InteractiveFly: GeneBrief

polyA-binding protein: Biological Overview | References


Gene name - polyA-binding protein

Synonyms - cytoplasmic poly(A)-binding protein 1, PABPC1

Cytological map position - 55B8-55B9

Function - RNA-binding protein

Keywords - regulation of translation initiation, nonsense-mediated mRNA decay

Symbol - pAbp

FlyBase ID: FBgn0265297

Genetic map position - 2R:14,027,583..14,033,743 [+]

Classification - Poly-adenylate binding protein, unique domain and four RNA recognition motifs

Cellular location - cytoplasmic and nuclear



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs with premature translation termination codons (PTCs). The mechanisms by which PTCs and natural stop codons are discriminated remain unclear. This study shows that the position of stops relative to the poly(A) tail (and thus of PABPC1) is a critical determinant for PTC definition in Drosophila. Indeed, tethering of PABPC1 downstream of a PTC abolishes NMD. Conversely, natural stops trigger NMD when the length of the 3' UTR is increased. However, many endogenous transcripts with exceptionally long 3' UTRs escape NMD, suggesting that the increase in 3' UTR length has co-evolved with the acquisition of features that suppress NMD. Evidence is provided for the existence of 3' UTRs conferring immunity to NMD. PABPC1 binding is sufficient for PTC recognition, regardless of cleavage or polyadenylation. The role of PABPC1 in NMD must go beyond that of providing positional information for PTC definition, because its depletion suppresses NMD under conditions in which translation efficiency is not affected. These findings reveal a conserved role for PABPC1 in mRNA surveillance (Behm-Ansmant, 2007).

Translation plays an important role in the regulation of gene expression and is implicated in the control of cell growth, proliferation, and differentiation. In eukaryotes, initiation is the rate-limiting step of translation in most circumstances and is a major target for regulation. The 5' cap structure (m7GpppN, where m is a methyl group and N is any nucleotide) of the mRNA is recognized by the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is comprised of three subunits: (1) eIF4E, the cap binding protein; (2) eIF4A, a bidirectional ATP-dependent RNA helicase; and (3) eIF4G, a modular scaffolding protein, which possesses binding sites for eIF4E and eIF4A and recruits the 40S ribosomal subunit to the mRNA via its interaction with eIF3. The 3' poly(A) tail of the mRNA is bound by the poly(A) binding protein (PABP). PABP is a phylogenetically conserved protein that functions in mRNA stability and translation. PABP is an essential protein: in Saccharomyces cerevisiae, deletion of the PAB1 gene is lethal and a P-element insertion in the Drosophila PABP gene is embryonic lethal (Sigrist, 2000). PABP is an 630-amino-acid (aa) protein containing four RNA recognition motifs (RRMs) arranged in tandem and a proline-rich C-terminal domain. RRMs 1 and 2 are the major contributors to the poly(A) binding activity of PABP (Deardorff, 1997; Kuhn, 1996). PABP directly interacts with eIF4G, leading to circularization of the mRNA by bridging the 5' and 3' extremities (closed-loop model) (Munroe, 1990; Sachs, 2000). The closed-loop model explains the synergistic enhancement of translation by the 5' cap structure and the 3' poly(A) tail of the mRNA. By joining the 5' and 3' ends of the mRNA, circularization may facilitate recycling of ribosomes, initiation complex formation, or the 60S ribosome-joining step (Roy, 2004 and references therein).

Nonsense-mediated mRNA decay (NMD) is a conserved mRNA quality control mechanism (surveillance) that ensures the fidelity of gene expression by detecting and degrading mRNAs containing premature translation termination codons (PTCs, nonsense codons). In this way, NMD safeguards cells from accumulating potentially deleterious truncated proteins (reviewed by Conti, 2005; Lejeune, 2005; Amrani, 2006; Behm-Ansmant, 2007 and references therein).

The NMD pathway serves not only to degrade mRNAs containing PTCs (as a consequence of mutations or errors in transcription or mRNA processing), but also regulates the expression of about 3%-10% of the transcriptome in Saccharomyces cerevisiae, Drosophila melanogaster and human cells. These natural NMD targets play a role in biological processes as diverse as transcription, cell proliferation, the cell cycle, telomere maintenance, cellular transport and organization, and metabolism (reviewed by Rehwinkel, 2006; Behm-Ansmant, 2007).

NMD is triggered by premature translation termination, which leads to the assembly on the mRNA of a so-called surveillance complex. The surveillance complex comprises the conserved NMD effectors UPF1, UPF2 and UPF3, and couples the premature translation termination event to mRNA decay by interacting with both eukaryotic translation termination factors (i.e. eRF1 and eRF3) and with mRNA degradation enzymes (Conti, 2005; Lejeune, 2005; Amrani, 2006; Behm-Ansmant, 2007).

Stop codons are recognized as premature depending on their location relative to downstream sequence elements (DSEs) and associated proteins (Conti, 2005; Lejeune, 2005; Amrani, 2006). In mammals, these downstream sequences are represented by exon-exon boundaries. Indeed, stop codons located at least 50-55 nt upstream of an exon-exon boundary are generally defined as premature, whereas most PTCs downstream of this point do not elicit decay (Behm-Ansmant, 2007).

Exon-exon boundaries are marked by the exon junction complex (EJC), which is deposited during splicing 20-24 nt upstream of a splice junction (Le Hir, 2000). Current models for mammalian NMD (reviewed by Lejeune, 2005) postulate that UPF3 associates with the EJC within the nucleus and recruits UPF2 following the export of the mRNA to the cytoplasm. When translating ribosomes encounter a stop codon upstream of an EJC, the recruitment of UPF1 by translation release factors leads to an interaction with the UPF2 and UPF3 proteins bound to the downstream EJC, and thus to the assembly of the surveillance complex and to mRNA degradation {Behm-Ansmant, 2007).

In S. cerevisiae and D. melanogaster, PTC recognition occurs independently of splicing, and different models have been proposed to explain what distinguishes premature from natural stops in these organisms. One model proposes that mRNAs harbor loosely defined DSEs with a function analogous to that of mammalian exon junctions (Amrani, 2006). Alternative models suggest that a generic feature of the mRNA, such as the poly(A) tail, or a mark deposited during the cleavage and polyadenylation reaction could provide positional information to discriminate premature from natural stop codons (Behm-Ansmant, 2007).

Yet another model, the 'faux 3' UTR model', proposes that premature translation termination is intrinsically aberrant because the stop codon is not in the appropriate context (Amrani, 2004; Amrani, 2006). According to this model, natural 3' UTRs are marked by a specific set of proteins that influence translation termination. Termination is efficient at natural stops because terminating ribosomes are able to interact with these 3' UTR-bound proteins. In contrast, translation termination would be impaired or too slow at premature stops, because of the inability of the terminating ribosome to establish these interactions. In this case, the surveillance complex is assembled, leading to the rapid degradation of the mRNA (Amrani, 2004; Amrani, 2006; Behm-Ansmant, 2007 and references therein).

In support of the 'faux 3' UTR model', experiments in S. cerevisiae have shown that translation termination is aberrant at premature stop codons, and prematurely terminating ribosomes are not released efficiently (Amrani, 2004; Amrani, 2006). This effect is abolished by flanking the nonsense codon with a normal 3' UTR. Moreover, tethering the poly(A)-binding protein (Pab1p) downstream of a PTC, which is likely to mimic a normal 3' UTR, leads to efficient translation termination and abolishes NMD (Amrani, 2004; Amrani, 2006). This suggests that proximal Pab1p binding defines natural stops in S. cerevisiae. Consistently, most S. cerevisiae 3' UTRs are homogeneous in length (ca. 100 nt), and aberrant transcripts with exceptionally long 3' UTRs (due to errors in 3'-end processing) are regulated by NMD (Behm-Ansmant, 2007).

In multicellular organisms, 3' UTR length distribution ranges from a few to several thousand nucleotides, raising the question of whether the faux 3' UTR model could account for PTC recognition. This study has investigated the mechanism of PTC recognition in Drosophila. The cytoplasmic poly(A)-binding protein (PABPC1) provides positional information discriminating premature from natural stops in this organism. Consistently, natural stops can be made to trigger NMD by increasing the 3' UTR length. However, a large proportion of naturally occurring transcripts with exceptionally long 3' UTRs are not NMD substrates, suggesting that some of these 3' UTRs may have evolved features to avoid NMD. Supporting this possibility, it is shown that 3' UTRs specifying rapid decay also confer immunity to NMD. Finally, it is demonstrated that depletion of PABPC1 inhibits NMD, revealing a new role for this protein in mRNA surveillance (Behm-Ansmant, 2007).

This study shows that PABPC1 plays a critical role in the mechanism by which premature stops are distinguished from natural stops. A synthetic poly(A) tail of 45 adenosine residues (or tethering of PABPC1) is sufficient to confer sensitivity to NMD on unadenylated RNAs, indicating that the role of PABPC1 in NMD is independent of 3'-end cleavage and polyadenylation. Finally, it is shown that NMD is impaired in cells depleted of PABPC1. These findings, together with previous studies in S. cerevisiae (Amrani, 2004; Amrani, 2006), reveal a conserved role for PABPC1 in NMD. More generally, the observation that natural stops are redefined as premature by increasing 3' UTR length suggests that NMD is likely to have presented a selective constraint on the evolution of eukaryotic 3' UTRs (Behm-Ansmant, 2007).

What could be the role of PABPC1 in PTC recognition? As proposed by the faux 3' UTR model, proximal PABPC1 binding is likely to increase the efficiency of translation termination and of ribosome release (Amrani, 2004). Indeed, PABPC1 interacts with eRF3, and tethering of eRF3 downstream of a PTC also abolishes NMD, albeit less effectively than does tethering PABPC1 (Amrani, 2004). PABPC1 may also recruit other translational regulators that stimulate translation termination or proteins that prevent NMD when translation terminates at the physiological stop codon. Thus, increasing the distance of the stop codon to PABPC1 for a given mRNA would impair these interactions (as is the case for PTCs located towards the 5' end of the transcript) and translating ribosomes may not terminate translation efficiently, leading to the recruitment of the NMD machinery (Behm-Ansmant, 2007).

Similar analyses to those described in this study have been performed in human cells. In most cases, the positional dependence of nonsense codons to trigger NMD exhibited a clear boundary effect, with PTCs upstream of the boundary eliciting mRNA degradation efficiently, whereas those close or downstream of the boundary being inefficient triggers of NMD. In contrast to the results in Drosophila, however, in mammalian cells the boundary corresponds to the 3'-most exon-exon junction. Indeed, for many reporters, nonsense codons should be located at least 50-55 nt upstream of the boundary to destabilize the transcript (an effect referred as to the '50 nt boundary rule' (Behm-Ansmant, 2007).

