InteractiveFly: GeneBrief

gawky: Biological Overview | References

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 RNA-binding protein GW182, which interacts with the AGOs and is required for miRNA function. Depletion of Drosophila 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 and NOT1, components of a deadenylase complex, or the DCP1:DCP2 decapping protein 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 (see Drosophila ), 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. (1) DCP1, GW182, and its paralog TNRC6B associate with AGO1 and AGO2 in human cells; (2) 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). (3) 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).

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. This study characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which was named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, since human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5'-3' mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development (Schneider, 2006).

The GW182 protein is a critical component of cytoplasmic RNP bodies that have been shown to function in mRNA degradation, storage, and, recently, microRNA (miRNA)- and siRNA-based gene silencing (Eystathioy, 2003; Yang, 2004; Ding, 2005; Jakymiw, 2005; Liu, 2005a; Rehwinkel, 2005). GW182 was named for the presence of multiple glycine (G)-tryptophan (W) amino acid pairs in the N-terminal region of a 182-kD protein with a predicted C-terminal RNA recognition motif (RRM). It localizes into cytoplasmic GW bodies (GWBs; Eystathioy, 2002; Maris, 2005) that also contain factors involved in 5'-3' mRNA decay, including the exonuclease XRN1, decapping enzymes DCP1 and DCP2, and the LSm1-7 decapping activator, pointing to a role for GWBs in regulating mRNA stability (Ingelfinger, 2002; Eystathioy, 2003; Cougot, 2004). These bodies may participate in additional roles in mRNA regulation, since they also contain the m7G cap-binding protein eIF4E and the eIF4E transporter but no other components of translation machinery. Importantly, intact GWBs are required for the functioning of the RNAi pathway in human cells potentially via direct interaction between GW182 (and the related TNRC6B protein) and Argonaute1 (Ago1) and 2 (Ago2; Jakymiw, 2005; Liu, 2005a,b; Meister, 2005; Schneider, 2006 and references therein).

GWBs are thought to be analogous to Saccharomyces cerevisiae cytoplasmic processing bodies (PBs). They are involved in mRNA decapping and 5'-3' exonucleolytic decay, and their integrity depends on the presence of nontranslating mRNAs. Both PBs and GWBs dissociate when polysomes are stabilized with drugs such as cycloheximide. However, despite similar compositions, there are functional differences between GWBs and PBs. GWBs increase in size and number in proliferating cells, whereas PBs increase in size and number during growth limitation and increased cell density. GWBs and PBs also differ in their responses to stress, as PBs increase in size and number in response to environmental stress. This is likely caused by decreased translation initiation because this response can be reproduced using a temperature-sensitive allele of Prt1p, a subunit of the eIF3 complex. In stressed mammalian cells, stalled preinitiation complex mRNAs are first targeted to stress granules (SGs), which may function as triage sites where mRNAs are sorted for future degradation, storage, or reinitiation of translation. Observation of interactions between SGs and GWBs in live cells suggest that transcripts may be exported from SGs to GWBs for degradation (Schneider, 2006 and references therein).

This study characterized the role of gawky (gw), the Drosophila melanogaster orthologue of the human GW182 gene family. GW localizes to punctate structures in the cytoplasm of Drosophila embryos and cultured S2 cells. Drosophila GWBs are electron-dense nonmembrane-bound cytoplasmic foci. These structures are targeted by human GW182 and its paralogues TNRC6B and TNRC6C in Drosophila cells. Unlike what is seen in some mammalian cells, only some foci colocalize with the previously identified GWB components LSm4, the Drosophila Xrn1 orthologue Pacman (PCM), and AGO2 (Ingelfinger, 2002; Eystathioy, 2003; Kedersha, 2005; Liu, 2005a; Sen, 2005). There is a requirement for the zygotic expression of full-length Drosophila GW during early embryonic nuclear divisions. This suggests a critical role for GWB-based cytoplasmic RNA regulation in Drosophila beginning with early embryo development (Schneider, 2006).

