InteractiveFly: GeneBrief

maternal expression at 31B: Biological Overview | References

A DEAD-box protein, Me31B, forms a cytoplasmic RNP complex with oocyte-localizing RNAs. During early oogenesis, loss of Me31B causes premature translation of oocyte-localizing RNAs within nurse cells, without affecting their transport to the oocyte. In early egg chambers that lack Me31B, at least two mRNAs in particles, OSK and Bicaudal-D mRNAs, are prematurely translated in nurse cells, though the transport of these RNAs to the oocyte is Me31B independent. These results suggest that Me31B mediates translational silencing of RNAs during their transport to the oocyte. These data provide evidence that RNA transport and translational control are linked through the assembly of RNP complex (Nakamura, 2001).

A visual screen was conducted with an ovarian GFP-cDNA library, in which fusion genes are expressed in germline cells during oogenesis. Transgenic flies were generated with this library and proteins were identified that distribute in a granular pattern during oogenesis. Screening ~3000 independent lines, one was isolated in which GFP signals were detected as cytoplasmic particles during oogenesis. The particles were dispersed in the cytoplasm of both nurse cells and oocytes but never detected within nuclei. The particles were frequently observed passing through ring canals, suggesting that the particles are assembled in nurse cell cytoplasm and transported to the oocyte (Nakamura, 2001).

The cDNA from this line was identified as me31B. In the cDNA fusion, almost the entire coding region of me31B, which lacks only the first four codons, was fused in frame with that of gfp. Me31B, a DEAD-box protein and therefore a putative ATP-dependent RNA helicase, was isolated as a gene expressed extensively during oogenesis. Me31B is a part of an evolutionally conserved DEAD-box protein group, which includes human RCK/p54 (71% identical), Xenopus Xp54 (73%), Caenorhabditis elegans C07H6.5 (76%), Schizosaccharomyces pombe Ste13 (68%) and Saccharomyces cerevisiae Dhh1 (68%). Furthermore, Me31B is phylogenetically close to two evolutionally conserved proteins, eIF4A and Dbp5/Rat8p but far from Vasa, which functions in germline development (Nakamura, 2001).

To examine distribution of the endogenous Me31B, antibodies were generated that specifically recognized Me31B. The distribution pattern of endogenous Me31B is identical to that of GFP-Me31B. No detectable signal in somatic follicle cells is observed at any stage of oogenesis. Me31B is first detected at a low level in germarium region 2B, where the signal is concentrated in the pro-oocytes. The signal remains concentrated in the oocyte until mid-oogenesis. In early egg chambers, a low level Me31B signal is detected in nurse cell cytoplasm. In both nurse cells and oocytes, the signal appears to be granular. Me31B signals in nurse cell cytoplasm become more evident from stage 5-6, when Me31B expression is drastically increased. In addition, Me31B is frequently enriched around nurse cell nuclei. Later, Me31B accumulates at the posterior pole of stage 10 oocytes. However, this posterior accumulation is transient, as revealed by uniform distribution of the signal in cleavage embryos. By cellular blastoderm stage, Me31B becomes undetectable in the entire embryonic region. No zygotic expression of Me31B was detected during embryogenesis (Nakamura, 2001).

Because Me31B is probably an RNA-binding protein that is transported to the oocyte, it was asked whether Me31B forms a complex with oocyte-localizing RNAs. Colocalization of OSK mRNA with Me31B was examined. OSK mRNA starts to accumulate in oocytes from germarium region 2B, with the concentration of OSK increasing over time. Posterior accumulation of OSK mRNA in the oocyte begins from stage 8 onwards. By fluorescent in situ hybridization, OSK mRNA exhibits particulate signals in the cytoplasm of both nurse cells and oocytes, and is frequently concentrated around nurse cell nuclei. This distribution pattern of OSK mRNA is essentially identical to that of Me31B, with colocalization present until OSK mRNA localizes to the posterior pole of stage 10 oocytes (Nakamura, 2001).

Colocalization of Me31B with other RNAs was also examined. Ovaries were doublestained for Me31B and BicD mRNA. In early egg chambers, BicD mRNA also produces particulate signals, and appears to localize in Me31B-containing particles. This colocalization becomes apparent from stage 5-6, when BicD mRNA expression is elevated. The oocyte-localizing RNAs examined [BCD, NOS, Oo18 RNA-binding (ORB), Polar granule component (PGC) and Germ cell-less (GCL)] all produce particulate signals in the cytoplasm of both nurse cells and oocytes, and colocalize with Me31B. In contrast, Vasa mRNA, which is not specifically transported to the oocyte, does not appear to be colocalized with Me31B. These results indicate that Me31B forms cytoplasmic particles that contain oocyte-localizing RNAs (Nakamura, 2001).