The 50 nt boundary rule is not absolute and in some mRNAs the spacing between the nonsense codon and the boundary can be smaller. Moreover, there are an increasing number of examples of PTCs that elicit NMD despite the absence of a downstream intron, although steady-state mRNA levels are only slightly affected in this case. These observations have been interpreted as an indication that in mammals, as in S. cerevisiae, PTC recognition depends on the distance to the poly(A) tail. However, although PABPC1 binds to mRNAs subject to NMD (Hosoda, 2006), a role for the poly(A) tail, and hence for PABPC1, in PTC-definition in mammals has not been demonstrated directly. On the contrary, available evidence indicates that PABPC1 is dispensable for NMD in human cells, provided that the PTC is located upstream of an exon-exon boundary. Indeed, a polyadenylation signal with a histone stem-loop structure does not affect NMD in human cells. In Drosophila cells mRNAs terminated with a histone stem-loop structure are immune to NMD (Behm-Ansmant, 2007).

Several lines of evidence point to a significant divergence between the NMD pathway in mammals and invertebrates. For instance, as a consequence of the splicing-dependent mechanism for PTC definition, mammalian EJC components are required for NMD. These include the proteins Y14, MAGOH, eIF4AIII, Barentsz and RNPS1 (Conti, 2005; Lejeune, 2005). The Drosophila orthologs are not required for NMD. Moreover, there are no clear orthologs of these proteins in S. cerevisiae. It has been argued that EJC components may not play a direct role in PTC recognition in mammals, but may act as enhancers of NMD, PTC recognition being dependent on the distance to the poly(A) tail (Buhler, 2006). However, even the observation that EJC components and exon-exon boundaries enhance NMD triggered by PTCs located upstream in mammals, but not in S. cerevisiae or D. melanogaster, points to clear differences in the mechanisms of NMD between these organisms (Behm-Ansmant, 2007).

Although the NMD machinery is conserved, the current results suggest that a change in the mechanism by which nonsense codons are defined occurred during evolution with a switch from PABPC1-dependent to a predominantly EJC-dependent mode. This may have created the opportunity for EJC components to functionally substitute for UPF2, as suggested by Gehring (2005). The release of functional constraints on NMD effectors may have in turn facilitated the acquisition of additional functions. Indeed, mammalian UPF1, SMG1 and SMG5-7 have been implicated in cellular processes distinct from NMD (reviewed by Rehwinkel, 2006; Behm-Ansmant, 2007 and references therein).

An important finding from these experiments is that replacing the cleavage and polyadenylation signal of nonsense mRNAs with a self-cleaving hammerhead ribozyme or a histone stem-loop structure suppresses NMD. These results are in agreement with those reported in S. cerevisiae, showing that nonsense ribozyme-terminated transcripts are not subject to NMD. However, in contrast to the results in S. cerevisiae, this study shows that immunity to NMD is not due to reduced translation efficiency, but may be at least in part due to the high decay rate of these mRNAs, which makes them resistant to any further acceleration of degradation. These short-lived, unadenylated mRNAs become susceptible to degradation by NMD when 45 or more adenosine residues are inserted upstream of the ribozyme cleavage site or the histone stem-loop structure, indicating that binding of PABPC1, independently of 3'-end processing, is sufficient to confer sensitivity to NMD (Behm-Ansmant, 2007).

For ribozyme-terminated mRNAs, binding of PABPC1 stabilizes the transcript and this may in turn be sufficient to confer sensitivity to NMD. In contrast, the reporters terminated with a histone stem-loop structure become NMD-sensitive upon PABPC1 binding, although a small effect on mRNA half-lives is observed. This suggests that PABPC1 may have additional roles in NMD. In agreement with this, depletion of PABPC1 inhibits NMD, under conditions in which translation efficiency is not affected. A possible additional role for PABPC1 could be that of facilitating the recruitment of the NMD machinery. However, this study failed to detect a stable interaction between PABPC1 and NMD factors by co-immunoprecipitation assays, suggesting that if these interactions occur they may be indirect or transient. Alternatively, PABPC1 could act in translation termination and indirectly on NMD (Behm-Ansmant, 2007).

The existence of a mechanism for PTC recognition based on the position of the stop relative to PABPC1 binding raises the question of how endogenous transcripts with exceptionally long 3' UTRs avoid NMD. There are several mechanisms by which mRNAs may escape NMD. Inherently unstable mRNAs are insensitive to NMD. Alternatively, long 3' UTRs may have sequence elements that are folded via RNA-RNA or RNA-protein interactions into structures that bring PABPC1 into close proximity with the natural stop. In this case, proximity to PABPC1 is not directly correlated to the length in nucleotides between the natural stop and the poly(A) tail (Behm-Ansmant, 2007).

An alternative mechanism to confer resistance to NMD may involve the presence of mRNA-stabilizing elements that prevent decay directly. Sequence elements that antagonize NMD have been reported in viral and S. cerevisiae transcripts. A sequence element that stabilizes unspliced Rous sarcoma virus RNAs also inhibits NMD when located downstream of a PTC (Weil, 2006). The S. cerevisiae PGK1, GCN4 and YAP1 mRNAs have sequence elements that prevent NMD in a heterologous context when positioned downstream of a PTC (Ruiz-Echevarria, 2000). The effects of the S. cerevisiae-stabilizing elements are mediated by the RNA-binding protein PUB1 (Ruiz-Echevarria, 2000). It is unclear whether PUB1 prevents decay or increases the efficiency of translation termination at nonsense codons located upstream of its binding site, but these two modes of action could be envisaged for different RNA-binding proteins. Immunity or susceptibility to NMD could be regulated if these proteins were to be expressed under specific physiological conditions or in a tissue-specific manner. In this way, NMD could contribute to the establishment of gene expression 'programs' in response to changes in physiological conditions or in specific tissues (Behm-Ansmant, 2007).

PABP and the poly(A) tail augment microRNA repression by facilitated miRISC binding

Polyadenylated mRNAs are typically more strongly repressed by microRNAs (miRNAs) than their nonadenylated counterparts. Using a Drosophila melanogaster cell-free translation system, this effect was found to be mediated by the poly(A)-binding protein (PABP). miRNA repression was positively correlated with poly(A) tail length, but this stimulatory effect on repression was lost when translation was repressed by the tethered GW182 silencing domain rather than the miRNA-induced silencing complex (miRISC) itself. These findings are mechanistically explained by a notable function of PABP: it promotes association of miRISC with miRNA-regulated mRNAs. It was also found that PABP association with mRNA rapidly diminished with miRISC recruitment and before detectable deadenylation. These data were integrated into a revised model for the function of PABP and the poly(A) tail in miRNA-mediated translational repression (Moretti, 2012).

Although the poly(A) tail and PABP are important effectors of miRNA function in many but not all experimental systems, the molecular mechanism(s) by which they contribute to miRNA-mediated silencing is unclear. This study used a combination of biochemical and functional assays in the D. melanogaster embryo in vitro system to discover a new aspect of this interplay and to show that stimulation of miRNA-mediated repression by the presence of a poly(A) tail is mediated by PABP (Moretti, 2012).

miRISC has been proposed to interfere with the interaction of PABP with eIF4G, causing a disruption of closed-loop formation and translational repression. The data reported in this study show a role of PABP in miRNA-mediated repression. It was found that the length of the poly(A) tail (and therefore the number of PABP molecules that it can accommodate) is positively correlated with the extent of miRNA-mediated repression. Analogously, for a reporter mRNA bearing only a single miRNA-binding site, the presence of the poly(A) tail is essential to confer repressibility; this reporter is not susceptible to repression in its nonadenylated form. Furthermore, the extent of repression of reporter mRNAs controlled by GW182-SD tethering is not influenced by the presence or absence of a poly(A) tail, although the poly(A) tail stimulates translation of these reporters; this result uncouples translatability and repressibility. Efficient repression by GW182 protein tethering to reporters lacking a poly(A) tail has also been observed with reporters bearing either a histone stem-loop structure or a hammerhead ribozyme cleavage site at their 3′ ends. These data all suggest that a physiological miRISC context is required for PABP and the poly(A) tail to stimulate repression (Moretti, 2012).

Deadenylation of target mRNAs is a widespread effect of miRNA-mediated repression and involves PABP. Several studies have suggested that PABP is displaced from the mRNA before deadenylation starts and proposed that PABP affinity for the poly(A) tail is modified either by specific domains of PABP itself or by mRNA-specific activators of deadenylation. In agreement with these studies, this study shows that miRNA-mediated repression (and repression achieved through GW182-SD tethering) causes lower target mRNA association with PABP. This observation supports the idea that the PABP-miRISC interaction could reduce the affinity of PABP for the poly(A) tail, rendering it more accessible to deadenylase. These experiments show that PABP dissociates from mRNAs in parallel with AGO1 association early in the establishment of silencing and notably before deadenylation begins. Therefore, PABP dissociation is not a mere consequence of deadenylation, and deadenylation could be facilitated by the loss of PABP. GW182 proteins can directly recruit components of deadenylase complexes (Braun, 2011; Chekulaeva, 2011; Fabian, 2011). Taken together, these observations support the notion that miRISC coordinates both PABP displacement and recruitment of the deadenylation machinery to efficiently promote deadenylation of miRNA targets (Moretti, 2012).

The above results also offer a mechanistic explanation for how PABP and the poly(A) tail stimulate miRISC action: they exert their stimulatory function, at least in part, by facilitating miRISC binding to target mRNAs. Does PABP facilitate miRISC binding by enhanced recruitment or by stabilization of the miRISC complex on a target mRNA? Without direct measurement of on and off rates of the miRISC complex it is not possible todiscriminate between these two possibilities. However, the first alternative is favored: if PABP stabilized miRISC on the mRNA over time, it would be expected to remain associated with the repressed mRNP to exert its effect. In contrast with this prediction, it was found that PABP dissociates rather early. If PABP supports miRISC recruitment, mRNAs that are already affected by miRNA-mediated repression would have been deadenylated, bear markedly lower amounts of PABP and therefore compete less efficiently for miRISC. Conversely, mRNAs that still have to be repressed will bear longer poly(A) tails and their associated PABP will promote miRISC recruitment. This effect would provide a mechanism for efficiently targeting miRISC complexes to mRNAs that should undergo repression. The RNA-binding protein HuR has also been proposed to stimulate miRNA-mediated repression by facilitating miRISC recruitment to target mRNAs. As GW182 mutants that do not interact with PABP are severely impaired in silencing (Huntzinger, 2010; Braun, 2011), it is speculated that PABP facilitates miRISC recruitment through its direct interaction with GW182, although bridging binding partners could also explain the data. PABP has been proposed to be dispensable for miRNA-mediated repression in vitro. Although the data support the notion that PABP is not absolutely required for miRNA-mediated repression in vitro, this study demonstrates an important function of PABP in silencing. The in vitro system referred to above relies on preloading of reporter-specific miRISC with exogenously supplemented miRNA duplexes. It is predicted that such an experimental system misses the function of PABP that is described in this paper (Moretti, 2012).