The results confirm that GW is homologous to human GW182 and that Drosophila GWBs are analogous to human GWBs and yeast PBs. GW localizes to rapidly moving and electron-dense, nonmembrane-bound cytoplasmic structures. Colocalization of GW to homologues of known GWB or PB components LSm4, AGO2, and PCM (Xrn1) shows that Drosophila GWBs are of similar composition to PBs and GWBs. Another similarity between GWBs and PBs is that Drosophila GWBs also require intact RNA to maintain their integrity. Functionally, human and Drosophila GW homologues are all targeted to the same foci when coexpressed in S2 cells. However, not all Drosophila GWBs contain the mRNA decay enzymes LSm4 and PCM or AGO2 associated with GWBs or PBs. There is an apparent lack of interdependence in functions of the nonsense-mediated decay, RNAi, and miRNA pathways in Drosophila S2 cells, as the depletion of proteins involved in one pathway did not affect the function of another (Rehwinkel, 2005). Thus, the variable composition of Drosophila GWBs provides evidence that there may be distinct functions for these cytoplasmic structures. It may be possible to discern functionally distinct classes of GWBs by analyzing relative localizations of other mRNA-processing proteins as they become known (Schneider, 2006).

There have been several exhaustive screens to identify zygotically transcribed genes that affect Drosophila precellular embryonic development. Currently, a total of seven genes are thought to be expressed before the cellular blastoderm stage. However, these screens focused on the X chromosome and autosomes two and three, but not four. It is proposed that gw represents an additional zygotically expressed gene required for successful completion of the early embryo development in Drosophila. The reduction in GW protein observed at 60-70 min AED suggests that maternally supplied GW is depleted. This would be subsequently replenished by zygotic gw transcription, as shown by rising mRNA levels beginning at 70-80 min AED, a time of rapid nuclear division that culminates in the cellularization and subsequent gastrulation steps of embryo development. Notably, increased levels of Drosophila GW expression are also observed during pupal development, which is another time of rapid cell proliferation. The increase in GW expression during periods of rapid cell division is consistent with elevated GW182 levels (Yang, 2004) observed in proliferating human cells (Schneider, 2006).

The function of GWBs described in mammalian cells suggests a potential role for these structures in Drosophila development. In many organisms, siRNA and miRNA, which are produced by Dicer-mediated cleavage of longer double-stranded or hairpin RNA precursors, regulate several developmental functions. For both siRNA and miRNA activity, the RNA-induced silencing complex (RISC) binds and selectively suppresses or degrades complementary target mRNA . Several recent studies have identified a link between GWBs and the RNAi pathway. RISC components Ago1-4 localize to GWBs (Liu, 2005b; Sen, 2005), as do reporter mRNAs targeted for miRNA-mediated translational repression (Liu, 2005b). In addition, intact GWBs are required for siRNA silencing (Jakymiw, 2005; Liu, 2005b). The effects of miRNA expression on Drosophila development were characterized in a screen of 46 embryonically expressed miRNAs. Injection of antisense RNA to block these miRNAs into 30-min AED embryos revealed 25 miRNAs with visible phenotypes affecting a variety of developmental processes. Blocking miR-9 resulted in several severe defects, including nuclear division and migration, actin cytoskeleton formation, and cellularization. A role for components of the RNAi machinery in the timing of heterochromatin formation and accurate chromosome separation has been reported in Schizosaccharomyces pombe and the trypanosome Trypanosoma brucei. Drosophila Ago2 mutants show several defects in early embryogenesis, including defects in centromeres, nuclear division, nuclear migration, and germ cell migration. However, homozygous Ago2 mutants are, for the most part, fertile and viable. Therefore, cytoplasmic-based RISC-mediated miRNA may have an effect on the control of timing of protein reorganization associated with cytoskeletal and mitotic events during early development (Schneider, 2006).

