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).
During the cleavage stage of animal embryogenesis, cell numbers increase dramatically without growth, and a shift from maternal to zygotic genetic control occurs called the midblastula transition. Although these processes are fundamental to animal development, the molecular mechanisms controlling them are poorly understood. This study demonstrates that Drosophila fragile X mental retardation protein (dFMRP) is required for cleavage furrow formation and functions within dynamic cytoplasmic ribonucleoprotein (RNP) bodies during the midblastula transition. dFMRP is observed to colocalize with the cytoplasmic RNP body components Maternal expression at 31B (ME31B) and Trailer Hitch (TRAL) in a punctate pattern throughout the cytoplasm of cleavage-stage embryos. Complementary biochemistry demonstrates that dFMRP does not associate with polyribosomes, consistent with their reported exclusion from many cytoplasmic RNP bodies. By using a conditional mutation in small bristles (sbr), which encodes an mRNA nuclear export factor, to disrupt the normal cytoplasmic accumulation of zygotic transcripts at the midblastula transition, the formation of giant dFMRP/TRAL-associated structures was observed, suggesting that dFMRP and TRAL dynamically regulate RNA metabolism at the midblastula transition. Furthermore, dFMRP associates with endogenous tral mRNA and is required for normal TRAL protein expression and localization, revealing it as a previously undescribed target of dFMRP control. It was also shown genetically that tral itself is required for cleavage furrow formation. Together, these data suggest that in cleavage-stage Drosophila embryos, dFMRP affects protein expression by controlling the availability and/or competency of specific transcripts to be translated (Monzo, 2006).
The data suggest that in cleavage-stage Drosophila embryos, dFMRP affects translational initiation of specific mRNA molecules within cytoplasmic RNP bodies by controlling their availability and/or modulating their competency to be translated. dFMRP does not measurably associate with polyribosomes under a wide range of conditions in cleavage-stage Drosophila extracts, similar to results obtained for Drosophila S2 cells, but in contrast to reports in other systems. Instead, dFMRP colocalize and cosediment was observed with TRAL and ME31B, known components of translationally quiescent cytoplasmic RNP bodies. Although dAGO2 cosediments with polyribosomes in cleavage-stage embryo extracts and could directly suppress translational elongation or termination, a similar role for dFMRP is unlikely. In fact, there is no indication that endogenous dFMRP directly interacts with dAGO2 in cleavage-stage Drosophila embryos, in contrast to their observed association in Drosophila S2 cell extracts. This discrepancy could result from a fundamental difference in RNA metabolism between S2 cells and cleavage-stage embryos undergoing the MBT (Monzo, 2006).
tral mRNA represents a previously undescribed in vivo target of dFMRP regulation. Although there is no direct evidence that dFMRP and TRAL form a stable complex in cleavage-stage embryos in vitro, dFMRP activity is clearly required for normal TRAL protein expression in vivo. Mislocalization of TRAL protein but not ME31B in both fmr1- and sbrts148 mutant embryos suggests that a specific functional relationship exists between dFMRP and TRAL. In fmr1- embryos, TRAL protein levels also are reduced 2-fold, indicating that TRAL does not simply get redistributed into abnormal structures, its rate of synthesis and/or degradation must also be affected. The co-IP of tral mRNA with dFMRP from WT embryo extracts demonstrates that dFMRP and tral mRNA form a stable RNP complex and suggests that dFMRP is involved in tral mRNA metabolism. Although it has not yet been determined whether dFMRP directly binds tral mRNA, this analysis of the tral mRNA sequence, by using the fast RNA motif/pattern searcher RNABOB identified a single G-quartet stem-loop structure within the tral 3' UTR, a motif that FMRP can bind with high affinity. Regardless of whether dFMRP binds tral mRNA directly, dFMRP could control the assembly of a translationally competent tral mRNP complex and/or its localized delivery for translation. The transient association of tral mRNA with cytoplasmic RNP bodies in a translationally quiescent state might be required for dFMRP to promote the assembly of a translationally competent tral mRNP. Alternatively, the restricted translation of tral mRNA, controlled by dFMRP-dependent localized release from cytoplasmic RNP bodies, might promote the normal assembly of a functional TRAL RNP complex. In either case, lower steady-state TRAL protein levels resulting from decreased synthesis and/or increased degradation in fmr1- embryos could be related to abnormal TRAL RNP complex assembly, observed as large structures by IF. Interestingly, the higher steady-state level of tral mRNA observed in fmr1- embryo extracts is reminiscent of the increased levels of another dFMRP target, pickpocket mRNA, observed in fmr1- embryo extracts and may reflect a common feature of dFMRP mRNA processing (Monzo, 2006).