Notably, the stimulatory effect of the poly(A) tail on AGO1 association was more pronounced for reporter mRNAs bearing 3′ UTRs of sickle (skl) or grim. Although additional miRNAs might bind skl and grim 3′ UTRs, this study observed a marked reduction (three- to five-fold) of AGO1 association upon incubation with anti-miR2 LNAs, indicating the importance of the miR2-RISC. Notably, the miR2-binding sites within these 3′ UTRs have a less favorable pairing with miR2 than the reaper 3′ UTR-binding motif present in the 1× and 6× reporters as indicated by the Z-score, an indicator of the folding energy of each miRNA-mRNA target pair. This inverse correlation suggests that the more pronounced stimulatory effect of PABP and the poly(A) tail on miRISC association results from the less favorable Watson-Crick base pairing. Potentially, the poly(A) tail and PABP contribute less to miRISC association with high-affinity binding sites showing strong Watson-Crick base pairing (Moretti, 2012).

On the basis of this data, an extended model is proposed for miRNA-mediated silencing in which PABP and the poly(A) tail augment binding of miRISC to the target mRNA during early phases of silencing. miRISC binding induces displacement of PABP and, upon recruitment of deadenylase complexes, deadenylation of target mRNA. This model does not challenge but rather extends earlier analyses of the functional importance of miRISC-PABP interaction and clarifies the complex interplay among miRISC, the translation initiation machinery and establishment of effective silencing (Moretti, 2012).

The novel gene twenty-four defines a critical translational step in the Drosophila clock

Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms. While attention has been focused on transcriptional and post-translational mechanisms, other post-transcriptional modes have been less clearly delineated. This study reports mutants of a novel Drosophila gene twenty-four (tyf; CG4857) that show weak behavioural rhythms. Weak rhythms are accompanied by marked reductions in the levels of the clock protein Period (Per) as well as more modest effects on Timeless (Tim). Nonetheless, Per induction in pacemaker neurons can rescue tyf mutant rhythms. Tyf associates with a 5'-cap-binding complex, poly(A)-binding protein (PABP), as well as per and tim transcripts. Furthermore, Tyf activates reporter expression when tethered to reporter messenger RNA even in vitro. Taken together, these data indicate that Tyf potently activates Per translation in pacemaker neurons to sustain robust rhythms, revealing a new and important role for translational control in the Drosophila circadian clock (Lim, 2011).

It remains unclear how TYF controls translation of its target RNAs. Specific effects on Per and Tim were observed but not on other clock components, and Tyf was found to interact with translation components such as the eIF4E-containing cap-binding complex and PABP. It is proposed that RNA-binding translational repressors associate with newly transcribed per RNA, temporarily postpone translation and thus delay Per feedback repression on its own transcription. Such a delay could contribute to the observed lag between protein and RNA particularly in pacemaker neurons, although post-translational mechanisms may also contribute, at least in the eyes. Tyf, which does not have a known RNA-recognition motif, could then be recruited to target transcripts by these translational repressors, releasing them to stimulate initiation of per translation. It has not been possible to biochemically or genetically link Tyf to RNA-binding proteins FMR, LARK, or the translation regulator Thor/4E-BP, which have been shown to contribute to circadian clock function. Nonetheless, TYF association with eIF4E and their similar polysome profiles implicate TYF as a novel translation initiation factor. In addition, the effects of TYF may be more evident on poorly adenylated transcripts on the basis of in vitro data. Of note, the Drosophila homologue of the clock-regulated deadenylase nocturnin has been shown to be important in dorsal neurons for circadian light responses but neither a LN function nor an RNA target has been described. Nevertheless, unique features of Tyf-regulated transcripts may mediate the highly selective TYF effects on clock components in vivo (Lim, 2011).

Post-transcriptional regulation on per RNA has been considered to be modulatory to clock function. The identification of a critical role for Tyf highlights an important role for Per translation in the Drosophila neural clockwork. It will be of interest to determine if proteins functionally analogous to Tyf serve similarly important and specific functions in the mammalian clock (Lim, 2011).

The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, this study characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases (Huntzinger, 2013).

Recent studies indicate that translational repression of miRNA targets precedes deadenylation and decay. This study shows that these two functional outcomes of miRNA regulation are linked and both require the interaction of GW182 proteins with PABP and deadenylases (Huntzinger, 2013).

The interaction of GW182 proteins with PABP has been well documented using biochemical and structural studies, and the PAM2 motif is highly conserved among vertebrate and insect GW182 proteins. Despite conservation, the study of the role of PABP in silencing in different systems has led to conflicting conclusions. For example, several studies have reported that the PABP–GW182 interaction is important for silencing in Drosophila and human cells and in cell-free systems that recapitulate silencing. Furthermore, PABP depletion prevented miRNA-mediated deadenylation in cell-free extracts from mouse Krebs-2 ascites cells, and mutations in the PAM2 motif of TNRC6C reduced the rate of deadenylation in tethering assays. In addition, a study in Drosophila cell-free extracts wherein silencing is mediated through endogenous preloaded miRISCs indicated that PABP stimulates silencing by facilitating the association of miRISC complexes with mRNA targets. It was also shown that on miRISC binding, PABP progressively dissociated from the mRNA target, in the absence of deadenylation (Huntzinger, 2013).

In contrast to the studies mentioned above, studies in zebrafish embryos and in a Drosophila cell-free assay wherein miRISCs are loaded with exogenously supplemented miRNA duplexes indicate that PABP is dispensable for miRNA-mediated silencing. Intriguingly, efficient silencing in zebrafish embryos required the GW182 PAM2 motif. Moreover, the observation that multiple and non-overlapping fragments of Drosophila GW182 (including N-term fragments that do not interact with PABP) silenced mRNA reporters in tethering assays was interpreted as evidence that the interaction of GW182 proteins with PABP is not required for silencing. This study shows that unlike in tethering assays, N-term fragments of GW182 fail to restore the silencing of a majority of the reporters tested in complementation assays. Thus, tethering assays bypass the requirement for PABP binding, and may not faithfully recapitulate silencing. Furthermore, the observation that PABP dissociates from the poly(A) tail of miRNA targets in the absence of deadenylationprovides one explanation for the occurrence of silencing in extracts in which PABP has been depleted or displaced from the poly(A) tail using an excess of Paip2 (Huntzinger, 2013).

In summary, these results confirm and further extend previous observations that a single amino acid substitution in the PAM2 motif of human TNRC6 proteins abolishes PABP binding and impairs silencing activity, despite the interaction of this mutant with deadenylases. Furthermore, Drosophila GW182 N-term protein fragments that bind deadenylases, but not PABP, failed to complement the silencing of eight of the nine reporters tested, although they are active in tethering assays. These results provide evidence for a role of PABP in silencing in human and Drosophila cells. However, it is possible that PABP becomes dispensable for silencing depending on cellular conditions or the nature of the specific mRNA target, as shown, for example, for the F-Luc-Nerfin-1 reporter when silencing is mediated by miR-9b (Huntzinger, 2013).

The SDs of human TNRC6 proteins directly interact with CNOT1 through tryptophan-containing motifs in the M1, M2 and C-term regions of the S. This study shows that these motifs contribute additively to CNOT1 binding and silencing activity in human cells. Indeed, when at least two motifs are simultaneously mutated, CNOT1 binding is strongly reduced and silencing activity impaired (Huntzinger, 2013).

The interaction between GW182 and deadenylases is conserved in Drosophila; however, in contrast to human SDs, the Drosophila SD is not sufficient for NOT1 binding. This study shows that in addition to the SD, the Q-rich region is required for full NOT1 binding activity. Thus, although Drosophila GW182 has lost the CIM-2 motif, this protein has acquired additional motifs that can interact with NOT1. This study also shows that in contrast to the human proteins, Drosophila GW182 can interact with NOT2 and PAN3 via N-term sequences. Consequently, Drosophila GW182 can recruit deadenylases in multiple ways. Considering that (1) NOT1 interacts with NOT2, (2) the PAN2–PAN3 complex interacts with PABP and (3) the CCR4–NOT and PAN2–PAN3 complexes form a larger multiprotein complex in vivo, the current observations indicate a high degree of connectivity and redundancy within the GW182 interaction network, which could explain why mutations in individual motifs do not abolish partner binding or silencing activity, but a combination of two or more mutations is required to abrogate binding and silencing activity (Huntzinger, 2013).

In addition, the ability of Drosophila GW182 N-term fragments to bind deadenylases also explains why these fragments are potent triggers of translational repression and mRNA degradation in tethering assays, whereas the corresponding fragments of the human proteins exhibit only residual activity. As discussed previously, despite their activity in tethering assays, Drosophila GW182 N-term fragments failed to complement the silencing of several of the reporters tested. The reason for the different activities of these fragments in tethering and complementation assays remains unknown (Huntzinger, 2013).

This study has demonstrated that silencing (i.e. translational repression and target degradation) requires the interaction between GW182 proteins and both PABP and deadenylases. Several lines of evidence support this conclusion. First, the TNRC6C SD, which is sufficient for PABP and deadenylase binding, rescues silencing when fused to a minimal ABD. Similarly, the minimal fragment of Drosophila GW182 that rescues silencing comprises the Q+SD region, which also binds both deadenylases and PABP. Second, the Drosophila GW182 N-term fragments that bind deadenylases but not PABP are generally inactive in complementation assays. Third, mutations that specifically disrupt TNRC6 binding to PABP or deadenylase impair silencing, and mutations that disrupt deadenylase binding exhibit a stronger deleterious effect. Silencing activity is abolished when these mutations are combined. Finally, silencing is inhibited in human cells overexpressing the CNOT1 Mid domain together with a catalytically inactive CNOT7 mutant. In combination with the previously published data, these results indicate that silencing minimally requires an AGO, a GW182 protein, PABP and deadenylases, thus defining the minimal interaction network required for silencing. The findings do not rule out that additional interactions are potentially required to achieve maximal repression, depending on the cellular context or the mRNA target. For example, the P-GL motif is highly conserved and important for silencing in zebrafish embryos. This motif may mediate interactions with additional partners (Huntzinger, 2013).

