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).

The Ataxin-2 protein is required for microRNA function and synapse-specific long-term olfactory habituation

Spinocerebellar ataxia type 2 (SCA-2) is an autosomal dominantly inherited neurodegenerative disease caused by trinucleotide (CAG) expansion in the ATXN2 gene resulting in the lengthening of polyglutamine stretch in the encoded protein ataxin-2. Ataxin-2 is a large conserved protein that carries an Sm-domain and a PAM-2 motif on the C-terminal side of the Sm-domain. The PAM-2 motif mediates interaction with the C-terminal helical domain of the poly(A) binding protein (PABP; see Drosophila pAbp) and is present in several PABP-interacting proteins. Levels of mutant ataxin-2 (with expanded polyglutamine stretch) are higher in brain tissue of patients with SCA-2 compared with that of wild-type ataxin-2 in normal individuals. Further, abnormal expression of Ataxin-2 has been shown to be deleterious in Drosophila (Satterfield, 2002). However, the biological function of ataxin-2 and the mechanism by which lengthening of the polyglutamine stretch in ataxin-2 leads to the disease are not clear (Tharun, 2008 and references therein).

Several studies implicate ataxin-2 in mRNA decay and regulation of translation suggesting that deregulation of these processes could be related to the disease. Ataxin-2 homologs from multiple organisms have been shown to interact with PABP consistent with the presence of PAM-2 motif in Ataxin-2. Depletion of Ataxin-2 homolog in C. elegans affects germline development and this seems to be due to the deregulation of translational repression by GLD-1 and MEX-3 of their mRNA targets. Reducing the expression of ATXN2 in mammalian cells using siRNAs impairs the formation of stress granules which are the sites where untranslated mRNAs are localized during stress. Finally, both in human cells and in Drosophila, Ataxin-2 is associated with the polysomes (Satterfield, 2006). These observations suggest that Ataxin-2 may have a role in translational repression in vivo. Interestingly, enhancement and suppression of Ataxin-2 expression in human cells leads to decrease and increase in the levels of PABP, respectively, without affecting the levels of PABP mRNA. Given that PABP is a key translation factor, it remains to be seen if the effects on translation caused by alterations in Ataxin-2 expression are related to the changes in the levels of PABP. Ataxin-2 overexpression also leads to decrease in the number of P-bodies in human cells. This seems to be related to the ability of Ataxin-2 to interact with Dhh1/RCK/p54, which is one of the decay factors important for P-body assembly in human cells. Importantly, reduction in P-bodies was caused by overexpression of Ataxin-2 irrespective of whether the polyglutamine stretch in Ataxin-2 was normal or long. These observations together suggest that deregulation of mRNA decay and/or translational repression resulting from abnormal expression of Ataxin-2 may be one of the reasons for the disease phenotype and the expanded polyglutamine stretch could contribute to increased expression of the mutant Ataxin-2 in the diseased individuals (Tharun, 2008 and references therein).

Studies in Drosophila suggest that Ataxin-2 is required for microRNA function and synapse-specific long-term olfactory habituation. Local control of mRNA translation has been proposed as a mechanism for regulating synapse-specific plasticity associated with long-term memory. Glomerulus-selective plasticity of Drosophila multiglomerular local interneurons observed during long-term olfactory habituation (LTH) requires the Ataxin-2 protein (Atx2) to function in uniglomerular projection neurons (PNs) postsynaptic to local interneurons (LNs). PN-selective knockdown of Atx2 selectively blocks LTH to odorants to which the PN responds and in addition selectively blocks LTH-associated structural and functional plasticity in odorant-responsive glomeruli. Atx2 has been shown previously to bind DEAD box helicases of the Me31B family, proteins associated with Argonaute (Ago) and microRNA (miRNA) function. Robust transdominant interactions of atx2 with me31B and ago1 indicate that Atx2 functions with miRNA-pathway components for LTH and associated synaptic plasticity. Further direct experiments show that Atx2 is required for miRNA-mediated repression of several translational reporters in vivo. Together, these observations (1) show that Atx2 and miRNA components regulate synapse-specific long-term plasticity in vivo; (2) identify Atx2 as a component of the miRNA pathway; and (3) provide insight into the biological function of Atx2 that is of potential relevance to spinocerebellar ataxia and neurodegenerative disease (McCann, 2011).