In conclusion, it is believed that a system of cytoplasmic RNP bodies exists in cleavage-stage embryos that associates with maternal and zygotic mRNAs to mediate their degradation or processing for subsequent release for translation during the MBT. A large proportion of these cytoplasmic RNP bodies contain dFMRP. It is likely that the cleavage furrow formation defect observed in fmr1- mutants is the result of disrupting TRAL function. Indeed, tral- embryos have a cellularization phenotype that resembles that of fmr1- embryos. A similar requirement has also been found for the Caenorhabditis elegans homolog of tral, car-1, in cleavage furrow formation. However, as with Fragile X syndrome, it is possible that the altered expression of many targets is responsible for the full fmr1- cellularization phenotype (Monzo, 2006).
In order to better understand the role of Tral in regulating membrane trafficking, the identification of Tral-associated proteins was attempted by immunoprecipitating Tral from Drosophila embryo extract using Tral antibody. By colloidal blue staining, three major bands were found that specifically coimmunoprecipitated with Tral: p147, p70, and p50. Using mass spectrometry, p147 was identified as the eIF4E binding protein Cup, and p70 as poly(A) binding protein (PABP). p50 was found to be a mixture of the RNA binding protein Ypsilon Schactel (Yps) and the RNA helicase Me31B. To confirm the identities of the Tral-associated proteins, Tral was immunoprecipitated from ovarian extracts and immunoblotted for Cup, Yps, and Me31B. Me31B, Yps, and Cup all specifically coimmunoprecipitate with Tral, indicating that these proteins are bona fide components of the complex. Because Me31B, Yps, and Cup have been previously shown to be part of an RNA-protein complex, the ability of each protein to coimmunoprecipitate with Tral was tested in RNase-treated ovarian extracts. It was found that while the association of Tral with Me31B, Yps, and Cup is RNase resistant, the association of Yps with Cup is sensitive to RNase treatment, indicating the presence of RNA in the complex (Wilhelm, 2005).
Previous work has shown that Me31B, Cup, and Yps colocalize in vivo. In order to demonstrate that Tral is part of the Me31B-Cup-Yps complex in vivo, egg chambers were immunoprecipitated for Tral and Me31B as well as Tral and Cup. The particulate staining in nurse cells showed a high degree of overlap for both the Tral/Cup and Tral/Me31B double-labeled egg chambers. Furthermore, the temporal-spatial pattern of Tral localization within the oocyte is identical to that previously described for Cup, Me31B, and Yps. These results, together with the previously demonstrated colocalization of Me31B, Cup, and Yps, indicate that Tral, Cup, Me31B, and Yps all exist as a complex in vivo (Wilhelm, 2005).
Because Tral is present on discrete domains of the ER, it was next asked whether other components of the complex were also present on the ER. Colocalization studies of GFP-KDEL with either Me31B or Cup showed that Me31B and Cup are both present on discrete ER subdomains. This observation, together with the biochemical analysis of the Tral complex, demonstrates that Tral is part of an RNA-protein complex that is associated with the ER (Wilhelm, 2005).