The finding that deadenylase complexes, in particular, are required for miRNA-mediated translational repression has broad implications regarding post-transcriptional mRNA regulation. Indeed, in addition to the GW182 proteins, various sequence-specific mRNA-binding proteins, such as Nanos, Bicaudal-C and Pumilio, recruit the CCR4–NOT complex to their mRNA targets. Furthermore, the direct tethering of the subunits of the CCR4–NOT complex represses the translation of mRNA reporters lacking a poly(A) tail, suggesting that the CCR4–NOT complex promotes translational repression in the absence of deadenylation. Therefore, elucidating the mechanism by which the CCR4–NOT complex regulates the fates of mRNA targets promises to increase understanding of the mechanism underlying repression by miRNAs and diverse sequence-specific RNA-binding proteins (Huntzinger, 2013).

Therapeutic modulation of eIF2alpha phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2 (see Drosophila Ataxin-2), is that they localize to stress granules. Unexpectedly, this study found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2alpha phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. The efficacy of a small molecule inhibitor of eIF2alpha phosphorylation was investigated in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach (Kim, 2014)

GW182 proteins cause PABP dissociation from silenced miRNA targets in the absence of deadenylation

GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)-binding protein (PABP) and the CCR4-NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. This study demonstrates that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4-NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. These findings indicate that the recruitment of the CCR4-NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation (Zekri, 2013).

The Drosophila poly(A) binding protein-interacting protein, dPaip2, is a novel effector of cell growth

The 3' poly(A) tail of eukaryotic mRNAs and the poly(A) binding protein (PABP) play important roles in the regulation of translation. Recently, a human PABP-interacting protein, Paip2, which disrupts the PABP-poly(A) interaction and consequently inhibits translation, was described. To gain insight into the biological role of Paip2, the Drosophila Paip2 (dPaip2) was studied. dPaip2 is the bona fide human Paip2 homologue; it interacts with dPABP, inhibits binding of dPABP to the mRNA poly(A) tail, and reduces translation of a reporter mRNA by 80% in an S2 cell-free translation extract. Ectopic overexpression of dPaip2 in Drosophila wings and wing discs results in a size reduction phenotype, which is due to a decrease in cell number. Clones of cells overexpressing dPaip2 in wing discs also contain fewer cells than controls. This phenotype can be explained by a primary effect on cell growth. Indeed, overexpression of dPaip2 in postreplicative tissues inhibits growth, inasmuch as it reduces ommatidia size in eyes and cell size in the larval fat body. It is concluded that dPaip2 inhibits cell growth primarily by inhibiting protein synthesis (Roy, 2004; full text of article).

Two human proteins that interact directly with PABP have been identified: Paip1 and Paip2 (PABP-interacting proteins 1 and -2). Paip1 stimulates, while Paip2 represses, translation (Craig, 1998; Khaleghpour, 2001a). Paip2 inhibits translation by reducing the binding of PABP to the poly(A) tail and by competing with Paip1 for binding to PABP. Paip1 and Paip2 share two conserved PABP-interacting motifs (PAMs). PAM1 consists of a stretch of acidic amino acids in the middle of Paip2 (aa 22 to 75) and at the C terminus of Paip1 (aa 440 to 479), and it binds strongly to RRMs 2 and 3 and to RRMs 1 and 2 of PABP, respectively (Khaleghpour, 2001b; Roy, 2002). The second binding site, PAM2, also called the PABP C-terminal binding motif, resides in the C terminus of Paip2 (aa 106 to 120) (Khaleghpour, 2001a) and the N terminus of Paip1 (aa 123 to 137) (Roy, 2002). PAM2 consists of a short stretch of 15 aa and binds to the C terminus of PABP (within aa 546 to 619) with a lower affinity (10- and 200-fold for Paip1 and Paip2, respectively) than that of the PAM1-PABP interaction. PAM2 is also found in several additional proteins, including eukaryotic release factor 3 (eRF3), ataxin 2, and transducer of ErbB-2 (Tob). Thus, Paip2 and Paip1 might compete with some of these PAM2 binding partners to regulate PABP function. The Drosophila homologue of the human Paip2 (dPaip2), was isolated and characterized. Its ability to interact with Drosophila PABP (dPABP), inhibit translation, and interfere with dPABP poly(A) binding activity was demonstrated. Importantly, dPaip2 inhibits growth in flies (Roy, 2004).

dPaip2 reduces growth without altering patterning in several Drosophila tissues, including the larval fat body, eyes, wings, and wing-imaginal discs. dPaip2 strongly inhibits translation in vitro, as was shown for hPaip2. Thus, dPaip2 most likely inhibits growth by repressing translation. Translation is a major target of growth control, as cells need to increase their protein content before they can divide in order to ensure daughter cell survival. Deregulation of translation has often been associated with growth defects. For example, in Drosophila, a collection of mutations in genes encoding ribosomal proteins (known as Minute mutations) have low overall growth rates and are delayed in development. Interference with the formation of the eIF4F complex at the 5' end of the mRNA by the translation suppressor d4E-BP results in a reduced-growth phenotype. Mutations in the translation initiator factors deIF4E and deIF4A caused a more dramatic larval growth arrest phenotype, which is similar to that seen upon amino acid starvation. It is well established that nutrient starvation causes inhibition of translation by affecting discrete translational-control pathways (Roy, 2004 and references therein).

A number of signaling pathways have been implicated in the promotion of cell growth. Nutrient availability plays a key role in growth, and the insulin-signaling pathway coordinates cellular metabolism with nutritional conditions. The insulin-signaling pathway promotes translation via stimulation of S6K and inactivation of the translational repressor 4E-BP. Ectopic overexpression in Drosophila of positive components of the insulin-signaling pathway, for example, dInr or dPI3K, or mutations in negative regulators, such as dPTEN and dTSC1/2, cause dramatic increases in cell size and, to a lesser extent, increases in cell numbers (reviewed in references. Moreover, mutations in these same positive signaling components, or ectopic overexpression of the negative regulators, such as a highly active version of d4E-BP, primarily reduce cell size and have significantly weaker effects on cell numbers (with the exception of dS6K, which affects only cell size). In addition, increased insulin signaling stimulates transition through the G1/S phases of the cell cycle, but the overall doubling time of these cells is unchanged due to a compensatory lengthening of the G2/M phases. Hence, this pathway seems primarily to stimulate mass accumulation, creating an imbalance between growth and proliferation signals, which results in an alteration of cell size. The reasons for this imbalance remain unclear. dMyc and dRas also control cell growth. Ectopic overexpression of dMyc or activated dRas (dRasV12) promotes growth and results in increased cell size and numbers. Conversely, loss of the dMyc gene inhibits growth and results in fewer and smaller cells. Interestingly, dRas appears to upregulate dMyc at a posttranscriptional level. Similar to the insulin-signaling pathway, overexpression of dMyc and dRas affects growth in an unbalanced fashion, as they also shorten the G1 phase of the cell cycle and the G2 phase is lengthened to compensate. However, these proteins have weaker effects on cell size than components of the insulin-signaling pathway (Roy, 2004 and references therein).

In contrast to the insulin-signaling pathway and to dMyc and dRas, cooverexpression of dCdk4 and dCyclin D promotes growth and accelerates cell cycling. This results in a balanced, proportional cell growth in which cell size remains unchanged while cell numbers increase. Consistent with this finding, deletion of the Cdk4 gene represses growth without altering cell size and leads to a decrease in cell numbers. Interestingly, the phenotypes observed upon loss of the Cdk4 gene are similar to the phenotype of dPaip2 overexpression. In dividing cells of the wing discs, dPaip2 decreases cell numbers without reducing cell size, while in nonproliferating cells of the larval fat body and of the eye, dPaip2 overexpression decreases cell size. These tissue-specific effects are consistent with an inhibition of growth. In proliferating cells, the reduced translation capacity of the dPaip2-overexpressing cells likely affects all phases of the cell cycle equally. The reduced growth of these cells results in longer cell doubling times, but growth remains coordinated with proliferation and cell size is not affected. Consistent with a primary effect on growth, the nonproliferating cells of the eye and the larval fat body are reduced in size owing to impairment in translation. It is unclear at present why d4E-BP overexpression creates an imbalance between growth and proliferation signals, which leads to an alteration in cell size, while dPaip2 does not. The different sensitivities of some mRNAs to these translational inhibitors might be responsible for the different phenotypes (Roy, 2004 and references therein).

What is the molecular mechanism by which dPaip2 mediates its effect on cell growth? A likely possibility is that dPaip2 reduces growth by inhibiting the interaction of dPABP with the poly(A) tail, thus disrupting the mRNA 5'-3' loop and inhibiting translation. eIF4F disproportionately stimulates the translation of mRNAs containing extensive secondary structures in their 5' UTRs, which mainly encode growth factors and their receptors, cyclins, and other mitogens. For example, the level of cyclin D1 increases when eIF4E is overexpressed. Inasmuch as PABP stimulates the translation of a subset of mRNAs by activating eIF4F, dPaip2 inhibition of translation might disproportionately affect the same subset of mRNAs. Cyclin D and Cdk4 are interesting candidates. It would be important to link dPaip2 and cyclin D/Cdk4 translation. In addition, the translation of some mRNAs might be especially sensitive to the level of dPABP or dPaip2 (Roy, 2004).

The phenotypes observed upon overexpression of dPaip2 in different tissues are less dramatic than would have been expected from the in vitro translation experiments (~0% inhibition of translation). It is conceivable that dPaip2 levels in vivo are tightly regulated to prevent deleterious interference with PABP function (PABP's gene is essential). There is one Drosophila homologue of Paip1 (dPaip1) that has not been studied yet. Since Paip1 stimulates translation, it is possible that it counteracts the effects of overexpression of the repressor dPaip2. dPaip2, like Paip1, eRF3, ataxin-2, and Tob, interacts with the C-terminal domain of PABP through its conserved PAM2 site. The different PAM2-containing proteins might compete with Paip2 to modulate the activity of PABP and consequently attenuate the effects of dPaip2 overexpression. Furthermore, since hPaip2 is a phosphoprotein, it is possible that dPaip2 is also a phosphoprotein and that its activity is controlled by its phosphorylation state (Roy, 2004).