In the mammalian brain, single neurons form up to 100,000 different synapses whose weights may be regulated independently during learning. In principle, the synapse-specificity of short-term plasticity may be explained simply by the restriction of signaling events to active synapses. However, synapse-specific long-term plasticity, which depends on products of nuclear gene expression that would be available in a cell-wide manner, clearly depends on distinct synaptic tags that mark only active synapses (McCann, 2011).

Several lines of evidence suggest that activity-regulated local translation of synaptic mRNAs normally stored in a repressed state contributes to the synapse-specificity of long-term plasticity. Consistent with this idea, several translational control molecules, such as fragile X mental retardation 1 protein, Staufen, cytoplasmic polyadenylation element binding protein/Orb2, and the Gld2 polyA polymerase, are required in Drosophila for long-term but not short-term memory. In the context of identified synapses, local protein synthesis is required for cAMP response element-binding protein (CREB)-dependent, synapse-specific long-term plasticity in cultured Aplysia sensorimotor synapses: In this system, postsynaptic translation may trigger a retrograde signal, which in turn stimulates local translation at presynaptic terminals. However, in vivo, the requirement for and regulation of local protein synthesis at synapses remains poorly understood, in part because of the paucity of preparations in which behavioral learning arises from plasticity within a defined, experimentally convenient, neural circuit (McCann, 2011).

Recent work has shown that long-term olfactory habituation (LTH), a phenomenon in which sustained exposure to an odorant results in a decreased behavioral response, arises through plasticity of synapses between local interneurons (LNs) and projection neurons (PNs) in the Drosophila antennal lobe (Das, 2011; Sachse, 2007). Although LTH requires the transcription factor CREB2 to function (globally) in a multiglomerular class of LNs, LTH is odorant selective and associated with glomerulus-selective (and hence local) structural and physiological plasticity. In screening candidate RNA-binding proteins for potential roles in PNs during LTH, Ataxin-2 (Atx2), a molecule of considerable interest for its known involvement in the human neurodegenerative disease spinocerebellar ataxia-2 (SCA2), was identified. Expansion of a polyglutamine tract in human Atx2 from about 22 (normal) to >32 (pathogenic) glutamines causes degeneration of cerebellar Purkinje cells. While Atx2 has been implicated in many different biological functions, it is generally believed to function in RNA regulation. Evidence for this role comes from biochemical and cell biological studies of the protein or its evolutionarily conserved orthologs in Caenorhabditis elegans, Saccharomyces cerevisiae, and Drosophila melanogaster (McCann, 2011).

In C. elegans, Atx2 is required in postembryonic germline cells for appropriate translational control of GLD-1- and MEX-3-target mRNAs. Atx2 binds the RNA regulatory proteins, polyA-binding protein (PABP) and Me31B/RCK/ Dhh1p/CGH-1, through domains also required for its observed assembly with polyribosomes. At a cell biological level, Atx2 function has been shown to regulate the assembly of P-bodies and stress granules, distinct cytoplasmic messenger ribonucleoprotein particles that contain translationally repressed mRNAs, together with the translational repressor Me31B/RCK/Dhh1p (McCann, 2011 and references therein).

Significantly, both the proteins and cytoplasmic structures with which Atx2 associates have been linked to translation repression by microRNAs (miRNAs), a class of small, noncoding RNAs that bind complementary sequences in mRNA 3'UTRs and repress translation via the RNA-induced silencing complex (RISC). Furthermore, miRNAs and miRNA components have been linked either to long-term memory in Drosophila or to sensorimotor synapses (McCann, 2011).

This study shows that (1) Atx2 functions in olfactory projection neurons for LTH as well as associated glomerulus-selective physiological and structural plasticity; (2) Atx2 functions in LTH with the known miRNA-pathway proteins Argonaute 1 (Ago1) and Me31B; and (3) Atx2 is part of a general machinery required for efficient miRNA-mediated translational repression (McCann, 2011).