Because mutations in tral have such striking effects on morphology of COPII foci, attempts were made to define the relationship between these foci and components of the Tral complex. Using GFP-Sar1 as a marker for COPII complex formation, it was found that while some COPII sites are not associated with the Tral complex, a number of sites either colocalize with or are bordered by the Tral complex. These observations are highly suggestive of a direct role in regulating exit site function, as recent work has implicated the regions around COPII sites in exit from the ER (Wilhelm, 2005).
In order to explain how an RNA-protein complex might regulate ER exit site function, it was hypothesized that Tral regulates the transcripts for COPII components on the surface of the ER. If this were true, then Tral complexes should contain the messages of COPII components. To test this hypothesis, Tral was immunoprecipitated from ovarian extracts, RNA was isolated from the pellet, and the presence of a variety of transcripts were assayed for by RT-PCR. This experiment showed that the transcripts for the COPII components sar1 and sec13 were enriched in Tral immunoprecipitates, while bcd, grk, and sec23 messages were not. Because Tral is biochemically associated with the sar1 message and mutations in tral cause profound disruption of the distribution of Sar1 protein, sar1 mRNA is a likely regulatory target of the Tral complex (Wilhelm, 2005).
One trivial way that this regulation could occur is by general derepression of a maternal pool of the sar1 message causing overexpression of Sar1 protein and the accumulation of large Sar1 foci. However, the levels of GFP-Sar1 are equivalent in tral1 heterozygotes and tral1 hemizygotes. This argues against bulk changes in translation or stability of the sar1 message and suggests that any regulation of the sar1 message is likely to be restricted to a subset of the transcript pool (Wilhelm, 2005).
In order to better understand where Tral might be acting during oogenesis, The distribution of Tral was examined in the egg chamber. Within the nurse cells, Tral was present in discrete particles throughout oogenesis. However, within the oocyte, Tral showed a dynamic distribution -- first being localized to the posterior of the oocyte during stages 1-6, accumulating briefly at the anterior during stages 7-8, followed by weak accumulation along the oocyte cortex with substantial enrichment at the posterior pole during stages 9-10 (Wilhelm, 2005).
Live imaging of GFP-Tral in nurse cells demonstrated that while there are a number of motile Tral particles, a substantial fraction of the particles are immobile and appear to be tethered to a large reticular structure reminiscent of the ER. This result, together with the finding that tral mutations interfere with the ER-Golgi trafficking of Grk and Yl, suggested that Tral might function on the surface of the ER. To test this, the ER was visualized by using a transgenic Drosophila line that expresses GFP fused to the KDEL ER retention signal (GFP-KDEL) only in the germline. While it was found that Tral protein colocalizes with the ER of the oocyte, the dense accumulation of ER membranes within the oocyte made close examination of the sites of colocalization difficult. Therefore analysis focused on Tral particles within the nurse cells, where ER membranes do not completely fill the cytoplasm. Immunostaining for Tral and GFP-KDEL revealed that the numerous Tral particles within the nurse cells are all associated with subdomains of the ER. These subdomains are typically sites of concentrated ER and are often present at the end of ER tubules. Given the Grk and Yl secretion defects observed in tral mutants, the localization of Tral to discrete domains of the ER suggests that Tral acts directly to regulate ER exit site function (Wilhelm, 2005).
Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. This study shows that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic 'P bodies,' including the RNA-degradative enzymes Decapping protein 1 (Dcp1p) and Xrn1p/Pacman and crucial components of miRNA (Argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways. Drosophila Me31B, a DEAD-box helicases, is shown to participate (1) with an FMRP-associated, P body protein (Scd6p/Trailer hitch) in FMRP-driven, Argonaute-dependent translational repression in developing eye imaginal discs; (2) in dendritic elaboration of larval sensory neurons; and (3) in bantam miRNA-mediated translational repression in wing imaginal discs. These results argue for a conserved mechanism of translational control critical to neuronal function and open up new experimental avenues for understanding the regulation of mRNA function within neurons (Barbee, 2006).
