InteractiveFly: GeneBrief

eukaryotic translation initiation factor 4A: Biological Overview | References

Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, the requirements for translation initiation factors during dendrite pruning were assessed. The canonical cap-binding complex eIF4F was found to be dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. It is proposed that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes (Rode, 2018).

Pruning, the developmentally controlled degeneration of synapses and neurites without loss of the parent neuron, is an important mechanism used to specify neuronal connections or to remove developmental intermediates. In holometabolous insects like Drosophila, the nervous system is remodeled during metamorphosis in response to the steroid hormone ecdysone. In the peripheral nervous system (PNS), the sensory class IV dendritic arborization (c4da) neurons completely prune their long and branched larval dendrites at the onset of the pupal phase, while their axons stay intact. C4da neuron dendrite pruning involves the specific destabilization of the dendritic cytoskeleton and plasma membrane and phagocytosis of severed dendrites by surrounding epidermal cells (Rode, 2018).

Ecdysone induces c4da neuron dendrite pruning through the hormone receptors EcR-B1 and ultraspiracle (Usp), which activate the transcription of pruning genes. Among these are headcase, a pruning gene of unknown function, and SOX14, an HMG box transcription factor that activates transcription of MICAL, encoding an actin-severing enzyme. Regulation of MICAL expression also involves the ubiquitin-proteasome system at a posttranscriptional level (Rode, 2018).

In addition to transcriptional activation of target genes, several lines of evidence suggest that ecdysone also regulates global translation rates through activation of the translation inhibitor eukaryotic initiation factor 4E-binding protein (4E-BP). In the Drosophila fat body, this occurs transcriptionally through FOXO, while in c4 da neurons, ecdysone inhibits the insulin and Target of Rapamycin (TOR) pathway to activate 4E-BP posttranslationally (Rode, 2018).

4E-BP inhibits translation initiation, the rate-limiting step of protein synthesis, by sequestering the cap-binding protein eIF4E. During canonical translation initiation, eIF4E binds to the 7-methylguanosine (m7Gppp) cap of eukaryotic mRNAs and then forms the so-called eIF4F complex by recruiting eIF4G, an adaptor that binds the 43S preinitiation complex (PIC), containing the 40S small ribosomal subunit, and the helicase eIF4A, which is thought to resolve hairpin structures in the 5' UTRs of mRNAs. This enables the 43S complex to scan 5' UTRs for the initiation codon, where it is joined by the large ribosomal subunit and translation can start. While eIF4A's role has been mainly linked to 5' UTR hairpins, it can also stimulate translation of mRNAs with unstructured 5' UTRs. Moreover, eIF4A is more abundant than eIF4E, suggesting that it has functions beyond the eIF4F complex (Rode, 2018).

Activated 4E-BP binds to eIF4E and prevents eIF4F assembly, thus inhibiting ribosome recruitment to mRNAs and globally dampening translation rates under stress or during development. Interestingly, 4E-BP affects translation of some mRNAs more than others. To explain this, eIF4E-independent translation initiation mechanisms have been proposed. One such mechanism could depend on internal ribosome entry sites (IRESs) that bypass the requirement for the m7Gppp cap. For example, the mRNAs of the Drosophila cell death factors reaper and hid may contain IRES sequences in their 5' UTRs that allow them to be translated under stress (Rode, 2018).

Alternative cap recognition mechanisms have also been proposed under conditions of high 4E-BP activity. In particular, the initiation factor eIF3, a 13-subunit complex, could provide a mechanism for eIF4E-independent initiation (Lee, 2015; Lee, 2016). It binds to the small ribosomal subunit as part of the 43S PIC, and it is thought to act downstream of eIF4G in mRNA recruitment. However, eIF3 dependence varies between mRNAs, and eIF3 can even suppress translation of some targets (Lee, 2015). Importantly, it was recently shown that translation of some 4E-BP-resistant mRNAs depends on an eIF3-based cap recognition activity in the eIF3d subunit that is stimulated by hairpin motifs in the 5' UTR (Lee, 2016). Other eIF3 subunits have also been shown to interact with the cap (Rode, 2018).