When tested in a Y-maze apparatus, flies exposed to either 15% CO2 or 5% ethyl butyrate (EB) for 30 min show a reduced aversive response that lasts less than 1 h (short-term habituation, STH). In contrast, flies exposed to 5% CO2 or 20% EB for 4 d show reduced aversion for days (LTH) and reduced odor-evoked responses in respective odor-responsive PNs, together with CREB-dependent growth of odor-responsive glomeruli (V and DM2/DM5, respectively). In this well-defined behavioral and synaptic context, it was asked whether PNs require Atx2 for LTH and associated synapse-specific structural plasticity. Expression of a UAS-Atx2RNAi construct in GH146-expressing neurons responsive to EB but not to CO2 (GH146Gal4/UASAtx2RNAi) completely blocked LTH to EB without altering LTH to CO2. Atx2 knockdown in GH146-expressing PNs blocked LTH to EB but had no effect on either STH to EB or the EB-avoidance response. Similarly, knockdown of Atx2 in the CO2-responsive VPN (VPNGal4;UASAtx2RNAi/+) selectively blocked LTH to CO2 without altering either STH to CO2 or the naive olfactory response to CO2. Thus, Atx2 is selectively required in glomerulus-specific PNs for odorant-selective LTH (McCann, 2011).

Two observations argue that Atx2 functions in adult neurons for LTH. First, both baseline behavioral responses to odorants and STH are normal in animals after Atx2 knockdown in PNs, indicating relatively normal development of the olfactory system. Second and more direct evidence is the selective block in LTH to EB seen following adult-specific knockdown of Atx2 in EB-responsive PNs using the TubGal80ts system (McCann, 2011).

Atx2 knockdown in odor-responsive PNs blocks not only olfactory LTH but also the LTH-associated increase in the volume of behaviorally relevant glomeruli. Thus, following 4 d of EB exposure, GH146Gal4/UAS-Atx2RNAi flies, which do not show LTH, also show no increase in the volume of either the DM5 glomerulus, previously shown to mediate the aversive response to this odorant, or the EB-responsive DM2 glomerulus. In contrast, the same GH146Gal4/UAS-Atx2RNAi flies show normal LTH to CO2 and robust increases in the volume of the VPN glomerulus in response to 4-d CO2 exposure as observed in control flies. Similarly, VPNGal4;UAS-Atx2RNAi/+ flies do not show LTH to CO2 or associated growth of the V glomerulus but display normal EB-induced LTH and EB-associated growth of DM5. Thus, Atx2 is required in specific PNs for the glomerulus-selective structural plasticity that accompanies odorant-selective LTH (McCann, 2011).

Normal LTH to EB is associated with an experience-dependent reduction in the EB-evoked physiological response of DM2 and DM5 PNs. This reduction can be measured in vivo by imaging odor-evoked calcium transients in PNs of flies expressing the genetically encoded calcium sensor, GCaMP3 (McCann, 2011).

To test whether this LTH-associated physiological plasticity requires Atx2 function in PNs, EB-evoked calcium fluxes were imaged and quantified in PN dendrites of 4-d EB-exposed and mock-exposed GH146Gal4, UAS-GCaMP3/UAS-Atx2RNAi flies (which do not show LTH to EB), and these results were compared with normally habituating GH146Gal4,UAS-GCaMP3/+ controls. In DM2 and DM5 of GH146Gal4, UAS-GCaMP3/UAS-Atx2RNAi flies, 4-d EB exposure caused significantly less change in EB (McCann, 2011).

Biochemical interactions of Atx2 orthologs in Drosophila and other organisms point to an interesting potential mechanism through which Atx2 regulates synapse-specific long-term plasticity required for LTH. In particular, Atx2 binding to Me31B and PABP orthologs, which in turn interact with other core miRNA-pathway proteins, GW182 and Argonaute, suggests that Atx2 may regulate miRNA-mediated translational repression directly. Could the function of Atx2 in LTH reflect a role in the miRNA pathway (McCann, 2011)?