The putative C. elegans GW protein orthologue Ain-l localizes to cytoplasmic foci with a composition similar to PBs and GWBs and forms complexes with ALG-1 (argonaute-like gene) Dicer-1 and miRNAs. However, C. elegans Ain-1 and RNAi components dicer-1, alg-1, and alg-2 function in the heterochronic pathway that regulates developmental timing in many postembryonic cell lineages (Grishok, 2001; Ding, 2005), while xrn1 is required in embryogenesis (Newbury, 2004) for ventral epithelial closure (Schneider, 2006).

The phenotypes associated with blocking Drosophila GW function suggest that functional GWBs are required for the completion of nuclear divisions during early embryonic development. These effects, although similar to Drosophila Ago2 mutants, are far more severe. Injection of anti-AGO2 antibody into early embryos caused a reduction in number and enlargement in the size of the embryonic nuclei detected by NLS-GFP. The more severe defects resulting from GW depletion may be caused by the nature of the Ago2 mutation, which does not completely block protein function, or may be the consequence of additional functions of Drosophila GWBs (which are not related to AGO2) and, by extension, RISC function (Schneider, 2006).

Drosophila GW is expressed throughout development and is required for the viability of cultured Drosophila cells. The data suggest that one function of GWBs is to coordinate the regulation of embryonic development in a posttranscriptional fashion. Subsets of eukaryotic mRNAs involved in the same cellular processes are often associated with specific RNA-binding proteins, depending on growth conditions. In one proposed model, RNP particles like GWBs coordinately regulate mRNAs encoding functionally related proteins, which is analogous to the operon-based coordination of prokaryotic gene expression (Keene, 2005). Thus, mRNAs with similar cis-elements would be recognized and trafficked by a common RNP to collectively regulate their translation or degradation. These data provide evidence that Drosophila GWBs mirror human GWB composition and function, providing an excellent model for genetic dissection of the potential role of GWBs in regulating mRNAs during development (Schneider, 2006).

A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing: Regulation of silencing by Ago1

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 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, 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).

P-body formation is a consequence, not the cause, of RNA-mediated gene silencing; Dicer-2 is required for P-body integrity

P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. This study shows that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity (Eulalio, 2007).

The first proteins found in P bodies are those functioning in the degradation of bulk mRNA. In eukaryotes, this process is initiated by removal of the poly(A) tail by deadenylases. There are several deadenylase complexes in eukaryotes: the PARN2-PARN3 complex is thought to initiate deadenylation, which is then continued by the CAF1-CCR4-NOT complex. Following deadenylation, mRNAs are exonucleolytically digested from their 3' end by the exosome, a multimeric complex with 3'-to-5' exonuclease activity. Alternatively, the cap structure is removed by the decapping enzyme DCP2 after deadenylation, rendering the mRNA susceptible to 5'-to-3' degradation by the major cytoplasmic exonuclease XRN1 (Eulalio, 2007).

Decapping requires the activity of several proteins generically termed decapping coactivators, though they may stimulate decapping by different mechanisms. In the yeast Saccharomyces cerevisiae, these include DCP1, which forms a complex with DCP2 and is required for decapping in vivo, the enhancer of decapping-3 (EDC3 or LSm16), the heptameric LSm1-7 complex, the DExH/D-box RNA helicase 1 (Dhh1, also known as RCK/p54 in mammals), and Pat1, a protein of unknown function that interacts with the LSm1-7 complex, Dhh1, and XRN1. In human cells, DCP1 and DCP2 are part of a multimeric protein complex that includes RCK/p54, EDC3, and Ge-1 (also known as RCD-8 or Hedls), a protein that is absent in S. cerevisiae (Eulalio, 2007).

The decapping enzymes, decapping coactivators, and XRN1 colocalize in P bodies. Additional P-body components in multicellular organisms include the protein RAP55 (also known as LSm14; Drosophila homolog - Trailer hitch), which has a putative role in translation regulation, and GW182, which plays a role in the microRNA (miRNA) pathway (Eulalio, 2007).