Given that ecdysone inhibits eIF4E-dependent translation, this study asked whether there are mechanisms that ensure the translation of ecdysone target mRNAs. To this end, the requirements for translation initiation factors during c4da neuron dendrite pruning was assessed. The canonical eIF4F components eIF4E and eIF4G were found not to be required for c4da neuron dendrite pruning, while the helicase eIF4A and the eIF3 complex are. Both eIF4A and eIF3 are required for Mical expression, and this specificity is conferred by the 5' UTR of Mical mRNA. Further biochemical analyses suggest that eIF4A regulates the interaction between eIF3 and the Mical 5' UTR. It is proposed that eIF4A/eIF3 constitute a 4E-BP bypass mechanism that ensures the adequate translation of ecdysone-induced genes in c4da neurons (Rode, 2018).

Developmental control of translation rate is required under various conditions. One well-characterized regulatory mechanism is through 4E-BP, which inhibits assembly of the cap-binding eIF4F complex. Despite the obvious need for global translation control during development, it is also clear that there must be exceptions to such regulation. Several lines of evidence suggest that global, eIF4E-dependent translation is downregulated by ecdysone during the pupal phase and that this is important for c4da neuron dendrite pruning. How downregulation of eIF4E-dependent translation contributes to dendrite pruning is not clear. TOR activity (and hence eIF4E-dependent translation) is associated with neurite regrowth after pruning in a Drosophila model for neuronal remodeling, and beta-actin mRNA was identified as a 4E-BP target in vertebrate neurons. General suppression of eIF4E-dependent translation may, therefore, serve to prevent precocious neurite growth or neurite stabilization through increased actin polymerization (Rode, 2018).

Despite the need for translation downregulation during dendrite pruning, ecdysone-induced mRNAs must still be efficiently translated. This study found that c4 da neuron dendrite pruning does not depend on the eIF4F subunits eIF4E and eIF4G, but instead on eIF3 and eIF4A. In keeping with a specific effect on dendrite-pruning genes, Mical mRNA was identified as the crucial target for eIF3 and eIF4A. The data suggest that this specificity is encoded in the 5' UTR of Mical mRNA, as a UAS-GFP reporter containing the Mical 5' UTR showed consistently stronger dependence on eIF4A and eIF3 than a regular UAS-GFP reporter. The important role of the Mical 5' UTR is also supported by the observation that Sox14 overexpression (which induces endogenous Mical mRNA) did not rescue the pruning defects induced by eIF4A RNAi, while overexpression of Mical from a UAS transgene (and thus lacking the endogenous 5' UTR) did. It is tempting to speculate that eIF3-eIF4A recognition signals may be abundant in 5' UTRs of ecdysone-induced genes (Rode, 2018).

Several lines of evidence indicate that translation initiation of pupal pruning factors in c4 da neurons is still cap dependent: for one, overexpression of a cap-binding-deficient eIF3d mutant causes dominant dendrite-pruning defects, and in vitro translation of a 5' UTRMical reporter mRNA depends on a functional cap. While physical interactions were observed between eIF3 and a 5' UTR Mical reporter mRNA in S2 cells, cap binding by eIF3 could not be directly demonstrated in vivo. eIF3 does not bind to the isolated cap structure, and a biochemical cap-binding assay for eIF3 would require crosslinking eIF3 with a purified mRNA with a radioactively labeled cap. To further investigate developmental control of translation initiation in the future, it would be interesting to set up such an assay to address whether the Mical mRNA cap is also recognized via eIF3d or another eIF3 subunit (Rode, 2018).