To address this question, the possibility of strong dominant genetic interactions between atx2^X1 and mutations affecting core components of the miRNA pathway was examined. First, LTH and STH were examined in ago1^K08121/+; atx2^X1/+ double-heterozygote animals, and these behaviors were compared with those of single-heterozygote controls. The results were striking. Although STH to EB and CO2 was normal in double heterozygote ago1^K08121/+; atx2^X1/+ animals, LTH to both EB and CO2 was completely abolished. In contrast, control +/+; atx2^X1/+ and ago1^K08121/P[atx2+]; atx2^X1/+ animals showed normal LTH to both odorants (McCann, 2011).

The observation that the atx2 genomic rescue construct P[atx2+] restored normal LTH to ago1^K08121/+; atx2^X1/+ flies also shows that altered LTH in the double-heterozygote flies is caused specifically by a defect in atx2. In a similar experiment LTH and STH were examined in me31B^δ2/+; atx2^X1/+ double-heterozygote animals exposed to EB or CO2. Again, these double heterozygotes showed no LTH but normal STH. The defects in LTH were not observed in +/+; atx2^X1/+ or me31B^δ2 /P[atx2+]; atx2^X1/+ animals, further confirming the involvement of atx2. LTH-associated structural plasticity also was blocked in ago1^K08121/+; atx2^X1/+ and me31B^δ2/+; atx2^X1/+ double heterozygotes. Thus, although the V and DM5 glomeruli of +/+; atx2^X1/+ flies showed the expected growth following 4 d of CO2 or EB exposure, respectively, both the EB-evoked increase in DM5 volume and the CO2-induced increase in V was abolished in ago1^K08121/+; atx2^X1/+ and me31B^δ2/+; atx2^X1/+ double heterozygotes. In every instance, the defect in structural plasticity was restored by a wild-type genomic atx2+ transgene: Both ago1^K08121/ P[atx2+]; atx2^X1/+ flies and me31B^δ2/P[atx2+]; atx2^X1/+ flies showed normal odor-induced structural plasticity (McCann, 2011).

These data indicate that Atx2 functions in odorant-selective LTHas well as in glomerulus-selective structural plasticity through a pathway that depends on Ago1 and on Me31B, two proteins previously linked with miRNA-driven translational control. Consistent with this hypothesis, RNAi-based knockdown of Me31B in EB-responsive PNs mimics the effects of Atx2 knockdown, causing a specific defect in LTH to EB (McCann, 2011).

The observed genetic interactions of atx2 mutations with me31B and ago1 mutations point to a likely role for the Atx2 protein in regulating Ago1- and Me31B-dependent, miRNA-mediated translational repression in vivo. To examine this possibility, it was asked if Drosophila Atx2 is required for miRNA-mediated translational repression in wing imaginal discs, a tissue in which the function and activities of endogenous miRNAs can be analyzed conveniently (McCann, 2011).

To reduce levels of endogenous Atx2 in identified subpopulations of wing imaginal disc cells, either a patched Gal4-driven RNAi construct (UAS-Atx2RNAi) was used against atx2 or the Flippase recognition target-Flippase (FRT-FLP) recombinase system to generate genetic-mosaic animals carrying clusters of homozygous atx2^X1/atx2^X1 mutant cells in the wing imaginal discs of hs-flp;+/+; FRT82B, atx2^X1/FRT82B, arm-lacZ. Homozygous mutant atx2^X1/atx2^X1 cells were identified using either an Atx2 antibody or a surrogate, anti-lacZ staining, which here labels all cells except the atx2^X1/atx2^X1 mutant clones generated by mitotic recombination (McCann, 2011).

To examine Atx2 function in the miRNA pathway, such clones were generated in a genetic background that included any one of a number of transgenically encoded, miRNA-dependent translational reporters, and how loss of Atx2 affected GFP expression of these reporters was assessed. Reporters for head involution defective (hid), bantam, mir-12, costal-2, and sickle were used. The hid, sickle, and costal-2 reporters consist of the 3' UTR of hid, sickle, or costal-2, respectively, placed downstream of GFP-coding sequences under the control of a tubulin promoter (McCann, 2011).