Several observations now indicate that P bodies, maternal granules, and a major subclass of neuronal RNP are similar in underlying composition and represent a conserved system for the regulation of cytoplasmic mRNAs. Known RNA transport and translational repressors shared between maternal and neuronal staufen granules now include, Stau, Btz, dFMR1, Pum, Nos, Yps, Me31B, Tral, Cup, eIF4E, Ago-2, and Imp. Strikingly, in human cells, the Me31B homolog RCK/p54, the Tral homolog RAP55, the four human argonaute proteins, eIF4E, and a eIF4E-binding protein analogous to Cup, 4E-T, are all found in P bodies. In yeast, homologs of Me31B (Dhh1p) and Tral (Scd6p) are also known to be in P bodies, and Dhh1p in particular plays a role in recruiting RNA-decapping proteins and exonucleases to these RNPs. Consistent with the above observations in yeast, the enzymes involved in mRNA hydrolysis including the 5′ to 3′ RNA exonuclease Xrn1p/Pcm and the RNA-decapping enzyme DCP1 are present on Drosophila neuronal staufen RNPs and maternal RNA granules. These data unequivocally demonstrate tight spatial proximity of components mediating various RNA regulatory processes in Drosophila neurons (Barbee, 2006).
The large collection of proteins and processes common to P bodies, staufen granules, and likely maternal RNA granules suggests that they share an underlying core biochemical composition and function, which would then be elaborated in different biological contexts. For example, one anticipates that proteins involved in mRNA transport will be more prevalent in maternal and neuronal RNPs, which need to be transported for their biological function (Barbee, 2006).
An interesting aspect of neuronal staufen RNPs described in this study is the diversity of translational repression systems that are present within them. (1) In Me31B, these RNPs contain a protein that works in general translation repression of a wide variety of mRNAs and can also affect miRNA-based repression. (2) In Ago-2, they contain a component specific to miRNA/RNAi-dependent repression. (3) Neuronal staufen granules also contain UPF1, which was originally thought to be solely involved in mRNA degradation. However, because UPF1 can act as a translation repressor and physically interacts with Stau, a reasonable hypothesis is that UPF1 might work in neuronal granules, in conjunction with Stau, to repress the translation of a subset of mRNAs. The presence of multiple mechanisms for translation repression colocalizing in granules in Drosophila neurons may allow for differential translation control of subclasses of mRNA in response to different stimuli (Barbee, 2006).
Evidence accumulating in the literature suggests that there is a potential diversity of RNA granule types in neurons. Observations in Drosophila neurons are most consistent with a model in which a major subclass of neuronal RNP, in which various translational repressor and mRNA turnover proteins colocalize, is related to other compositionally distinct, diverse RNPs. A major subclass of staufen-containing RNP is indicated by data showing substantial colocalization among various proteins analyzed. Diversity is indicated by the lack of 100% colocalization: for instance, 55% of staufen-positive particles in wild-type neurons do not contain detectable dFMR1 (Barbee, 2006).
Two types of observations suggest that the apparent subclasses of particles containing Stau or dFMR1, but not both, are related to the particles in which they colocalize: (1) these two types of RNPs are clearly compositionally related to particles that contain both proteins; (2) this is supported by the observation that colocalization can be substantially increased under some conditions. Overexpression of either dFMR1 or Stau:GFP increases colocalization between Stau and dFMR1 from 45% in wild-type neurons to more than 80%. Concurrent with increased frequency of colocalization, Stau:GFP or dFMR1 induction increases apparent particle size (or brightness) and reduces the total number of particles. The increase in colocalization and brightness, as well as reduction in particle number, is most easily explained by growth and/or fusion of related RNPs. Significantly, similar effects on mammalian neuronal granule size and number have been reported following overexpression of Stau or another granule protein, RNG105. Thus, the underlying regulatory processes appear conserved between Drosophila and mammalian neurons (Barbee, 2006).