Sox14 expression seemed resistant to the inhibition of either eIF4E or eIF3, but this study found that these pathways can mediate Sox14 expression in a redundant fashion. Sox14 is upstream of the Cul-1 ubiquitin ligase that activates 4E-BP in c4da neurons. Its mRNA may be adapted to this position in the pruning pathway, as it could still use the regular eIF4F pathway early during the pupal phase and the eIF3 pathway later. Mical translation may only start when 4E-BP activity is already high, hence explaining its strong eIF3 dependence (Rode, 2018).

Translation of long mRNAs is sensitive to the eIF4A cofactor eIF4B (Sen, 2016), and eIF4A dependence is also in part conferred by sequences in the coding region. eIF4B manipulation did not cause dendrite pruning defects, but the Mical construct used to rescue the pruning defects induced by eIF4A knockdown lacks an internal region non-essential for pruning. It is, therefore, possible that internal regions of the long Mical mRNA also contribute to its dependence on eIF4A (Rode, 2018).

The strong similarities between the phenotypes caused by the manipulation of eIF4A and eIF3 suggested that these two factors cooperate functionally. eIF4A and eIF3 can be found in an eIF4A ATPase-dependent complex and that eIF4A clamping on the mRNA prevents eIF3 release from a 5' UTRMical reporter mRNA. Two recent in vitro studies found functional interactions between eIF4A and eIF3 in the context of canonical eIF4F-dependent translation initiation (Yourik, 2017, Sokabe, 2017): first, eIF3 stimulates eIF4A ATPase activity via its eIF3g subunit to promote PIC maturation (Yourik, 2017); and, second, eIF4A ATPase activity was required to reposition the eIF3j subunit within the PIC during maturation (Sokabe and Fraser, 2017). this study now demonstrate genetically that eIF4A has an eIF3-related function independently of eIF4F. These data showing that eIF3 and eIF4A interact in an ATPase-dependent manner and that eIF4A helicase activity is required for dendrite pruning are consistent with both the above proposals (Rode, 2018).

Taken together, these data suggest that eIF3-eIF4A are part of a bypass mechanism that ensures translation of crucial ecdysone-induced mRNAs in the absence of an eIF4E-dependent translation initiation during developmental neuronal remodeling in the Drosophila PNS (Rode, 2018).

eIF4A inactivates TORC1 in response to amino acid starvation inactivates TORC1 in response to amino acid starvation

Amino acids regulate TOR complex 1 (TORC1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC1 recruitment to lysosomes where TORC1 is activated by binding Rheb. How the absence of amino acids inactivates TORC1 is less well understood. Amino acid starvation recruits the TSC1/TSC2 complex to the vicinity of TORC1 to inhibit Rheb; however, the upstream mechanisms regulating TSC2 are not known. This study identified the the eIF4A-containing eIF4F translation initiation complex (composed of three subunits: eIF4E, eIF4A and eIF4G) as an upstream regulator of TSC2 in response to amino acid withdrawal in Drosophila. TORC1 and translation preinitiation complexes bind each other. Cells lacking eIF4F components retain elevated TORC1 activity upon amino acid removal. This effect is specific for eIF4F and not a general consequence of blocked translation. This study identifies specific components of the translation machinery as important mediators of TORC1 inactivation upon amino acid removal (Tsokanos, 2016).

To maintain homeostasis, biological systems frequently use a combination of two distinct mechanisms that converge and counteract each other. For instance, the level of phosphorylation of a target protein depends not only on the rate of phosphorylation by the upstream kinase, but also on the rate of dephosphorylation by the phosphatase. Both the activating kinase and the inactivating phosphatase can be regulated separately. Likewise, the activity of TORC1 in response to amino acid levels appears to reflect a balance between activating and inactivating mechanisms that converge on Rheb. When amino acids are re-added to cells, TORC1 is activated via Rag or Arf1 GTPase-dependent recruitment to the lysosome where TORC1 binds Rheb (Kim, 2008; Sancak, 2008). In contrast, when amino acids are removed from cells, TORC1 activity drops in part by blocking this activation mechanism and in part via a distinct inactivation mechanism whereby TSC2 is recruited to the vicinity of TORC1 to act on Rheb (Demetriades, 2014). The existence of this distinct and counteracting mechanism is highlighted by the fact that in the absence of TSC2, both Drosophila and mammalian cells do not appropriately inactivate TORC1 in response to amino acid removal (Demetriades, 2014). The upstream mechanisms regulating TSC2 in response to amino acid withdrawal, however, are not known. This study has identified the translational machinery, and in particular components of the eIF4F complex, as one upstream regulatory mechanism working via TSC2 to inactivate TORC1 upon amino acid withdrawal (Tsokanos, 2016).