In conclusion, dPaip2 is an inhibitor of cell growth, most likely because of its ability to repress translation. This study also highlights the importance of regulating PABP function in translation and growth. This system should serve as a basis to identify regulators of dPaip2 activity by screening for genetic interacting partners (Roy, 2004).

The Bin3 RNA methyltransferase is required for repression of caudal translation in the Drosophila embryo

Bin3 was first identified as a Bicoid-interacting protein in a yeast two-hybrid screen. In human cells, a Bin3 ortholog (BCDIN3) methylates the 5' end of 7SK RNA, but its role in vivo is unknown. This study shows that in Drosophila, Bin3 is important for dorso-ventral patterning in oogenesis and for anterior-posterior pattern formation during embryogenesis. Embryos that lack Bin3 fail to repress the translation of caudal mRNA and exhibit head involution defects. bin3 mutants also show (1) a severe reduction in the level of 7SK RNA, (2) reduced binding of Bicoid to the caudal 3' UTR, and (3) genetic interactions with bicoid, and with genes encoding eIF4E, Larp1, polyA binding protein (PABP), and Ago2. 7SK RNA coimmunoprecipitated with Bin3 and is present in Bicoid complexes. These data suggest a model in which Bicoid recruits Bin3 to the caudal 3' UTR. Bin3's role is to bind and stabilize 7SK RNA, thereby promoting formation of a repressive RNA-protein complex that includes the RNA-binding proteins Larp1, PABP, and Ago2. This complex would prevent translation by blocking eIF4E interactions required for initiation. These results, together with prior network analysis in human cells, suggest that Bin3 interacts with multiple partner proteins, methylates small non-coding RNAs, and plays diverse roles in development (Singh, 2011).

The human homolog of Bin3, also called BCDIN3 or methylphosphate capping enzyme (MePCE), was shown to methylate the 5' γ-phosphate on 7SK RNA and to stabilize 7SK RNA in cell culture. This study found that Bin3 associates with and stabilizes 7SK RNA in ovaries and embryos. And, as in human cells, Bin3 activity was specific for 7SK RNA and did not affect U3 RNA or another RNA pol III product, U6 RNA, both of which are methylated by distinct mechanisms. It seem likely, therefore, that Drosophila Bin3 has a similar biochemical activity to its human counterpart despite differing in size and sequence outside the AdoMet binding domain and the highly conserved Bin3-homology domain. Prior attempts to demonstrate protein-arginine methyltransferase activity of Bin3 were negative, consistent with Bin3 methylating RNA rather than protein. In Drosophila, there are two other Bin3-like genes, CG11342 and CG1239, but each is more divergent from the human BCDIN3 within the conserved motif architecture (24% and 39% identity, respectively) than Bin3. It is possible that CG1239, which is expressed in early embryos, could have partially overlapping functions with Bin3 that might contribute to the incomplete penetrance of the bin3 mutations (Singh, 2011).

Putative Bin3 orthologs containing the two conserved motifs are found in at least 70 eukaryotic organisms ranging from the yeast, Schizosaccharomyces pombe to humans, and including Caenorhabditis elegans, Arabidopsis thaliana, Xenopus laevis, and Danio rerio. It is not known what any of these genes do, with the possible exception of the zebrafish bin3 gene which was shown by morpholino knockdown to be important for anterior development and to display RNA splicing defects. Similar defects were sought in splicing of bicoid, caudal, eIF4E, d4EHP, and a control gene, taf1, known to show alternative splicing. No splicing defects were found using a sensitive qRT-PCR approach. It is possible that the splicing defects in zebrafish result from aberrant 5' capping of non-coding RNAs important for splicing (Singh, 2011).

Mammalian 7SK RNA has been studied extensively, but Drosophila 7SK RNA has only been annotated, and prior to this study has not been characterized. This study shows that 7SK RNA is highly expressed in ovaries and embryos and is regulated by Bin3 in a manner similar to that in humans (by BCDIN3). 7SK RNA can be coimmunoprecipitated with Bin3 and Bicoid and may work as a scaffold in translation repression. This is the first indication that 7SK RNA has a function apart from its role in the regulation of the pTEFb transcription elongation factor. While this study focused on Bicoid-dependent regulation, it is likely that 7SK RNA also functions in transcription elongation in other stages of development. Indeed, it was found that Drosophila 7SK RNA mutants showed larval lethality at later stages of development (Singh, 2011).

Bin3 seems to play no role in Bicoid's gene activation function, but instead is crucial for Bicoid-dependent repression of caudal mRNA. Bin3 seems to stabilize Bicoid at the caudal BRE via a mechanism that involves 7SK RNA. As suggested by genetic interaction data, the Bicoid/Bin3/7SK RNA complex may include Larp1, PABP, and Ago2, and target the eIF4E initiation factor (Singh, 2011).

La-related proteins are not restricted to control of transcription elongation. In C. elegans, a Larp1 homolog was shown to be important for downregulation of translation of mRNAs in the Ras-MAPK pathway and to localize to P-bodies, known sites of mRNA degradation, while in mammalian cells, LARP4B plays a stimulatory role in translation initiation. In Drosophila, it has been shown that Larp1 associates directly with PABP independent of RNA and double mutants show enhanced lethality, suggesting that Larp1 facilitates mRNA translation. It is not surprising, therefore, that genetic interactions were observed between bin3 and larp1, as well as with pAbp in the context of caudal translation regulation. Note that it is though that PABP (and Larp1) plays a negative role in translation initiation, as does PABP in the repression of msl-2 mRNA by Sex-lethal (Singh, 2011).

In human cells, BCDIN3 and LARP7 interact cooperatively with 7SK RNA forming a stable core complex that associates transiently with HEXIMS, hnRNPs and the P-TEFb elongation complex (see Drosophila Hexim). An emerging theme is that 7SK RNA serves as a scaffold for stable association of protein partners. In fact, there is evidence that 5' γ-methylation of 7SK RNA by BCDIN3 may occur co-transcriptionally, but that the modified RNA remains associated with both BCDIN3 and LARP7, which bind 7SK RNA cooperatively. It is proposed, therefore, that Bin3 and Larp1 are associated with 7SK RNA at the caudal BRE, but that 5'-methylation does not necessarily occur there. Consistent with the idea of cooperative binding to 7SK RNA, it was found that larp1 mutation enhanced the bin3 mutant phenotype (Singh, 2011).

Some of the phenotypes observed for bin3 mutants were also observed in mutants of the microRNA miR-184, including oogenesis defects and a cellularization defect. This was the rationale behind including ago2 in the genetic analysis. However, no effect was found of bin3 mutation on levels of several miRNAs, including miR-184, it was surprising to observe a genetic enhancement (albeit mild) of the bin3 phenotype when combined with an ago2 mutation. Ago2 has been shown to bind eIF4E and interfere with mRNA circularization mediated by PABP. However, this occurs in the context of the miRNA/RISC complex, so whether and how Ago2 participates in Bicoid-Bin3 repression is not clear, but it could potentially involve the 7SK RNA component (Singh, 2011).

Finally, no interaction was detected between bin3 and D4EHP, which encodes a previously identified partner of Bicoid important for repressing caudal translation. D4EHP interacts with Bicoid and is thought to directly bind the m7G cap of caudal mRNA, thereby displacing eIF4E and blocking all subsequent steps of initiation. Perhaps the Bin3 mechanism works redundantly with the D4EHP mechanism or perhaps Bin3 helps recruit D4EHP, and so that mutation of bin3 would preclude binding of D4EHP to the initiation complex. Thus, bin3 mutation would be epistatic to the D4EHPCP53 mutation. Further investigation will be needed to determine relationship between these two pathways (Singh, 2011).

Bin3 is unlikely to be a dedicated Bicoid interactor and probably has roles as an RNA methyltranferase in many distinct pathways throughout development. In adults, quantitative trait transcript analysis linked bin3 with sleep-wake cycling. While studying Bin3's role in embryonic patterning, strong oogenesis defects were observed, particularly in bin3 null mothers, although other allelic combinations also revealed similar defects, especially at 29°C. Specifically, bin3 loss-of-function mutants showed dorsalized egg shell phenotypes. Conversely, bin3 overepressing lines showed strong ventralized egg shell patterns that appear to result from a failure of the dorsal appendage primordium to resolve into two domains along dorsal midline. These defects are similar to those of early D-V patterning mutations in the grk pathway, and probably do not result from defects that occur in later during morphogenesis step (Singh, 2011).

bin3 loss-of-function mutants resembled mutations in capicua, squid, cup and fs(K10), among others, while bin3 overexpressing lines resembled grk and pAbp mutations. Interestingly, mechanisms for translation repression of unlocalized grk mRNA feature prominently in the D-V patterning pathway, with squid and cup playing a critical role in repression via interaction with eIF4E, and PABP55 being important for release of that repression. Staining of bin3 mutant ovaries revealed a delocalized signal for Gurken protein but not for grk mRNA. Given the role of Bin3 in translation regulation, and the egg shell phenotypes of bin3 mutations, it seems plausible that Bin3 plays a role in negative regulation of grk translation (Singh, 2011).

Results presented in this study show that Bin3 plays a critical role during both oogenesis and embryonic development. In embryos, Bin3 is required for Bicoid to establish the Caudal protein gradient. Bin3 binds 7SK RNA and likely works by methylating 7SK RNA and stabilizing a repressive complex that assembles on the Bicoid-response element in the 3' UTR of caudal mRNA. Bin3's role during oogenesis is less clear, but based on the observed eggshell phenotypes in bin3 mutants, and gurken expression, Bin3 could play a similar role to help ensure that grk mRNA is translated only in the anterior-dorsal region of the oocyte (Singh, 2011).

Squid, Cup, and PABP55B function together to regulate gurken translation in Drosophila

During Drosophila oogenesis, the proper localization of gurken (grk) mRNA and protein is required for the establishment of the dorsal–ventral axis of the egg and future embryo. Squid (Sqd) is an RNA-binding protein that is required for the correct localization and translational regulation of the grk message. Cup and polyA-binding protein (PABP) interact physically with Sqd and with each other in ovaries. cup mutants lay dorsalized eggs, enhance dorsalization of weak sqd alleles, and display defects in grk mRNA localization and Grk protein accumulation. In contrast, pAbp mutants lay ventralized eggs and enhance grk haploinsufficiency. PABP also interacts genetically and biochemically with Encore. These data predict a model in which Cup and Sqd mediate translational repression of unlocalized grk mRNA, and PABP and Enc facilitate translational activation of the message once it is fully localized to the dorsal–anterior region of the oocyte. These data also provide the first evidence of a link between the complex of commonly used trans-acting factors and Enc, a factor that is required for grk translation (Clouse, 2008).