The P-body marker GW182 localizes to cytoplasmic foci in Drosophila S2 cells together with the decapping enzyme DCP2 and the decapping coactivator DCP1, suggesting that these foci represent P bodies. To characterize D. melanogaster P bodies further, antibodies were raised to the Drosophla orthologs of two proteins found in human-cell P bodies. These correspond to Ge-1 and Tral (LSm15), which is closely related to human RAP55 (or LSm14) (see Tanaka, 2006). Both antibodies stained the cytoplasm diffusely and also stained discrete cytoplasmic foci with a diameter ranging from 100 nm to 300 nm. The antibody signals are specific, as they are lost in cells in which the cognate proteins were depleted. The foci are present in about 95% of the cell population and are readily detectable because the concentration of Tral or Ge-1 in these foci is significantly higher than that in the surrounding cytoplasm (Eulalio, 2007).

The distribution of green fluorescent protein (GFP)-tagged versions of proteins found in P bodies was examined in yeast and/or human cells. These include DCP1, DCP2, GW182, Me31B (the D. melanogaster ortholog of S. cerevisiae Dhh1 and vertebrate RCK/p54), CG5208 (the D. melanogaster homolog of S. cerevisiae Pat1, referred to as HPat hereafter), and EDC3 (also known as LSm16). All of these proteins formed cytoplasmic foci that costained with the anti-Tral or anti-Ge-1 antibodies. Importantly, the expression of the GFP-tagged proteins did not significantly alter the number and size of endogenous P bodies. Together, these results indicate that the localization of decapping enzymes and decapping coactivators into P bodies is evolutionarily conserved. The localization of GW182 in Drosophila P bodies is in agreement with the proposal that GW-bodies and P bodies overlap, as reported for mammalian cells (Eulalio, 2007).

The localization of proteins implicated in translational regulation was examined in Drosophila oocytes whose corresponding transcripts are detectable in S2 cells, in particular, Smaug and the dsRNA binding protein Staufen. Smaug is a translational repressor that also promotes deadenylation of bound mRNAs by recruiting the CAF1-CCR4-NOT1 complex (Zaessinger, 2006). Both proteins localized to P bodies with endogenous Tral. Strikingly, P bodies increased in size in cells expressing Staufen at high levels but not in cells overexpressing GFP fusions of Smaug, suggesting that Staufen promotes P-body formation. Drosophila Staufen, Tral, DCP1, DCP2, XRN1, and Me31B have also been detected in RNP granules in neuronal cells and/or in oocytes, indicating that P bodies and other RNP granules observed in neuronal cells or during development share common components (Eulalio, 2007).

P-body formation requires nontranslating mRNPs and/or mRNPs undergoing decapping. A conserved feature of P bodies in human and yeast cells is that their formation depends on RNA and is enhanced in cells in which the concentration of nontranslating mRNAs or of mRNAs undergoing decapping increases. These observations indicate that mRNAs must exit the translation cycle to localize to P bodies. In agreement with this, it was observed that Drosophila P bodies decline when cells are treated with RNase A or with cycloheximide (which inhibits translation elongation and stabilizes mRNAs into polysomes). In contrast, P-body sizes increase in cells treated with puromycin, which causes premature polypeptide chain termination and polysome disassembly. Both puromycin and cycloheximide inhibit protein synthesis in S2 cells, as judged by the reduction of F-Luc and R-Luc activities after the treatment of cells transiently expressing these proteins with these drugs (Eulalio, 2007).

The size of Drosophila P bodies also depends on the fraction of mRNAs undergoing decapping, in agreement with the results reported for yeast and human cells. Indeed, blocking mRNA decay at an early stage, for instance, by preventing deadenylation in cells in which NOT1 (a component of the CAF1-CCR4-NOT deadenylase complex) is depleted, leads to the dispersion of P bodies, whereas P bodies are on average more prominent in cells from which DCP2 or XRN1 is depleted (in which decapping and subsequent 5'-to-3' mRNA decay are inhibited) (Eulalio, 2007).