While it remains unclear how FMRP, Stau, or RNG105 enhance granule growth or fusion, it is conceivable that individual mRNAs first form small RNPs whose compositions reflect specific requirements for translational repression of the mRNAs they contain. These small RNPs exist in dynamic equilibrium with larger RNPs in which multiple, diverse translational repression complexes are sequestered. Induction of factors that promote granule assembly could push the equilibrium toward mRNP sequestration within large granules. A requirement of this dynamic model, which postulates interactions among different types of RNP, is that the RNPs themselves can change in composition during transport to synaptic domains. This is supported by FRAP analyses showing rapid exchange of Stau:GFP between cytosol and granule (Barbee, 2006).
Additional types of RNPs have also been described in neurons. For example, polysomes apparently arrested in translation have been observed near dendritic spines, and these RNPs show no obvious similarity to large, ribosome-containing particles, termed neuronal RNA granules. In addition, a potentially distinct RNP containing Stau, kinesin, and translationally repressed RNAs, but not ribosomes, has been purified from the mammalian brain. More recently, it has been shown that RNPs containing stress-granule markers TIA-1 and TIA-R as well as pumilio2 are induced by arsenate-treatment of mammalian cultured neurons. Interestingly, as previously shown for somatic cells, these large stress granules appear tightly apposed to domains containing DCP1 and Lsm1, markers of P bodies. Determining the temporal and compositional relatedness of such varied RNPs, their pathways of assembly as well as their functions, is a broad area of future research not only in neuroscience but also in cell biology (Barbee, 2006).
These diverse types of biochemical compartments for individual mRNAs suggest that neural activity or other developmental signaling events would influence translation in two steps: first, by desequestering mRNPs held within large granules and, then, by derepressing quiescent mRNAs in individual mRNPs. Thus, RNPs described in this study could have a complex precursor-product relationship with other RNPs, including polysomes discovered by now-classical studies at dendritic spines (Barbee, 2006).
Despite the complexity revealed by the diversity of neuronal RNPs, the importance and significance of the observed colocalization of Me31B, Tral, argonaute, and dFMR1 in staufen-positive neuronal RNPs is most clearly demonstrated by functional analyses revealing biological pathways in which these proteins function together (Barbee, 2006).
Several independent lines of evidence are consistent with a function for Me31B in neuronal translational repression as part of a biochemical complex that includes dFMR1. (1) Subcellular localization studies indicate that Me31B and Tral localize to dFMR1-containing RNPs especially prominent at neurite branch points in cultured Drosophila neurons. (2) Me31B, Tral, and dFMR1 coimmunoprecipitate from Drosophila head extract, thus confirming the physical association of three proteins. (3) Loss-of-function alleles of either Me31B or Tral suppress the rough eye phenotype seen when dFMR1 is overexpressed in the sev-positive photoreceptors. (4) Overexpression of Me31B in sensory neurons leads to altered branching of terminal dendrites, a phenotype also seen with overexpression analyses of Nos, Pum, and dFMR1. (5) Reduction of Me31B expression in sensory neurons by RNAi results in abnormal dendrite morphogenesis and tiling defects, phenotypes similar to that observed following loss of nanos, pum, or dFmr1 function. Significantly, the effect of Me31B on dendritic growth is correlated with its ability to function in translational repression. These five independent lines of evidence provide considerable support for Me31B (and Tral) function in neuronal translation control processes. While the site of functional interaction between dFMR1, Me31B, and Tral (soma or neuronal processes) is not identified here, the importance of the physical interactions is clearly demonstrated (Barbee, 2006).
Several observations also argue that Me31B acts, at least in part, within neurons to promote translation repression and/or mRNA degradation in response to miRNAs. This possibility was first suggested by the physical and genetic interactions of Me31B with dFMR1, a protein that has been implicated in the miRNA-mediated repression. Using direct assays for miRNA-mediated function in vivo, this study shows that Me31B is required for efficient repression by the bantam miRNA in developing wing imaginal discs. This identifies Me31B as a protein required for efficient miRNA-based repression (Barbee, 2006).