The subcellular localization of TORC1 plays an important role in its regulation. A significant body of evidence shows that TORC1 needs to translocate to the lysosome or Golgi to become reactivated following amino acid starvation and re-addition. Whether active TORC1 then remains on the lysosome, or whether it can move elsewhere in the cell to phosphorylate target proteins, is less clear. Several findings in the literature, as well as the data presented in this study, indicate that active TORC1 can leave the lysosome, yet remain active: (1) Upon amino acid re-addition in starved cells, the Rag GTPases are necessary for mTORC1 lysosomal localization and reactivation. In contrast, Rag depletion in cells growing under basal conditions, replete of serum and amino acids, does not cause a strong drop in mTORC1 activity, although it causes a similar delocalization of mTORC1 away from lysosomes. Hence, under these conditions, mTORC1 is non-lysosomal, but still active to a large extent. (2) Similarly, particular stresses such as arsenite treatment can cause TORC1 to localize away from the lysosome, yet remain active. (3) The Rag GTPases tether TORC1 to the LAMTOR complex present on the lysosome. Amino acid restimulation, which activates TORC1, actually decreases binding between Rag GTPases and LAMTOR, suggesting that active Rag-bound TORC1 complexes can leave the lysosome and reside elsewhere in the cell. Additional mechanisms also contribute to the delocalization of the Rag GTPases away from lysosomes (4) Active TORC1 phosphorylates target proteins such as 4E-BP and S6K, which are physically associated with translation preinitiation complexes. Indeed, this study reports physical interactions between the TORC1 complex and translation preinitiation complexes, in agreement with what has also been observed by others. Therefore, either translation preinitiation complexes need to translocate to lysosomes to meet TORC1, or TORC1 needs to come off the lysosome to meet translation preinitiation complexes in the cytoplasm. (5) Using proximity ligation assay, an interaction was observed between Raptor and eIF4A, which does not colocalize with either lysosomes or endoplasmic reticulum, suggesting that it takes place in the cytoplasm. (6) In agreement with these PLA data, antibody staining of cells in the presence of amino acids with anti-TOR antibody reveals an accumulation of TOR on lysosomes, as well as a more diffuse, non-lysosomal TORC1 localization throughout the cytoplasm. (7) A recent report employing a FRET-based probe detects mTORC1 activity at lysosomes as well as in the cytoplasm and nucleus. Taken together, these data suggest that although TORC1 is activated on the lysosome, it then in part translocates to other sites in the cell including the cytoplasm to phosphorylate target proteins (Tsokanos, 2016).

Upon amino acid withdrawal, both cytoplasmic and lysosomal fractions of active TORC1 need to be inactivated. The data presented in this study suggest that upon amino acid removal, inactivation of TORC1 happens in part via an eIF4A-dependent mechanism acting on TSC2 to inactivate Rheb in the cytosol. In agreement with this, TORC1 inactivation upon amino acid removal can be rescued by supplying cells with dominantly active, but not wild-type Rheb. It has been previously reported that a pool of TSC2 is also recruited to lysosomes upon amino acid removal (Demetriades, 2014). This study shows in Drosophila cells, upon amino acid removal, some TSC2 accumulates in lysosomes, whereas some remains in the cytosol. Therefore, TSC2 is likely recruited to all subcellular sites where active TORC1 is located to inactivate it. Indeed, Rheb and TSC2 have been observed at several subcellular compartments. Since Rheb localizes to many endomembranes in the cell, Rheb that is not bound to TORC1 could potentially remain active, to provide a pool for subsequent TORC1 reactivation (Tsokanos, 2016).