Several lines of evidence show that P bodies do not serve as storage sites for the effectors of posttranscriptional process but are sites where mRNA degradation and silencing can take place. For instance, P-body formation is RNA dependent, and decay intermediates, siRNAs, and miRNAs and their targets are detected in P bodies. Moreover, the size and number of P bodies depends on the fraction of mRNAs undergoing decapping. However, the question of whether mRNA decay and silencing require the environment of microscopic, wild-type P bodies to occur or whether these processes can also occur outside of P bodies in soluble protein complexes remains open. This study shows that formation of large P bodies visible in the light microscope as observed in wild-type cells is not required for several processes associated with P-body components, including NMD, mRNA decay, and RNA-mediated gene silencing (Eulalio, 2007).

The question addressed in this study was whether the environment of macroscopic P bodies is required for posttranscriptional regulation. P bodies are defined as the large cytoplasmic foci visible by light microscopy in wild-type cells. These foci are on average 100 to 300 nm in diameter and are readily detected as bright cytoplasmic dots because the concentration of proteins in these foci is significantly higher than in the surrounding cytoplasm. Nevertheless, most P-body components are also detected diffusely throughout the cytoplasm. For a limited number of examples that have been analyzed, it has been shown that P-body components are not confined to these structures but dynamically exchange with the cytoplasmic pool. Quantitative information regarding the fractionation of P-body components between P bodies and the cytoplasm is still lacking, but given the volume of P bodies relative to that of the cytoplasm, it is likely that the diffuse cytoplasmic fraction is significantly larger. This suggests that posttranscriptional processes are likely to occur and may even be initiated in the diffuse cytoplasm or in soluble protein complexes that aggregate to form P bodies. Whether these processes take place in submicroscopic aggregates or soluble protein complexes in the absence of detectable microscopic P bodies remains to be solved. However, it is considered that aggregates or large multiprotein assemblies that are not detectable by light microscopy cannot be defined as bodies (Eulalio, 2007).

Translation factors or ribosomes are generally not present in P bodies (with the exception of cap binding protein eIF4E), indicating that mRNAs leave the translation cycle prior to entering P bodies. Consistently, releasing mRNAs from polysomes leads to increases in P-body sizes and numbers, whereas the stabilization of mRNAs into polysomes disrupts P bodies. These observations suggest that a critical step in P-body formation is the release of mRNPs from a translationally active state associated with polysomes to a translationally inactive state. This paper has shown that releasing mRNAs from polysomes by puromycin treatment is not sufficient to elicit P-body formation and that functional silencing pathways or proteins generically termed decapping coactivators are required for P-body assembly. These proteins include Me31B (Dhh1 in yeast), HPat (Pat1 in yeast), Ge-1, and the LSm1-7 complex (Eulalio, 2007).

What could be the role of these proteins in P-body formation? Me31B is an RNA helicase which could facilitate rearrangements in mRNP composition upon release from polysomes. The role of HPat is unclear, but the yeast ortholog interacts with Dhh1, XRN1, and the heptameric LSm1-7 complex. Coimmunoprecipitation assays indicate that the interaction between Dhh1 and Pat1 orthologs (i.e., Me31B and HPat) is conserved in Drosophila. Finally, the LSm1-7 complex associates with deadenylated mRNAs and stimulates decapping. Clearly, many details regarding the precise molecular function of these proteins remain to be discovered, but their requirement for P-body assembly indicates that mRNAs that are not actively translated do not enter into P bodies by default: the activity of a defined set of proteins is required. Alternatively, nontranslating mRNAs may enter silencing pathways, and this would also lead to changes in mRNP composition due to the recruitment of Argonaute proteins and binding partners, which include P-body components such as GW182, decapping enzymes, and RCK/p54 (Eulalio, 2007).

Once P-body components are bound to an RNP, P-body formation may then be triggered by protein-protein interactions. Indeed, proteins required for P-body assembly are known to interact to form multimeric protein complexes. Consistently, in addition to the interactions mentioned above, DCP1, DCP2, Ge-1, RCK/p54, and EDC3 form a multimeric protein complex in human cells. The absolute requirement of RNA for P-body formation could be explained if affinities between these proteins increased upon RNA binding. Additionally, proteins like GW182 and Ge-1 are multidomain proteins that could bind more than one RNP simultaneously, bringing into close proximity several components and thus nucleating the formation of P bodies (Eulalio, 2007).