The complicated and redundant phenotypes observed in me31B^- egg chambers in mid-oogenesis are unlikely to be the primary effect of loss of me31B function. Earlier phenotypes of me31B^- egg chambers were examined using a FLP/FRT system to generate homozygous germline clones that are marked by the loss of Vas-GFP fusion protein. Based on Hoechst and phalloidin staining, me31B^- egg chambers are morphologically normal until stage 4-5. From stage 6 onwards, oocytes in me31B^- egg chambers fail to grow normally. At this stage, these egg chambers begin to degenerate. In early me31B^- egg chambers, Exu signal is concentrated to the oocytes. Distributions of OSK and BicD mRNAs in me31B^- egg chambers were examined. Both OSK and BicD mRNAs also accumulate in the oocytes of me31B^- egg chambers until the chambers degenerate. Particulate signals for these RNAs are detectable in nurse cell cytoplasm in these egg chambers. These results indicate that Exu, OSK and BicD mRNAs can be transported to the oocyte even in the absence of Me31B. It is concluded that in early egg chambers, Me31B is dispensable for the transport of the molecules that form a complex with Me31B (Nakamura, 2001).

Whether loss of Me31B affects translation of OSK and BicD mRNAs was examined. Ovaries were immunostained with an anti-Osk antiserum. Although OSK mRNA is expressed during almost all stages of oogenesis, its translation is repressed to keep Osk protein level very low during early oogenesis. In me31B^- egg chambers, Osk signal is significantly increased compared with that in the neighboring me31B⁺ egg chambers (Nakamura, 2001).

A similar increase of BicD signal in me31B^- egg chambers is more evident. In wild-type egg chambers, BicD protein, like BicD mRNA, is highly concentrated in the oocytes starting from germarium region 2B. In the egg chambers lacking me31B, increased BicD signal is detected in nurse cell cytoplasm. These results suggest that loss of Me31B in germline cells causes derepression of OSK and BicD mRNA translation during their transport to the oocyte (Nakamura, 2001).

Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis

Translational control is a critical process in the spatio-temporal restriction of protein production. In Drosophila oogenesis, translational repression of oskar1 (osk) RNA during its localization to the posterior pole of the oocyte is essential for embryonic patterning and germ cell formation. This repression is mediated by the osk 3' UTR binding protein Bruno (Bru), but the underlying mechanism has remained elusive. An ovarian protein, Cup, is required to repress precocious osk translation. Cup binds the 5'-cap binding translation initiation factor eIF4E through a sequence conserved among eIF4E binding proteins. A mutant Cup protein lacking this sequence fails to repress osk translation in vivo. Furthermore, Cup interacts with Bru in a yeast two-hybrid assay, and the Cup-eIF4E complex associates with Bru in an RNA-independent manner. These results suggest that translational repression of osk RNA is achieved through a 5'/3' interaction mediated by an eIF4E-Cup-Bru complex (Nakamura, 2004).

In a search for new components of the oskar RNP complex, this study identified the 147-kD protein of this complex as the product of the female sterile gene cup. Surprisingly, cup is required both for translational repression and localization of oskar mRNA. Cup was found to bind to eukaryotic initiation factor 4E (eIF4E) and is necessary to recruit the localization factor Barentsz to the complex. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. Because of its interactions with both the localization and translational control complexes, it is proposed that Cup is a likely regulatory target for the coupling machinery (Nakamura, 2004).

During localization, osk RNA forms cytoplasmic granules in both nurse cells and the oocyte. The granules contain several proteins, including the DEAD-box protein Maternal expression at 31B (Me31B), the Y-box protein Ypsilon schachtel (Yps), and Exuperantia (Exu). Genetic evidence has shown that Exu is involved in the proper localization of bcd and osk RNAs in oogenesis, although the molecular function of Exu remains unknown. Both Yps and Me31B are involved, directly or indirectly, in the translational silencing of osk RNA in oogenesis. Yps antagonizes Orb, a positive regulator of osk RNA localization and translation. In egg chambers lacking me31B, osk RNA is prematurely translated in early oogenesis (Nakamura, 2001). These data indicate that the granules are maternal ribonucleoprotein (RNP) complexes containing proteins required for both RNA localization and translational control. The complex is highly enriched in eIF4E and a germline protein, Cup. Cup is required to repress osk translation. Evidence is provided that Cup-mediated translational repression is achieved by preventing the assembly of the eIF4F complex at the 5' end of osk RNA, and that Cup acts together with Bru to repress osk translation (Nakamura, 2004).