This study has taken a direct approach to identify proteins that interact with Sqd protein in ovaries. Using an Sqd antibody, immunoprecipitations out of ovarian extracts were performed, proteins were isolated that specifically interacted with Sqd, and those proteins were identified by mass spectrometry. Four of the Sqd-interacting proteins were positively identified in the mass spectrometry analysis: Cup, PABP55B, Imp, and Hrb27C/Hrp48. The remaining bands were not identified with certainty. Imp and Hrb27C/Hrp48 are two factors that have previously been shown to be involved in RNA localization, and both Hrb27C/Hrp48 and Imp bind to grk mRNA. The identification of these two factors confirmed that the immunoprecipitation method could successfully identify functional Sqd interactors (Clouse, 2008).

One of the Sqd interactors identified in the mass spectrometry analysis was the novel 150-kDa protein Cup. cup mutants display egg chambers with nurse cell nuclear morphology defects and eggs with open chorions. Cup interacts with several factors known to be required for osk localization and translation, such as Exu, Yps, eIF4E, Me31B, and Bruno and independent studies have shown that osk mRNA is prematurely translated in cup mutants. Cup co-localizes with the cap-binding protein, eIF4E, and eIF4E is not properly localized to the oocyte posterior pole in cup mutants. Cup competes away eIF4G, another translation initiation factor, for binding to eIF4E, thereby repressing translation. Together, these data are consistent with the following model for Cup-mediated translational repression; Cup represses the translation of RNAs containing BREs through interactions with Bruno. In this complex, Cup binds directly to eIF4E and interferes with eIF4G binding to eIF4E. Because eIF4G binding to eIF4E is a prerequisite for translation initiation, Cup represses translation by blocking this interaction. Direct biochemical data supporting this model have recently been obtained (Chekulaeva, 2006). It is proposed that Cup represses grk translation by a similar mechanism prior to its localization to the dorsal–anterior of the oocyte (Clouse, 2008).

Cup activity is used by several transcript-specific factors to mediate translational repression of that RNA in a developmentally appropriate context. For instance, Cup is required to mediate the translational repression of the nanos (nos) transcript. Cup has been shown to interact with Nos protein and co-localizes with Nos in the germarium. cup and nos also interact genetically, as heterozygosity for cup suppresses nos-induced phenotypes in early oogenesis. Later in development, Cup binds to Smaug, a factor that specifically binds to nos RNA and is required for its translational repression in embryos. In this example, Cup is required for Smaug to interact with eIF4E and mediate nos repression. Consistent with this biochemical model, Smaug-mediated translational repression is less efficient in cup mutants (Clouse, 2008).

This study as shown that Cup is also required for grk translational repression. This contrasts with previous reports that grk expression is normal in cup mutants, but these earlier reports used relatively weak cup alleles and monitored Grk levels by immunofluorescence. In contrast, in this study alleles were used that allowed assessment of the eggshell phenotype in cup mutants, providing the most sensitive assay for defects in Grk levels. These analyses showed that the different cup alleles vary greatly in phenotypic strength and range of phenotypes (Clouse, 2008).

Using two different alleles of cup from two distinct genetic backgrounds, it was shown that cup mutants lay dorsalized eggs, display defects in Grk protein accumulation, and display less efficient grk mRNA localization. Furthermore, Cup interacts biochemically with Sqd and Hrb27C/Hrp48 in ovarian extracts. Finally, heterozygosity for cup is able to enhance the moderate dorsalization observed in weak allelic combinations of sqd. Together, these data strongly support a model in which Cup functions with Sqd and Hrb27C/Hrp48 to mediate the translational repression of the grk message (Clouse, 2008).

Once grk mRNA is properly localized to the future dorsal/anterior of the oocyte, translational control must be switched from repressive to promoting. In many cellular situations, this activation is accomplished by binding of PABP to polyA tails of transcripts. In fact, PABP55B contains four RNA-recognition motifs (RRMs) that directly bind to polyA tails. PABP55B also has a C-terminal polyA domain that is used for oligomerization of PABP55B on polyA tails. Once PABP55B is bound to RNA, it binds to eIF4G, and this interaction helps to increase the affinity of eIF4G for eIF4E. With this increased affinity, eIF4G is able to effectively compete with Cup for binding to eIF4E, and translation is able to begin (Clouse, 2008).

There are at least three polyA-binding proteins in the Drosophila genome (CG5119 at 55B, CG4612 at 60D, and CG2163 at 44B), which are predicted to function as general translation factors, so it is conceivable that PABP55B could regulate a subset of RNAs. CG2163 has also been designated as PABP2 and has been shown to have essential roles in germ line development and in early embryogenesis (Benoit, 2005). This study has shown that PABP55B mediates the translational activation of fully localized grk mRNA. Specifically, heterozygous pAbp55B mutants lay ventralized eggs in certain genetic combinations, and heterozygosity for pAbp55B also enhances the weakly ventralized phenotype of grk heterozygotes, consistent with a role in translational activation of grk (Clouse, 2008).

PABP55B binds to Enc in ovarian extracts, and that this interaction may be direct and not bridged by an RNA molecule. Furthermore, heterozygosity for pAbp55B is able to enhance the weakly ventralized phenotype of enc mutants raised at 25 °C. Taken together, the biochemical and genetic interactions suggest that PABP55B and Enc function together to mediate the translational activation of grk mRNA once it is localized to the dorsal–anterior of the oocyte (Clouse, 2008).

Previously, Enc has been shown to be required for activation of grk translation in mid-oogenesis. An effect on osk mRNA localization has also been previously observed in enc mutants, but it is unclear at what level this process is affected, or whether this effect is direct. In addition, Enc has been shown to interact with subunits of the proteasome early in oogenesis. Because of its large size and its ability to interact with several different proteins, Enc may play multiple roles during oogenesis. Considering the function of Enc in grk translational activation and its localization to the dorsal–anterior region of the oocyte, It is hypothesized that Enc could function as a scaffolding protein that helps to mediate the transition from translational repression to activation of grk mRNA (Clouse, 2008).

Cup functions with Sqd in a protein complex that mediates the translational repression of grk mRNA before it is properly localized. It is clear from the analysis of mutants such as spn-F and encore, in which mislocalized grk mRNA is translationally silent, that these two steps can be uncoupled. It is proposed that once the RNA has reached the future dorsal–anterior region of the oocyte, PABP, Sqd, and Enc facilitate the translational activation of grk mRNA, PABP is shown associating with the complex once it is fully localized; however, it is possible that PABP associates with the grk transport complex in an inactive form that is remodeled following its anchorage at the dorsal–anterior of the oocyte (Clouse, 2008).

Previous studies have shown that Bruno (Bru) binds directly to Cup protein and is required for the translational repression of osk. Bru binds to specific sequence elements in the osk 3′ UTR called Bruno Response Elements (BREs), and mutations in these BREs have been shown to reduce Bru binding and result in ectopic Osk accumulation in the oocyte. Similarly, Bru has also been shown to bind to grk mRNA and to Sqd protein. Overexpression of bru cDNA leads to ventralization of the eggshell, consistent with reduced Grk protein expression in the oocyte. Furthermore, disrupting bru expression in certain genetic contexts has been shown to result in excess Grk protein in the oocyte, consistent with Bru being required to mediate grk translational repression. In light of the results presented in this study, it is proposed that this phenotype is the result of Bru-mediated repression of grk translation by Cup (Clouse, 2008).

The mechanism of grk translation and the trans-acting factors required for translational control largely parallel the mechanism employed by osk RNA, so an important question to be answered is how these two different RNAs are differentially transported and translationally regulated in distinct parts of the oocyte at the appropriate stage in oogenesis. Since the same group of trans-acting factors is involved in the expression of both RNAs, the specificity could be provided by cis-acting sequences within the RNA molecules themselves that affect the activity of common trans-acting factors. Alternatively, RNA-specificity could be generated by as-yet unidentified trans-acting factors. Given that Enc functions in grk translational activation, but is not required for osk translational activation, it is possible that Enc is providing some degree of specificity to the commonly used machinery that mediates translational control of multiple, unrelated transcripts. Currently, Enc is the only factor known to function uniquely in the translational activation of grk mRNA, and these results provide the first evidence of a link between this factor and the general translational control machinery that is used by multiple RNAs in oogenesis (Clouse, 2008).

Ataxin-2 and its Drosophila homolog, ATX2, interact with PABP and physically assemble with polyribosomes

Mutations resulting in the expansion of a polyglutamine tract in the protein ataxin-2 give rise to the neurodegenerative disorders spinocerebellar ataxia type 2 and Parkinson's disease. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration are unknown. This study demonstrates that ataxin-2 and its Drosophila homolog, ATX2, assemble with polyribosomes and poly(A)-binding protein (PABP), a key regulator of mRNA translation. The assembly of ATX2 with polyribosomes is mediated independently by two distinct evolutionarily conserved regions of ATX2: an N-terminal Lsm/Lsm-associated domain (LsmAD), found in proteins that function in nuclear RNA processing and mRNA decay, and a PAM2 motif, found in proteins that interact physically with PABP. The PAM2 motif mediates a physical interaction of ATX2 with PABP in addition to promoting ATX2 assembly with polyribosomes. These results suggest a model in which ATX2 binds mRNA directly through its Lsm/LsmAD domain and indirectly via binding PABP that is itself directly bound to mRNA. These findings, coupled with work on other ataxin-2 family members, suggest that ATX2 plays a direct role in translational regulation. These results raise the possibility that polyglutamine expansions within ataxin-2 cause neurodegeneration by interfering with the translational regulation of particular mRNAs (Satterfield, 2006; full text of article).