Recently, miRNA-based regulation has been shown to be important for the control of spine growth in hippocampal neurons and to be a target of protein-degradative pathways involved in long-term memory formation in Drosophila. Thus, the data predict that Me31B will be important in modulating miRNA function pertinent to development of functional neuronal plasticity. More generally, because Me31B homologs in yeast and mammals have been shown to function in P body formation in somatic cells, the requirement for Me31B in miRNA function provides evidence to support a model in which formation of P bodies is required for efficient miRNA-based repression in varied cell types and biological contexts (Barbee, 2006).
The conclusion that staufen- and dFMR1-containing neuronal RNPs are similar in organization and function to P bodies has several implications for neuronal translational control. (1) The presence of diverse translational repression systems on these RNPs suggests that, like in P bodies, different classes of mRNAs will be repressed by different mechanisms. This may allow specific RNA classes to be released for new translation in response to different stimuli. Such diversity of control may allow synapses to remodel themselves differently, depending on the frequency and strength of stimulation (e.g., LTD or LTP). (2) FRAP experiments indicate that both P bodies and staufen granules are dynamic structures. This argues that, like P bodies, staufen granules are in a state of dynamic flux, perhaps in activity-regulated equilibrium with the surrounding translational pool. (3) The presence of mRNA-degradative enzymes on staufen granules suggests regulation of mRNA turnover may play an important role in local synaptic events. For example, if synaptic signaling were to induce turnover of specific mRNAs at a synapse, then stimulated synapses could acquire properties different from unstimulated ones that retain a 'naive' pool of stored synaptic mRNAs. Finally, these observations imply that the proteins known to function in translation repression within P bodies will play important roles in modulating translation in neurons. Thus, it is anticipated that proteins of mammalian or yeast P bodies such as Edc3p, Pat1p, the Lsm1-7p complex, GW182, and FAST will be present on and influence assembly and function of neuronal granules (Barbee, 2006).
While screening P element insertions generated by the Berkeley Drosophila Genome Project (BDGP) gene disruption project for uncharacterized genes required for embryonic axis formation, a female sterile P element insertion, KG08052, was identified that exhibited defects in the dorsal-ventral patterning of the eggshell. The KG08052 insertion site lies within the first intron of CG10686, suggesting that disruption of this gene, trailer hitch, is responsible for the dorsal-ventral patterning defect. Quantitation of the dorsal-ventral patterning defect in eggs laid by females homozygous for the KG08052 insertion (tral1) revealed that 80% of eggs have either no dorsal appendages or display a single fused appendage -- a phenotype indicative of ventralization of the eggshell. Females hemizygous for tral1 showed an enhancement of the dorsal appendage phenotype, with 100% of eggs showing either 0 or 1 dorsal appendages, indicating that tral1 is a strong hypomorphic allele (Wilhelm, 2005).
In order to further characterize the tral locus, two additional insertions were obtained in the tral locus: e03082 (tral2), a PiggyBAC transposon insertion in the 5′UTR of tral and d09277 (tral3), a P element insertion in the first intron of tral. Ninety-three percent of eggs laid by tral1/tral2 mothers display a ventralized eggshell phenotype consistent with tral2 being a strong hypomorphic mutation. In contrast, while tral3/tral1 mothers are sterile, only 12% of their eggs had ventralized eggshells, indicating that tral3 is a weak hypomorphic allele of tral. Because the tral locus is quite close to the citron kinase gene (dck), complementation tests were performed between tral and dck to determine whether the phenotype was due to the insertions affecting both genes. The lethal allele, dck1, fully complements tral1, indicating that eggshell ventralization in tral mutants is not due to disruption of dck. Thus, tral1, tral2, and tral3 constitute an allelic series with respect to the strength of the ventralization phenotype and do not disrupt the closest neighboring gene, dck (Wilhelm, 2005).