RNAs targeted by silencing pathways nucleate P bodies. In this study, it is shown that both the RNAi and miRNA pathways contribute to the generation of a pool of nontranslating mRNPs and/or of mRNPs committed to decay which are required for P-body formation. Nevertheless, silencing can occur in the absence of microscopic P bodies. The results provide support to previous models proposing that silencing is initiated in the cytoplasm and that the localization of the silencing machinery into P bodies is a consequence, rather than the cause, of silencing (Eulalio, 2007).

An unexpected observation from these studies is that AGO2 and Dicer-2, which function in siRNA-mediated gene silencing in Drosophila, are required for P-body integrity. The role of these proteins in P-body assembly is unlikely to be structural, because P bodies are restored upon puromycin treatment in cells from which AGO2 or Dicer-2 is depleted. The most likely explanation for the requirement of these proteins is, therefore, that silencing by siRNAs also generates RNPs that elicit P-body formation. The requirement for AGO2 could be at least partially explained by the observation that the expression levels of a small subset of endogenous miRNA targets are affected in AGO2-depleted cells, suggesting that some miRNAs may be loaded into AGO2-containing RNA-induced silencing complexes. Furthermore, the AGO1 and AGO2 genes interact, although it is unclear how this interaction affects the activities of these proteins (Eulalio, 2007).

The requirement for Dicer-2 in P-body assembly, however, suggests that endogenous siRNA targets also contribute to P-body formation. Because the levels of dsRNA synthesis from endogenous loci that could provide precursors for the production of endogenous siRNAs are currently unknown, the fraction and origin of transcripts regulated by endogenous siRNAs cannot be estimated. Nonetheless, a possible source of endogenous dsRNAs is the bidirectional transcription of pseudogenes and transposable elements, in agreement with the role of the RNAi pathway as a defense mechanism against RNA viruses and mobile genetic elements (Eulalio, 2007).

The essential role of silencing pathways in P-body formation in Drosophila, and presumably in human cells, raises the question of how P bodies are assembled in S. cerevisiae, which lacks silencing pathways. One possibility is that other posttranscriptional processes generate nontranslating mRNPs required to nucleate P bodies. For instance, the NMD pathway contributes to P-body assembly in yeast cells, because depletion of Upf2 or Upf3 leads to increases in P-body size and number in a Upf1-dependent manner, whereas similar experiments with Drosophila cells do not affect P bodies (Eulalio, 2007).

With the exception of the proteins involved in silencing, the composition of P bodies and the effects of drugs such as cycloheximide and puromycin on P-body size and number are strikingly similar in yeast, Drosophila, and human cells, raising the question of what the role of these structures accounting for their conservation in eukaryotic cells could be. The results show that the environment of microscopic P bodies is not essential for mRNA decay or silencing but do not exclude that the formation of P bodies confers a kinetic advantage. Moreover, the results do not rule out a role for large P bodies in sequestering a specific set of nontranslating mRNPs and reinforcing their repression by shielding them from the translation machinery (Eulalio, 2007).

Finally, the conservation of P bodies may reflect a role for these structures in other cellular processes that is not yet fully appreciated. A role in some steps of retroviral or retrotransposon life cycles is suggested by the localizations of the antiretroviral proteins APOBEC3G and APOBEC3F in human cell P bodies and of the protein and RNA components of the retrovirus-like element Ty3 in yeast P bodies. A link between P bodies and the regulation of retrotransposition would be consistent with the role of RNAi pathways in silencing the expression of transposable elements. Because all known essential P-body components play roles in decapping and/or silencing and proteins playing an exclusively structural role in P-body assembly have not yet been identified, it is currently not possible to evaluate the role of P bodies for cell, tissue, or organism survival (Eulalio, 2007).