Given the evidence supporting a role for ataxin-2 in translational regulation, the question arises as to the mechanism by which ataxin-2 imposes this regulation. One possibility is that ataxin-2 directly influences the activity of PABP. PABP promotes translation by facilitating the interaction between the 5' and 3' ends of the mRNA, a process thought to promote the re-initiation of translation of terminating ribosomes (Kahvejian, 2001). PABP accomplishes this task by simultaneously binding to the poly(A) tail and to the PAM2 motif of eIF4G, a component of the 5' cap-binding translation initiation complex (see A model depicting how ATX2 might influence translation). Another PAM2 protein, Paip1, mimics the activity of eIF4G by simultaneously binding poly(A)-bound PABP and eIF4A, another component of the 5' cap-binding translation initiation complex. In contrast to eIF4G and Paip1, another PAM2 protein, Paip2, inhibits translation. Paip2 accomplishes this task by binding PABP and preventing its assembly onto the poly(A) tail (Kahvejian, 2001; Khaleghpour, 2001a; Roy, 2006). The finding that ATX2 is capable of assembling with poly(A)-bound dPABP indicates that, unlike Paip2, ATX2 does not prevent dPABP from assembling with the poly(A) tail. Assuming that ATX2 influences dPABP activity, it appears to do so while dPABP is assembled with the poly(A) tail, possibly by promoting or preventing the interaction between dPABP and the 5' cap-binding translation initiation complex. Further work will be required to elucidate the functional significance of the ATX2–dPABP interaction (Satterfield, 2006).

Although previous evidence indicates that ataxin-2 family members interact functionally with PABP, several observations indicate that ataxin-2 does not act solely through PABP. For example, in yeast, Pbp1 deletions suppress the lethality caused by deletion of Pab1, indicating that Pbp1p can perform a functional role in the complete absence of Pab1p (Mangus, 1998). Moreover, an ATX2 transgenic construct that encodes a protein lacking the PAM2 motif significantly extends the lethal phase of ATX2 mutant flies. Although ATX2 null mutants do not develop beyond the second instar larval stage, these mutants can be rescued to the adult stage of development using a wild-type ATX2 transgene. Use of ATX2 transgenes bearing a PAM2 deletion can also extend the lethal phase of ATX2 mutants to the pupal stage of development, although none of the partially rescued offspring survives to the adult stage of development. Given that the PAM2 motif is required for ATX2 to interact with dPABP, this observation indicates that ATX2 possesses a biological activity that is independent of its physical interaction with dPABP. The finding that the Lsm/LsmAD domain of ATX2 is capable of promoting its assembly with polyribosomes independently of the PAM2 motif, together with the observation that the Lsm/LsmAD domain represents the only other evolutionarily conserved sequence in ATX2, suggests that this domain is the source of the dPABP-independent activity of ATX2 (Satterfield, 2006).

Assuming that the Lsm/LsmAD domain is responsible for the dPABP-independent activity of ATX2 and that this activity serves a translational regulatory role, the question arises as to the precise mechanism by which this domain regulates translation. Although eukaryotic Lsm and the related Sm proteins are not currently known to regulate translation, one well-studied bacterial Sm protein, Hfq, does appear to regulate translation. Hfq functions as an RNA chaperone and regulates translation by stabilizing basepairing interactions between small non-coding RNAs (sRNAs) and their mRNA targets. These sRNA–mRNA interactions influence translation by altering the physical structure of the mRNA target. Although only limited sequence homology exists between Hfq and other Sm and Lsm proteins, the crystal structures of Staphylococcus aureus Hfq and eukaryotic Sm proteins are nearly identical, indicating that these proteins may function in a similar fashion. Furthermore, studies of eukaryotic Sm and Lsm proteins suggest that these proteins also function by mediating RNA–RNA interactions. The structural similarity of Sm and Lsm proteins raises the possibility that the Lsm/LsmAD domain of ATX2 might function, like Hfq, to regulate translation by mediating RNA–RNA interactions. An attractive potential target of ataxin-2 regulation in eukaryotes is the group of sRNAs known as microRNAs. MicroRNAs are known to play translational regulatory roles by basepairing with particular target mRNAs on polyribosomes. Studies are currently underway to investigate this possible mode of ATX2 regulation (Satterfield, 2006).

Previous work on several other polyglutamine disorders indicates that pathogenesis results from a reduction in transcriptional efficiency. Although the cytoplasmic localization of ataxin-2 indicates that this protein does not directly influence transcription, the finding that ATX2 physically assembles with polyribosomes, coupled with other work on ataxin-2 homologs, raises the possibility of a conserved mechanism of polyglutamine pathogenesis involving dysfunctional gene expression. In contrast to the transcriptional alterations associated with other polyglutamine diseases, polyglutamine expansions within ataxin-2 may adversely affect gene expression by impairing translation. Although polyglutamine expansion of human ataxin-2 does not detectably influence the binding of ataxin-2 to polyribosomes, it remains conceivable that polyglutamine expansions within ataxin-2 influence a function of ataxin-2 in translational regulation that lies downstream of polyribosome binding. The finding that polyglutamine expansions within ataxin-2 also cause a heritable form of Parkinsonism further suggests that altered translational regulation of particular targets might trigger the degeneration of dopaminergic neurons in the substantia nigra. As increasing evidence indicates that an overabundance of the protein alpha-synuclein plays an important role in the pathogenesis of Parkinson's disease, the current findings raise the interesting possibility that polyglutamine expansions within ataxin-2 lead to increased translation of alpha-synuclein. Future studies aimed at a better understanding of ataxin-2 function and the effects of polyglutamine expansions on that function will be required to directly address the hypothesis that translational dysregulation underlies ataxin-2-mediated neurodegeneration (Satterfield, 2006).

Polyglutamine genes interact to modulate the severity and progression of neurodegeneration in Drosophila

The expansion of polyglutamine tracts in a variety of proteins causes devastating, dominantly inherited neurodegenerative diseases, including six forms of spinal cerebellar ataxia (SCA). Although a polyglutamine expansion encoded in a single allele of each of the responsible genes is sufficient for the onset of each disease, clinical observations suggest that interactions between these genes may affect disease progression. In a screen for modifiers of neurodegeneration due to SCA3 in Drosophila, atx2, the fly ortholog of the human gene that causes a related ataxia, SCA2, was isolated. The normal activity of Ataxin-2 (Atx2), also called Sca2 in the literature, is critical for SCA3 degeneration, and Atx2 activity hastens the onset of nuclear inclusions associated with SCA3. These activities depend on a conserved protein interaction domain of Atx2, the PAM2 motif, which mediates binding of cytoplasmic poly(A)-binding protein (PABP). PABP also influences SCA3-associated neurodegeneration. These studies indicate that the toxicity of one polyglutamine disease protein can be dramatically modulated by the normal activity of another. It is proposed that functional links between these genes are critical to disease severity and progression, such that therapeutics for one disease may be applicable to others (Lessing, 2008; full text of article).

PABP is the only known protein to date that interacts directly with Atx2 through the PAM2 motif (Kozlov, 2001; Ralser, 2005; Satterfield, 2006); therefore, given the important role of the PAM2 motif, it was asked if PABP played a role in SCA3 neurodegeneration. Heterozygosity for the available pabp allele had no effect on Atx3 toxicity, although this allele is unlikely to be a complete loss of function (Sigrist, 2000). A deletion chromosome that removed the pabp gene was tested, comparing to appropriate control lines. Flies expressing pathogenic Atx3 that were heterozygous for this deletion showed dramatically enhanced photoreceptor loss. Control experiments confirmed that the deletion alone, in the absence of pathogenic Atx3, did not cause neurodegeneration. In contrast to the loss-of-function situation, overexpression of PABP significantly suppressed neurodegeneration. These observations indicated that PABP has the opposite activity as Atx2 with respect to Atx3-dependent neurodegeneration: whereas Atx2 enhances the toxicity of Atx3, PABP is protective (Lessing, 2008).

Whether PABP could modulate the degeneration induced by strong expression of Atx2 was tested. Decreased PABP function enhanced Atx2-dependent photoreceptor loss; likewise, up-regulation of PABP protected against photoreceptor degeneration. These studies suggest that the toxicity of Atx2 is mitigated by physical association with PABP, and they are consistent with PABP also playing a crucial role in the Atx2-Atx3 interaction. Together with results demonstrating the crucial role of the PAM2 motif, these data highlight the importance of the normal biological activity of Atx2 and of PABP in modulating the toxicity of pathogenic Atx3 (Lessing, 2008).

Thus the toxicity of pathogenic human Atx3 is critically dependent on Atx2 activity. Reduction of endogenous Atx2 function mitigated Atx3-induced neurodegeneration, and up-regulation of Atx2 synergistically enhanced degeneration. This study also revealed the roles in neural integrity played by the non-polyglutamine PAM2 motif of Atx2 and by PABP, which binds to Atx2 via this motif. These data are consistent with and expand upon clinical findings suggesting interactions between Atx2 and Atx3 in human disease. In the fly, endogenous Atx2 colocalized with pathogenic Atx3 in inclusions, as seen in human patients, with up-regulation of Atx2 enhancing Atx3 toxicity concomitant with a faster onset of inclusions and of SDS-insoluble complexes. These findings suggest that therapeutic approaches to modulate Atx2 activity may be effective against multiple disease situations, including SCA2 and SCA3 (Lessing, 2008).

Interestingly, normal Atx2 is toxic, causing degeneration when up-regulated. Previous animal models have demonstrated that normal protein products associated with SCA1 and Parkinson's disease - Ataxin-1 (see Drosophila Ataxin-1) and alpha-Synuclein, respectively - are also toxic when expressed at sufficiently high levels. Expansion of the polyglutamine domain in Ataxin-1 or Parkinson disease-associated missense mutations of alpha-Synuclein presumably lead to increased levels of the respective proteins, sufficiently high to elicit disease. Up-regulation of Drosophila Atx2 may cause degeneration for similar reasons. These studies further reveal that neuronal toxicity of Atx2 depends on its PAM2 motif - an observation with an interesting parallel to Ataxin-1, the protein that causes SCA1: an expanded polyglutamine repeat in Ataxin-1 is not sufficient to cause neurodegeneration in mouse models for SCA1, but rather pathogenic Ataxin-1 also requires its AXH domain to cause disease (Lessing, 2008).

The importance of the PAM2 motif for Atx2's toxicity and for the enhancement of Atx3 toxicity suggests a clue to the mechanism of the interaction. The PAM2 motif has been shown to bind specifically to the PABC domain, with PABP being currently the only known PABC-containing protein that interacts with Atx2. PABP is a ubiquitously expressed and essential protein that binds to the polyadenylated tails of mRNAs and is required for their translation. Furthermore, biochemical and genetic data support an interaction between Atx2 and PABP across many species (Ciosk, 2004; Satterfield, 2006; Mangus, 1998). Data from C. elegans indicate that loss of Atx2 can result in misregulated translation, and in yeast Atx2 negatively regulates PABP. Consistent with these findings, this study has shown that Atx2 and PABP have opposing activities in modulating the progression of SCA3 toxicity in flies (Lessing, 2008).