To confirm that tral1, tral2, and tral3 disrupt Tral expression, antibodies were raised to the first 130 amino acids of Tral and immunoblots of ovaries derived from various tral allelic combinations for Tral protein were probed. tral1 and tral2 in combination with either each other or with the deficiency Df(3L)ED4483 decreased tral expression to an undetectable level, consistent with the disruption of tral expression being responsible for the observed ventralization of the eggshell. Immunoblots of ovaries from tral3/tral1 or tral3/Df(3L)ED4483 females showed a decrease in Tral protein expression as compared to tral3 heterozygotes or a yw control but did not completely eliminate expression, consistent with tral3 being a weak hypomorphic allele of tral. Although rescue of the mutant phenotype would be necessary to rule out the possibility that a second site mutation is the cause of the observed phenotypes, the fact that tral1, tral2, and tral3 constitute an allelic series with respect to both strength of phenotype and expression of tral strongly argues that the observed phenotypes are due to decreases in tral expression (Wilhelm, 2005).
One of the key events in dorsal-ventral patterning is the localization of grk mRNA to the dorsal-anterior region. The localization of grk mRNA in turn causes the trafficking of Grk protein to be confined to dorsal-anterior endoplasmic reticulum (ER)-Golgi units. It is this localized secretion of Grk that instructs the dorsal follicle cells to assume a dorsal cell fate. These dorsal follicle cells then secrete the proper eggshell components to generate a dorsal appendage. The dorsal-ventral patterning defect of the tral mutants suggests that tral might regulate some aspect of the localization or secretion of Grk. In wild-type egg chambers, Grk protein is expressed homogeneously throughout the oocyte during stages 6–7 and then is found only in small puncta near the plasma membrane in the dorsal-anterior region of the oocyte during stages 8-10. These small Grk puncta are known to coincide with sites of exit from the ER (Herpers, 2004). In both tral1 homozygotes and tral1 hemizygotes, abnormally large Grk puncta were observed in 48% of homozygotes and 63% of hemizygotes during stages 6-8. This suggests that mutations in tral disrupt some aspect of Grk trafficking through the secretory pathway (Wilhelm, 2005).
Conceivably, tral mutants could affect Grk trafficking either by interfering with the proper localization/translational control of the grk message, by disrupting the microtubule cytoskeleton, or by blocking the normal trafficking of Grk protein through the secretory pathway. To test these possibilities, the localization of grk mRNA was assayed in tral mutant egg chambers by in situ hybridization. The localization of grk mRNA to the dorsal-anterior region of the oocyte during stages 8-10 is normal in tral mutant egg chambers. This result argues against defects in grk mRNA localization being responsible for the Grk trafficking defect observed in tral mutants (Wilhelm, 2005).
The fact that grk mRNA is correctly localized argues that the normal polarity of the microtubule cytoskeleton is intact in tral mutants; a polarized microtubule network is essential for grk mRNA localization. To confirm that the microtubule polarity is intact, whether the localization of Osk protein to the posterior is normal in tral mutant egg chambers was examined. Because the correct localization of Osk protein to the posterior requires both normal microtubule polarity and the proper localization of osk mRNA, this assay should reveal any functional defects in either the microtubule polarity or the transport of osk mRNA. Whereas large Grk puncta accumulate in the oocytes of tral mutants, Osk protein is present at the posterior of the oocyte. Consistent with this result, mutations in tral do not affect the normal anterior-posterior gradient of microtubule density in stage 9 egg chambers. Thus, mutations in tral do not affect either microtubule polarity or the transport of the grk and osk messages. This result may seem paradoxical, since grk signaling early in oogenesis is required to establish the microtubule polarity of the oocyte. However, the establishment of microtubule polarity is less sensitive to changes in the level of grk signaling than dorsal appendage formation. Because none of the tral alleles cause complete ventralization of the eggshell, it is not surprising that it has been possible to selectively affect dorsal appendage formation without altering the microtubule polarity of the oocyte (Wilhelm, 2005).