The 3' UTR of hid is repressed by endogenous bantam miRNA and that of sickle by miR-2b. The bantam and miR-12 reporters consist of two copies of the bantam target recognition sequence or four copies of the miR-12 target recognition sequence, respectively, also downstream of GFP-coding sequences (McCann, 2011).

Atx2-deficient cells had a noticeable reduction in the intensity of Me31B and Ago1 staining, suggesting that in vivo Drosophila Atx2 is necessary for maintaining Me31B particles potentially involved in miRNA-mediated translational repression. In addition, and consistent with a defect in miRNA function in vivo, cells lacking Atx2 showed distinctly elevated expression of specific miRNA reporters (McCann, 2011).

Clones of atx2^X1/atx2^X1 mutant cells showed clear up-regulation of the hid reporter compared with flanking atx2^X1/+ or +/+ cells. The increased hid reporter levels in atx2^X1/atx2^X1 mutant cells were not observed if similar clones also expressed a wild-type atx2 genomic transgene). This observed genetic rescue confirms that the increase in hid-reporter expression in atx2^X1/atx2^X1 cells is caused by the absence of atx2 and not by any other unknown potential mutation on the FRT82B,atx2^X1 chromosome. Thus, as predicted by its biochemical and genetic interactions with various miRNA-pathway components, Atx2 is required for optimal repression of a miRNA reporter in vivo (McCann, 2011).

Further experiments confirmed that this requirement reflects a broad requirement for Atx2 function for the repression of many different miRNA-target genes. Clones of atx2^X1/atx2^X1 cells also show increased expression of the sickle, bantam BandB��), and miR12 reporters. Given that the latter two reporters are regulated by artificial UTRs engineered to be repressed solely through the miRNA pathway, these data strongly argue that Atx2 is broadly required for miRNA function. In contrast to the other four miRNA reporters, costal-2 reporter expression was not increased detectably in atx2^X1/atx2^X1 cells. This result was surprising, because the costal-2 reporter is similar to the other reporters in being repressed by miRNAs through a mechanism that requires Dicer-1 (Dcr-1), the endonuclease involved in miRNA biogenesis. Therefore, Atx2 is required only for repression of a subset of miRNA targets (McCann, 2011).

A model is considered in which Atx2 functions in only one of two miRNA-repression pathways recently distinguished in Drosophila. Although produced by Dcr-1, miRNAs may repress translation by one of two alternative pathways: either through an Ago1-RISC that requires GW182 or through an Ago2 RISC via a poorly understood GW182-independent mechanism. To test the possibility that Atx2 would be required exclusively for the Ago1/GW182-dependent pathway, previously shown to be required for bantam miRNA function, how the various reporters were affected by knockdown of GW182 was examined (McCann, 2011).

To knock down GW182 in identified groups of cells, the FLP-out technique was used to overexpress a transgenic RNAi construct against GW182 in wing imaginal discs expressing hid, miR12, or costal-2 reporters. Cells expressing a GW182RNAi construct (labeled by anti-lacZ staining) showed visibly increased expression of the Atx2-sensitive hid and miR12 reporters but no up-regulation of the Atx2-insenstive costal-2 reporter (McCann, 2011).

To understand why the costal-2 reporter could be insensitive to GW182 (or Atx2) knockdown, the sequence of its 3' UTR was examined and it was found to contain not only target sites for miR12 and miR283, but also two binding sites for miR277. This finding is significant because, unlike the majority of Dcr1-dependent miRNAs, miR277 is loaded preferentially onto Ago2-RISC complexes because of the extensive base-pairing between its miRNA and miRNA* strands. Thus, these observations suggest that Atx2, although necessary for GW182-dependent repression through Ago1-RISC, may not be necessary for Ago2-RISC.dependent repression (McCann, 2011).

