detected in the mutant at stage 15 than in the wild-type. It was difficult to examine whether the dying cells were relevant to the assumed defect in the cell cycle (Kataoka, 2001).
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 ARETNF-RNA decay by miR16 is sequence specific. Just as with Dicer, a function of Ago family members in ARETNF-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 ARETNF-RNA. miR289 has a similar effect on the stability of AREIL-6-RNA and AREIL-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).
RNA silencing pathways are conserved gene regulation mechanisms that elicit decay and/or translational repression of mRNAs complementary to short interfering RNAs and microRNAs (miRNAs). The fraction of the transcriptome regulated by these pathways is not known, but it is thought that each miRNA may have hundreds of targets. To identify transcripts regulated by silencing pathways at the genomic level, mRNA expression profiles were examined in Drosophila melanogaster cells depleted of four Argonaute paralogs (i.e., AGO1, AGO2, PIWI, or Aubergine) that play essential roles in RNA silencing. Cells depleted of the miRNA-processing enzyme Drosha were also examined. The results reveal that transcripts differentially expressed in Drosha-depleted cells have highly correlated expression in the AGO1 knockdown and are significantly enriched in predicted and validated miRNA targets. The levels of a subset of miRNA targets are also regulated by AGO2. Moreover, AGO1 and AGO2 silence the expression of a common set of mobile genetic elements. Together, these results indicate that the functional overlap between AGO1 and AGO2 in Drosophila is more important than previously thought (Rehwinkel, 2006).
Using microarray analysis of Drosophila cells depleted of Drosha and Argonaute proteins, this study shows that transcripts whose levels are likely to be directly regulated by silencing pathways (up-regulated transcripts) represent less than 20% of the Drosophila S2 cell transcriptome. Computational predictions of miRNA targets indicate that more than 30% of the transcriptome is targeted by miRNAs. There are several possible explanations for these seemingly contradictory observations. First, it was shown that not all authentic targets change levels in a detectable manner. This indicates that although microarrays are a valuable tool to identify miRNA targets, many targets may escape detection using this approach. Second, some miRNAs and targets are expressed in a tissue-specific manner, so it is likely that only a subset of miRNA/target pairs is expressed in S2 cells. Finally, current models of miRNA function suggest that miRNAs expressed in a given cell type target transcripts that are already expressed at low levels but avoid housekeeping genes or genes that are expressed in these cells at high levels. These targets may escape detection by microarray analysis. Nevertheless, among transcripts regulated by the Argonaute proteins several were found that are expressed at relatively high levels, suggesting that miRNAs not only silence the expression of undesirable, low-abundance transcripts but may also play a role in fine-tuning the expression of abundant mRNAs (Rehwinkel, 2006).
AGO1 and AGO2 are thought to have nonoverlapping functions in Drosophila. This study shows that these proteins regulate the expression levels of a common set of miRNA targets. The observation that Drosha also regulates these transcripts strongly supports the idea that regulation is mediated by miRNAs. In agreement with this, it was observed that AGO2 can associate with endogenous miRNAs, although less efficiently than does AGO1. In this way, AGO2 may also regulate the expression levels of a subset of miRNA targets. Nonetheless, when miRNA function were assayed by overexpressing miRNAs together with luciferase-based mRNA reporters, it was observed that miRNA-mediated translational repression requires AGO1 but not AGO2. It is therefore possible that in this assay the fraction of miRNAs incorporated into AGO2-containing RISC is too small to observe changes in the expression levels of the reporter. Dicer-1 is involved in miRNA biogenesis and is also required for the assembly of RISC complexes, so these observations suggest that Dicer-1 may load AGO2-containing RISCs with miRNAs, at least to some extent (Rehwinkel, 2006).
A partial functional overlap between AGO1 and AGO2 is also suggested by the observation that these proteins regulate the expression of a common set of transposable elements. It remains, however, to be established whether this regulation occurs via similar mechanisms and whether it happens at the transcriptional or posttranscriptional level (Rehwinkel, 2006).
Apart from the common regulated transcripts, transcripts regulated exclusively by AGO2 but not by Drosha or AGO1 have also been identified, suggesting that AGO2 may regulate the expression of these transcripts by an miRNA-independent mechanism that might involve endogenous siRNAs (Rehwinkel, 2006).
The levels of hid and reaper mRNAs (two experimentally validated miRNA targets increase in cells in which the miRNA pathway is impaired. Moreover, by analyzing changes in mRNA levels, additional miRNA targets have been identified and validated in Drosophila. The observation that miRNA targets change levels following inhibition of the miRNA pathway lends further support to the idea that miRNAs can reduce the levels of the targeted transcripts and not just the expression of the translated protein. Along these lines, it has recently been shown that miRNAs can trigger a strong reduction in target levels in C. elegans. Among the 136 core transcripts, 21% are between 1.5- and 2-fold up-regulated, 73% exhibited changes in the 2- to 5-fold range, and 6% were at least 5-fold up-regulated in AGO1-depleted cells. Thus, although changes in transcript levels can be used to validate miRNA targets, the effects can be modest and, as mentioned above, not all targets can be identified using this approach (Rehwinkel, 2006).
In human cells, the Argonaute proteins localize to P-bodies. These are specialized cytoplasmic foci in which the enzymes involved in mRNA degradation in the 5'-to-3' direction colocalize (e.g., the DCP1:DCP2 decapping complex and the 5'-to-3' exonuclease XRN1. In addition, mRNA decay intermediates, miRNA targets, and miRNAs have been observed in P-bodies, suggesting a functional link between P-bodies and RNA silencing pathways. Consistent with this, it has been shown that P-body components play a crucial role in silencing pathways. In particular, the RNA-binding protein GW182 (a P-body component in metazoa) and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing in Drosophila cells. Likewise, human GW182 plays a role in silencing mediated by miRNAs and siRNAs. Finally, the C. elegans protein AIN-1, which is related to GW182, is also required for regulation of a subset of miRNA targets. Together with the observation that miRNAs inhibit cap-dependent but not cap-independent translation initiation, these observations suggest a model in which miRNA targets are stored in P-bodies after translation inhibition, where they are maintained in a silenced state by associating with proteins that prevent translation or possibly by removal of the cap structure. Decapping or simply the storage of miRNA targets in P-bodies may make these mRNAs susceptible to degradation, providing a possible explanation for the reduction in mRNA levels. In agreement with this, depletion of a 5'-to-3' exonuclease in C. elegans partially restores the levels of miRNA targets (Rehwinkel, 2006).
Nevertheless, not all authentic miRNA targets change expression levels. Thus, it is possible that the extent of the degradation depends on the number of miRNA binding sites and/or the stability of the miRNA:mRNA duplexes. It is also possible that the rate of mRNA decay triggered by miRNAs for some targets does not exceed the rate of transcription and that thus the steady-state levels of these targets remain unchanged. It would therefore be of interest to determine whether miRNAs generally cause a reduction in the half-life of targeted transcripts (Rehwinkel, 2006).
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 ago2dop 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 ago2dop 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 ago2dop 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 ago251B 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 ago2dop mutant embryos are simply caused by disturbing the function of Ago2 in RNAi. Unlike ago2dop1 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 ago2dop 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 ago2dop 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 ago2dop alleles. The model is favored that disrupting both Ago1 and Ago2 function is sufficient to cause the observed defects at the MBT because ago2dop1 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 ago2dop alleles with other components of RNA silencing pathways should be examined to further understand the genetic and molecular basis for the altered activity of Ago2dop 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 ago2dop 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 ago2dop 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 (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).
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