Search PubMed for articles about Drosophila GW182/Gawky

Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P. and Izaurralde, E. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20(14): 1885-98. Medline abstract: 16815998

Conti, E. and Izaurralde, E. (2005). Nonsense-mediated mRNA decay: Molecular insights and mechanistic variations across species. Curr. Opin. Cell. Biol. 17: 316-325. Medline abstract: 15901503

Cougot, N., Babajko, S. and Seraphin, B. (2004). Cytoplasmic foci are sites of mRNA decay in human cells. J. Cell Biol. 165: 31-40. Medline abstract: 15067023

Ding, L., Spencer, A., Morita, K. and Han, M. (2005). The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. Mol. Cell. 19: 437-447. Medline abstract: 16109369

Eulalio, A., Behm-Ansmant, I., Schweizer, D. and Izaurralde, E. (2007). P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol. Cell. Biol. 27(11): 3970-81. Medline abstract: 17403906

Eystathioy, T., et al. (2002). A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles. Mol. Biol. Cell. 13: 1338-1351. Medline abstract: 11950943

Eystathioy, T., et al. (2003). The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies. RNA. 9: 1171-1173. Medline abstract: 13130130

Giraldez A. J., Mishima Y., Rihel J., Grocock R. J., Van Dongen S., Inoue K., Enright A. J. and Schier A. F. (2006). Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312: 75-79. Medline abstract: 16484454

Grishok, A., et al. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell. 106: 23-34. Medline abstract: 11461699

Ingelfinger, D., et al. (2002). The human LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct cytoplasmic foci. RNA. 8: 1489-1501. Medline abstract: 12515382

Jakymiw, A., et al. (2005). Disruption of GW bodies impairs mammalian RNA interference. Nat Cell Biol. 7: 1267-1274. Medline abstract: 16284622

Kedersha, N., et al. (2005). Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J. Cell Biol. 169: 871-884. Medline abstract: 15967811

Keene, J. D. and Lager, P. J. (2005). Post-transcriptional operons and regulons co-ordinating gene expression. Chromosome Res. 13: 327-337. Medline abstract: 15868425

Liu, J., et al. (2005a). A role for the P-body component GW182 in microRNA function. Nat. Cell Biol. 7: 1261-1266. Medline abstract: 16284623.

Liu, J., et al. (2005b). MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat. Cell Biol. 7: 719-723. Medline abstract: 15937477

Maris, C., Dominguez, C. and Allain, F. H. (2005). The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 272: 2118-2131. Medline abstract: 15853797

Meister, G., et al. (2005). Identification of novel argonaute-associated proteins. Curr. Biol. 15(23): 2149-55. PubMed citation: 16289642

Newbury, S., and A. Woollard. (2004). The 5'-3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis. RNA 10: 59-65. Medline abstract: 14681585

Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. and Izaurralde, E. (2005). A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11(11): 1640-7. Medline abstract: 16177138

Rehwinkel, J., et al. (2006). Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster. Mol. Cell. Biol. 26: 2965-2975. Medline abstract: 16581772

Schneider, M. D., et al. (2006). Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development. J. Cell Biol. 174(3): 349-58. Medline abstract: 16880270

Sen, G. L. and Blau, H. M. (2005). Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat. Cell Biol. 7: 633-636. Medline abstract: 5908945

Tanaka, K. J., et al. (2006). RAP55, a cytoplasmic mRNP component, represses translation in Xenopus oocytes. J. Biol. Chem. 281(52): 40096-106. Medline abstract: 17074753

Wu, L., Fan, J. and Belasco, J. G. (2006). MicroRNAs direct rapid deadenylation of mRNA. Proc. Natl. Acad. Sci. 103: 4034-4039. Medline abstract: 16495412

Yang, Z., et al. (2004). GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation. J. Cell Sci. 117: 5567-5578. Medline abstract: 15494374

Zaessinger, S., Busseau, I. and Simonelig. M. (2006). Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4. Development 133: 4573-4583. PubMed citation: 17050620

date revised: 15 April 2008

Home page: The Interactive Fly © 2007 Thomas Brody, Ph.D.