To rule out that large Grk puncta are due to a defect in Grk translational control, the distribution of large Grk puncta was examined during stages 8-10. If there were a defect in translational repression of grk mRNA, Grk protein should accumulate broadly throughout the oocyte. While some large Grk puncta are mislocalized to the side of the nucleus facing away from the oocyte cortex, both normal sized and large Grk puncta are restricted to the dorsal-anterior region of the oocyte. Because a defect in translational control would be expected to yield high levels of Grk protein throughout the oocyte, this result argues that the large Grk foci are not due to a loss of translational repression of the grk message. Because the polarity of the microtubule cytoskeleton and the localization/translation of grk mRNA appear normal in tral mutant egg chambers, the hypothesis was tested that the formation of large Grk puncta in tral mutants is due to a defect in the trafficking of Grk (Wilhelm, 2005).
In a variety of systems, ER exit sites are closely associated with Golgi units, presumably due to the role of ER trafficking in establishing and maintaining the Golgi. Because previous work established that small Grk puncta are coincident with ER exit sites, also known as the transitional ER, the effects of tral mutants on the distribution of Grk and its association with the Golgi were examined (Herpers, 2004). In wild-type egg chambers, the majority of Grk protein is present in small puncta that are closely associated with an individual Golgi complex that is positive for the Golgi marker Lava lamp. However, in tral mutants, the large Grk puncta have lost their intimate association with the Golgi. This suggested that the formation of large Grk foci might be due to a defect in ER exit (Wilhelm, 2005).
The COPII complex, which is required for ER-to-Golgi trafficking, is known to label discrete sites on the ER. Furthermore, a number of experiments have implicated these COPII sites and the regions surrounding them in exit from the ER (Bevis, 2002; Mironov, 2003). Using GFP-Sar1 as a marker for COPII complex formation, the distribution of ER exit sites was examined in wild-type and tral hemizygous egg chambers. GFP-Sar1 is distributed in small puncta throughout the nurse cells and oocyte in wild-type egg chambers. However, this organization is severely disrupted in tral1/Df(3L)ED4483 egg chambers. In these egg chambers, the GFP-Sar1 is found in abnormally large puncta similar to those observed for Grk protein. Thus, tral is required for normal ER exit site distribution and morphology. The accumulation of Grk in large foci that are not correctly associated with the Golgi, together with the role of tral in organizing ER exit sites, argues that the disruption of ER exit sites in tral mutants leads to a functional defect in ER-Golgi trafficking. It is this disruption of ER-Golgi trafficking that likely underlies the failure in dorsal-ventral patterning observed in tral mutants (Wilhelm, 2005).
If tral plays a general role in ER exit site function, one would expect to observe defects in the trafficking of other secreted proteins. In order to test this, the effects of tral mutants on the trafficking of the vitellogenin receptor Yl were examined. Previous work on Yl has shown that in wild-type egg chambers, Yl protein is distributed homogeneously throughout the ER of the oocyte with an occasional small puncta until stage 8, when all of the Yl protein is transported to the plasma membrane (Schonbaum, 2000). Homozygous tral1 oocytes showed no obvious disruption of Yl trafficking. However, 75% of hemizygous tral1 oocytes showed Yl foci within the oocyte during stages 6-9. Therefore, tral is required for the trafficking of proteins besides Grk and likely plays a general role in promoting exit from the ER (Wilhelm, 2005).
It was next asked whether the Grk and Yl foci are distinct in tral1 hemizygous oocytes. Immunostaining for both Grk and Yl revealed that the large foci for each protein are separate. This suggests that the two proteins use separate trafficking pathways that both require tral. The observation that the trafficking of Grk is more sensitive to decreases in tral function than the trafficking of Yl is consistent with this idea (Wilhelm, 2005).
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date revised: 10 April 2008
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