This tentative model is supported by the observation that RNAi-induced, Ago-2-dependent eye phenotypes also are not sensitive to knockdown of Atx2. Knockdown of the caspase inhibitor Drosophila inhibitor of apoptosis (DIAP) by GMRGal4- driven expression of UAS-DIAPRNAi results in significantly smaller eyes because of increased apoptosis. The cell-death phenotype is suppressed if Ago2 levels are reduced by simultaneous expression of UAS-Ago2RNAi. However, similar coexpression of a functional UAS-Atx2RNAi (or UAS-GFPRNAi) does not alter phenotypes of GMRGal4;UASDIAPRNAi flies (McCann, 2011).

Taken together with prior evidence that Atx2 associates physically with Me31B and PABP, two proteins required for the Ago1-RISC pathway, these data indicate that Atx2 is part of a core pathway required for miRNA-mediated translational repression. However, Atx2 may be dispensable for repression by the Ago2-RISC pathway (McCann, 2011).

The circuit that underlies LTH has allowed experience-induced, synapse-specific plasticity to be examined in the context of behavioral memory. Previous pioneering work in cultured Aplysia sensorimotor synapses has led to a model in which CREB-dependent nuclear gene expression provides global (cell-wide) control of long-term facilitation. This facilitation can be restricted to specific synapses, in part through the synapse-specific local translation of stored mRNAs, which also is required for long-term plasticity. In the context of olfactory LTH, which is driven by the plasticity of inhibitory LN-PN synapses in the antennal lobe, previous work has shown that CREB function is required globally in a multiglomerular class of LNs for LTH to CO2 and EB. This study now shows that LTH additionally requires Atx2 in postsynaptic PNs for the glomerulus-restricted plasticity necessary for odorant-selective LTH. Knockdown of Atx2 in adult-stage PNs selectively blocks LTH without affecting either basal odorant response or STH. This distinctive phenotype also is shown following Me31B knockdown in PNs or in animals doubly heterozygote for atx2 and ago1 or atx2 and me31B. When taken together with independent observations that Atx2 is required for efficient miRNA function, these very strong genetic interactions point to a role for Atx2 in miRNA-mediated translational control in the regulation of long-term memory (McCann, 2011).

It is hypothesized that, under appropriate circumstances, NMDA receptor activation in PN dendrites may trigger local protein synthesis, perhaps through RISC degradation, giving rise to a retrograde signal from PNs to LNs that in turn stimulates or synergizes with the cell biological changes required for glomerulus- limited, long-term plasticity. The data do not demonstrate that Atx2 and Me31B function in local translation of synaptic mRNAs, but they do show a specific requirement for Atx2 and Me31B for miRNA function and synapse-specific LTH (but not STH) and thus provide a strong argument for local translation of synaptic mRNAs being the underlying mechanism by which these proteins regulate synapse-specific long-term plasticity in vivo (McCann, 2011).

The proposed need for postsynaptic translation and postulate of retrograde signaling are consistent with recent observations and models explaining long-term synaptic facilitation in Aplysia (Wang, 2009; Cai, 2008). In Drosophila, these models may be tested and elaborated through further genetic and in vivo studies and may lead to an understanding of the local and global mechanisms and their interactions that regulate long-term synaptic plasticity (McCann, 2011).

An important finding is that Atx2 is required for translational repression of four different miRNA reporters. Taken together with prior evidence, in particular that Atx2 binds two known components of the miRNA pathway, this finding indicates a wide and general requirement for Atx2 in miRNA-mediated translational repression (McCann, 2011).