To identify new proteins in the Me31B complex, ovarian extracts from wild-type females were immunoprecipitated on a preparative scale using an affinity-purified anti-Me31B antibody (α-Me31B). α-Me31B specifically coprecipitatesmany proteins from the extracts. Mass spectrometric analyses of these proteins revealed that both Exu and Yps, the known components in the Me31B complex (Nakamura, 2001), are present in the immunoprecipitates. The analyses also revealed that the 35 kDa protein was eIF4E and the 150 kDa protein is Cup, a germline-specific protein required for oogenesis. Cup is expressed from early oogenesis and present until the blastoderm stage of embryogenesis. Numerous cup alleles have been isolated as female sterile mutants, which show a wide range of phenotypes. However, the biochemical function of Cup has remained elusive (Nakamura, 2004).

To examine the association among Me31B, eIF4E and Cup in vivo, ovaries expressing a GFP-Me31B fusion protein were stained for eIF4E and Cup. The GFP-Me31B form cytoplasmic particles in the germline, and the distribution patterns of the fusion protein are indistinguishable from those of endogenous Me31B (Nakamura, 2001). α-eIF4E stains cytoplasmic particles that are positive for GFP-Me31B. This colocalization is observed throughout oogenesis. Cup colocalized with GFP-Me31B is also found throughout oogenesis. Thus, eIF4E, Cup, and Me31B all form a complex during oogenesis (Nakamura, 2004).

To better understand the interactions between Me31B, eIF4E, and Cup, ovarian extracts were immunoprecipitated by α-Me31B and α-eIF4E, and the precipitates were analyzed by Western blotting. α-Me31B coprecipitates eIF4E and Cup, and α-eIF4E coprecipitates Me31B and Cup, indicating that they all form a complex. However, in the presence of RNase during immunoprecipitation, α-Me31B fails to coprecipitate eIF4E or Cup. Thus, the Me31B-eIF4E and the Me31B-Cup interactions are indirect and probably mediated through RNA in the complex. In contrast, α-eIF4E coprecipitates Cup even in the presence of RNases, suggesting a direct interaction between eIF4E and Cup in vivo (Nakamura, 2004).

The interaction of Cup and eIF4E in vitro was studied using a GST pull-down assay. GST-eIF4E pulls down Cup synthesized in vitro. The association is unaffected by RNase. These results indicate that Cup associates with eIF4E in vitro and that the interaction is RNA independent (Nakamura, 2004).

The results show that Cup is an eIF4E binding protein that is involved in translational repression of osk RNA during oogenesis. The conserved YxxxxLφ motif in Cup is important for eIF4E binding and Cup and eIF4G are likely to bind the same surface of eIF4E. These results suggest that Cup competes with eIF4G for eIF4E binding, and hence inhibits translation initiation. Cup^Δ212 protein, which lacks the conserved eIF4E binding sequence, is unable to bind eIF4E in vivo, and fails to repress osk translation. These results strongly suggest that the Cup-eIF4E interaction is essential for the Cup-mediated repression of osk translation, although it is possible that other of Cup's functions are also affected in the cup^Δ212 mutant. Furthermore, Cup was found to interact with Bru in a yeast two-hybrid assay and that the Cup-eIF4E complex associates with Bru in an RNA-independent manner. Based on these results, it is speculated that the Bru-mediated repression of osk translation is operated, at least in part, through the interaction with Cup, which binds eIF4E and prevents the eIF4E-eIF4G interaction at the 5' end of osk RNA (Nakamura, 2004).

Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies

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).

Search PubMed for articles about Drosophila maternal expression at 31B

Barbee, S. A., et al. (2006). Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies. Neuron 52(6): 997-1009. Medline abstract: 17178403

Nakamura, A., Amikura, R., Hanyu, K. and Kobayashi, S. (2001). Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development 128(17): 3233-42. Medline abstract: 11546740

Nakamura, A., Sato, K. and Hanyu-Nakamura, K. (2004). Drosophila Cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis. Dev. Cell 6: 69-78. 14723848

date revised: 1 October 2007

Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.