Protein interaction studies indicate that Atx2 and Atx3 do not interact directly; in a survey of the interaction network of ataxia-associated proteins, Atx2 and Atx3 were separated by four nodes. However, the known function of PABP and the role of the PAM2 motif in localizing Atx2 to polyribosomes (Satterfield, 2006) together indicate that Atx2 and PABP modulate translation of specific transcripts. Since Atx2 is sufficient to cause neurodegeneration in the absence of pathogenic Atx3, Atx3 mRNAs cannot be the sole target of Atx2-PABP interactions, and additional transcript targets must be critical to normal neuronal integrity (Lessing, 2008).

Experiments in Drosophila demonstrate that the fly provides an outstanding complement to clinical observations and to vertebrate disease models. In this case, the fly has highlighted the significance of intriguing interactions between the genes that cause SCA2 and SCA3 diseases that can be supported by molecular and genetic findings. More specifically, these data indicate striking crosstalk between the pathways of normal Atx2 function and pathogenic Atx3 activity. Further understanding of both the Atx2 and Atx3 pathways may reveal insight into maintenance of neuronal integrity in a number of distinct disease situations (Lessing, 2008).

Postsynaptic translation affects the efficacy and morphology of neuromuscular junctions

Long-term synaptic plasticity may be associated with structural rearrangements within the neuronal circuitry. Although the molecular mechanisms governing such activity-controlled morphological alterations are mostly elusive, polysomal accumulations at the base of developing dendritic spines and the activity-induced synthesis of synaptic components suggest that localized translation is involved during synaptic plasticity. This study shows that large aggregates of translational components as well as messenger RNA of the postsynaptic glutamate receptor subunit DGluR-IIA are localized within subsynaptic compartments of larval neuromuscular junctions of Drosophila. Genetic models of junctional plasticity and genetic manipulations using the translation initiation factors eIF4E and poly(A)-binding protein showed an increased occurrence of subsynaptic translation aggregates. This was associated with a significant increase in the postsynaptic DGluR-IIA protein levels and a reduction in the junctional expression of the cell-adhesion molecule Fasciclin II. In addition, the efficacy of junctional neurotransmission and the size of larval neuromuscular junctions were significantly increased. These results therefore provide evidence for a postsynaptic translational control of long-term junctional plasticity (Sigrist, 2000).

Translational control is primarily exerted by regulation of the initiation step of translation, which appears to be controlled by the rate-limiting initiation factor eIF4E. In addition, the interaction of the 5' cap bound eIF4E with the 3' end of mRNAs through a complex of other initiation factors and the poly(A)-binding protein (PABP) has been shown to synergistically facilitate translation initiation. To assess the potential role of regulated translation during the development of the larval neuromuscular junctions (NMJs) in Drosophila, the subcellular expression pattern of eIF4E and PABP were analyzed in filet preparations of third instar larvae. Both antigens showed a weak and ubiquitous expression in the cytoplasm of all larval cells, and they colocalized in strongly immunopositive aggregates up to 2microm in length close to NMJs. The specific localization of eIF4E/PABP aggregates close to and partially overlapping with junctional profiles revealed that eIF4E/PABP aggregates are positioned subsynaptically within or adjacent to the subsynaptic reticulum (SSR). No evidence was found for presynaptic or axonal localization of such aggregates. Therefore, the almost exclusive subsynaptic distribution of the eIF4E/PABP aggregates within larval muscles indicates that there may be a functional relationship between NMJs and the appearance of nearby eIF4E/PABP aggregates (Sigrist, 2000).

Ultrastructural examinations of larval NMJs revealed polysomal accumulations within and close to the SSR. According to their variable size, subsynaptic location and frequency of detection, the larger of these polysomal clusters are likely to represent the eIF4E/PABP aggregates detected by light microscopy. In addition, smaller polysomal aggregates were widely distributed in discrete membranous compartments throughout the SSR, whereas presynaptic and axonal profiles were free of polysomes. It is therefore concluded that mRNAs are translated within subsynaptic compartments of larval NMJs and that local centres of concentrated, subsynaptic translation are identified by large junctional eIF4E/PABP aggregates (Sigrist, 2000).

To assess whether junctional translation is subject to regulation, the number was quantified of synaptic specializations (boutons) per NMJ that were labelled by one or more translation aggregates. Animals that overexpressed PABP in larval muscles and larvae that were mutant in pabp showed a significantly increased occurrence of subsynaptic eIF4E/PABP aggregates and an unaltered level of muscular PABP staining. In addition, the total PABP levels in crude larval protein extracts were unaltered in all analysed genotypes, even when PABP mRNA levels were significantly increased or reduced under genetic gain-of-function or loss-of-function conditions, respectively. Such a homeostasis of total PABP levels is a well described phenomenon for PABP, and in crude protein extracts it might have masked the significant local increase in the number of PABP aggregates observable within subsynaptic compartments of NMJs. Although the exact reason for this increase in the occurrence of eIF4E/PABP aggregates is unknown, a local perturbation of PABP levels owing to a previously described overshooting compensation of the PABP-homeostasis mechanism might facilitate formation of subsynaptic translation aggregates (Sigrist, 2000).

A similar increase in the frequency of postsynaptic translation aggregates was also observed in two mutants representing well established genetic models of long-term synaptic plasticity in Drosophila, the hyperactive K+-channel mutant eag, Sh and the cAMP-phosphodiesterase mutant dunce. Thus, increased neuronal activity levels (in eag, Sh) as well as elevated cellular cAMP levels (in dunce) are capable of inducing subsynaptic translation aggregate formation. These findings are consistent with the hypothesis that synaptic activity can control synaptic translation (Sigrist, 2000).

To identify potential substrates and targets of subsynaptic translation at larval NMJs, quantitative immunostainings were performed of several junctionally expressed proteins, including the synaptic vesicle protein synaptotagmin, the junctional anti-horseradish peroxidase (HRP) epitope, the cell-adhesion molecule Fasciclin II (FasII), the postsynaptic glutamate receptor subunit DGluR-IIA and the conventional myosin as a nonsynaptic protein. No obvious differences were detected in the expression levels of myosin, synaptotagmin and the junctional anti-HRP immunoreactivity in all analysed genotypes; however, animals that showed elevated numbers of subsynaptic translation aggregates consistently displayed increased junctional levels of DGluR-IIA and an altered junctional distribution of FasII, which was associated with a significant reduction of synaptic FasII levels as compared with control animals. A similar FasII phenotype has been described in the plasticity models eag, Sh and dunce, and it has been shown that presynaptic FasII downregulation is essential for increased junctional outgrowth. Intriguingly, in Aplysia the FasII homologue apCAM is also presynaptically downregulated after treatments that increase synaptic efficacy and growth of new synaptic connections. This synaptic apCAM regulation is thought to be achieved by a protein-synthesis-dependent activation of an endocytic apCAM internalization. Given that FasII has been detected in membranes of a subset of presynaptic vesicles, it seems possible that subsynaptic protein synthesis affects junctional FasII levels through similar mechanisms to those in Aplysia (Sigrist, 2000).

The postsynaptic DGluR-IIA immunoreactivities were significantly stronger in translationally sensitized animals. This strong increase of synaptic DGluR-IIA expression was not due to transcriptional upregulation of dglur-IIA; the total amounts of DGluR-IIA mRNAs were unaltered or even reduced in the analysed genotypes as compared with controls. In situ hybridization experiments revealed that DGluR-IIA mRNA surrounds individual type-I boutons, with prominent staining of terminal and branch-point boutons and weak or absent staining within the SSR of interbouton connectives. Thus, the subsynaptically localized DGluR-IIA mRNA represents a direct substrate for the junctional translation machinery. These results can not exclude an extrajunctional contribution to the observed synaptic DGluR-IIA increase, but they suggest that this phenotype is due to an increased subsynaptic synthesis of DGluR-IIA in genotypes with a higher occurrence of junctional eIF4E/PABP aggregates (Sigrist, 2000).

To analyse the functional consequences of genetically modified subsynaptic translation, the strength of neurotransmission at NMJs was assessed on muscle 6 of third instar larvae. The average amplitudes of miniature excitatory junctional currents (mEJCs) and thus the quantal sizes were indistinguishable in all analysed genotypes. This finding indicates either that the additional receptor subunits that are synaptically localized may be functionally silent (for example, through physiological silencing or intracellular localization or that the amount of glutamate released from an individual quantum is not sufficient to saturate the postsynaptic receptors. In contrast, postsynaptic responses evoked by stimulation of motor nerve axons were substantially larger in all mutants exhibiting increased levels of subsynaptic translation. Thus, the derived quantal content was significantly increased above control values, suggesting that the observed larger amplitudes of evoked junctional responses arise from an increased number of released presynaptic vesicles per action potential (Sigrist, 2000).

To investigate whether the increase in junctional efficacy was due to a change in the number of synaptic specializations, the number of junctional boutons per NMJ was quantified. Genotypes that displayed an increased occurrence of subsynaptic translation aggregates had significantly larger NMJs and reduced junctional FasII levels. In addition, the junctional sizes of the analysed animals correlated in a highly significant manner with their estimated quantal contents, suggesting that junctional efficacy and the morphological elaboration of NMJs are tightly coupled. On the basis of light microscopic examinations of DGluR-IIA labelled NMJs, the density of synapses within NMJs of all mutant animals appeared similar to that of controls or even higher, indicating that the total number of synapses increased proportionally with the junctional size. This finding indicates that the increased quantal content in animals with facilitated subsynaptic translation may be because of an increase in the number of vesicle release sites per given stimulus (Sigrist, 2000).

In summary, this study has shown that translational machinery and mRNAs are associated with the subsynaptic reticulum of NMJs and that genetic manipulations that affect the occurrence of subsynaptic translation aggregates are accompanied by changes in the levels of synaptic proteins, such as DGluR-IIA and FasII. These same manipulations also affected the function and morphology of NMJs, suggesting that subsynaptic translation can instruct junctional growth and synaptic reorganization and thereby long-term functional changes. These results further suggest that subsynaptic translation can be regulated by altered levels of neuronal activity, indicating that the regulation of postsynaptic translation participates in activity-dependent junctional plasticity. Thus, the inducible recruitment of postsynaptic protein synthesis appears to render individual synapses competent to instruct long-term changes in their functions and morphological organization. Given that localized protein synthesis has been shown to act in a synapse specific stabilization of long-term facilitation in central neurons of Aplysia, it emerges that synaptic translation might represent a common principle of long-term alterations of neuronal function and connectivity (Sigrist, 2000).


REFERENCES

Search PubMed for articles about Drosophila PABP

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Biological Overview

date revised: 15 July 2015

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