However, Atx2 is not required for silencing of the costal-2 reporter, an observation that may be may be explained by costal-2 reporter's repression by a possibly Atx2-independent RISC complex. Previous work has shown that miRNAs partition between two different silencing complexes, Ago1-RISC and Ago2-RISC; in contrast, siRNAs associate almost exclusively with Ago2-RISC. Ago1-RISC and Ago2-RISC silence mRNAs by different mechanisms: Ago1-RISC is characterized by its dependence on GW182. The specific pathway that produces an miRNA or siRNA does not require that small RNA to associate with a particular Ago protein. Thus, although bantam and miR-277 miRNAs are produced by Dcr1, bantam associates exclusively with Ago1-RISC, whereas miR-277 is loaded into the Ago2 pathway. This loading of miR-227 occurs because, in contrast to the bantam microRNA, which has several bulges and mismatches, the duplex precursor to miR-277 strongly resembles an siRNA precursor with a high degree of perfect matching. By demonstrating that loss of Atx2 causes up-regulation of GW182- or Ago1-dependent miRNA reporters, the results identify Atx2 as a frequently used component of the Ago1-GW182 RISC pathway. Loss of Atx2 does not affect repression of the GW182-insensitive costal-2 reporter, possibly repressed via the Ago2-RISC pathway. This observation, combined with the insensitivity of the RNAi pathway to Atx2 knockdown in the Drosophila eye, suggests that Atx2 may not be required for Ago2-RISC function (McCann, 2011).

Atx2's role in the Ago1-miRNA pathway raises the question as to how Atx2 influences miRNA-mediated translational repression. Uncovering Atx2's molecular mechanism of action is complicated by lack of consensus as to how miRNAs regulate gene expression. However, Atx2 is likely to function through its interactions with PABP [mediated by its PABP-interacting motif 2 (PAM2) domain] or Me31B [via its Like Sm (Lsm) and Like-Sm-associated domain (LsmAD) domains] (McCann, 2011).

Three possible models for Atx2 actions are considered. Under appropriate conditions, Atx2 interactions with PABP could help break eukaryotic initiation factor (eIF) 4G eIF4g-PABP interactions required for efficient translational initiation. In addition, directly or through interactions with Me31B, Atx2 may help recruit either of two deadenylase complexes that promote mRNA deadenylation and consequent repression (McCann, 2011).

The identification of Atx2 as a core component of the neuronal and nonneuronal miRNA repression machinery has implications for understanding spinocerebellar ataxias and some forms of amyotrophic lateral sclerosis. Several studies underline the importance of functional components of the miRNA repression machinery in the mammalian brain. It has been demonstrated that miRNA-regulated activities play a role in polyglutamine-induced neurodegeneration. In addition, other work has shown that Atx2 is required for pathogenic forms of Atx1 and Atx3 to induce neurodegeneration in Drosophila, suggesting a potentially common pathway for neuronal loss in different ataxias. Loss of Dcr1 function results in microcephaly and progressive neurodegeneration, consistent with a model in which miRNA function is required for maintaining the adult nervous system (Saba, 2010). Given Atx2's involvement in human neurodegenerative disease, the current findings may help illuminate some of the phenotypes and symptoms of SCA2 patients and also may illuminate possibly common pathways for neuronal loss in different neurodegenerative conditions. If altered miRNA function contributes to neurodegeneration in SCA2 or related diseases, then it is possible that these diseases arise because of altered regulation of a subset of Atx2-target mRNAs in neurons. The identification and study of such target mRNAs may contribute to further understanding and potential therapeutic strategies (McCann, 2011).

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

McCann, C., et al. (2011). The Ataxin-2 protein is required for microRNA function and synapse-specific long-term olfactory habituation. Proc. Natl. Acad. Sci. 108(36):vE655-62. PubMed Citation: 21795609

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

Satterfield, T. F., Jackson, S. M. and Pallanck, L. J. (2002). A Drosophila homolog of the polyglutamine disease gene SCA2 is a dosage-sensitive regulator of actin filament formation. Genetics 162: 1687-1702. PubMed Citation: 12524342

Satterfield, T. F. and Pallanck, L. J. (2006). Ataxin-2 and its Drosophila homolog, ATX2, physically assemble with polyribosomes. Hum. Mol. Genet. 15: 2523-2532. PubMed citation: 16835262

Tharun, S. (2008). Roles of Eukaryotic Lsm Proteins in the Regulation of mRNA Function. Int. Rev. Cell and Molec. Biol. 272: 149-189. PubMed Citation: 19121818

date revised: 15 October 2011

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