Upon inactivation, the data indicate that TORC1 remains bound to preinitiation complexes, in agreement with previous reports. This finding is reminiscent of the fact that Raptor is also recruited to stress granules, which are essentially stalled preinitiation complexes, in response to another stress-oxidative stress. Whether the Rag GTPases also remain bound to preinitiation complexes upon amino acid removal is unclear because some experiments showed a decrease in binding between Rag GTPases and initiation factors, and some did not (Tsokanos, 2016).

How could eIF4A affect TORC1 activity? The data indicate that the effects of eIF4A knockdown cannot be explained as a consequence of generally impaired translation, since other means of blocking translation do not have the same effects on TORC1 activity upon amino acid starvation. Instead, knockdown of any of the three members of the eIF4F complex gives this elevated TORC1 phenotype, indicating that it is specific for the eIF4F complex. The data are consistent with two interpretations: One option is that the eIF4F complex is specifically required to translate a protein that promotes TSC2 function. An alternate option is that the eIF4F complex acts directly on TSC2, regulating its activity. The latter is supported by the fact that eIF4A and TSC2 proteins are seen interacting with each other. Interestingly, eIF4A has been reported to have additional functions that are not translation-related (Tsokanos, 2016).

Some differences were noted between Drosophila cells and mammalian cells. The first is that overexpression of wild-type Rheb is sufficient to activate TORC1 upon amino acid removal in mammalian cells, whereas this is not the case in Drosophila cells. This could be due to a difference in the biology of the two cell types, or simply to a technical difference having to do with levels of Rheb overexpression. A second difference is that cycloheximide treatment is sufficient to maintain elevated TORC1 levels in HeLa or HEK293 cells upon amino acid removal, whereas this is not the case in Drosophila cells. This could be due to differences in rates of amino acid efflux and levels of autophagy in mammalian compared to S2 and Kc167 cells, causing intracellular amino acid levels to remain elevated in mammalian cells when both amino acid import from the medium and amino acid expenditure via translation are simultaneously blocked (Tsokanos, 2016).

A number of studies have looked at the involvement of Rheb in the cellular response to amino acids, with some disagreement on whether amino acids affect Rheb GTP-loading or Rheb-mTOR binding. The current data fit with previous reports that Rheb GTP-loading is affected by amino acids and with the conclusion that amino acids affect TORC1 activity via both a Rheb-dependent and a Rheb-independent mechanism (Tsokanos, 2016).

The data indicate a close physical relationship between TORC1 and the translational machinery. This is in part mediated by a direct interaction between the major scaffolding subunit of the initiation complex, eIF4G, and RagC and in part likely mediated by additional interactions between TORC1 and preinitiation supercomplexes as previously reported. Interestingly, TORC2 is also physically associated with the ribosome and requires ribosomes, but not translation, for its activation. Hence, both TORC1 and TORC2 have close physical connections to the translational machinery (Tsokanos, 2016).

Some side observations in this study are interesting and could constitute a starting point for further studies. For instance, eIF4A-knockdown cells inactivate TORC1 more robustly than control cells upon serum removal. Also, eIF2b knockdown causes S6K phosphorylation to decrease significantly in S2 cells. It is not known why this occurs. The latter might suggest that there are additional points of cross-talk between TORC1 and the translation machinery (Tsokanos, 2016).

How cells sense the presence or the absence of amino acids has been an open question in the field. The data presented in this study indicate that the translational machinery itself might sense the absence of amino acids. Indeed, the relevant parameter for a cell is likely not the absolute levels of intracellular amino acids, but rather whether the available amino acid levels are sufficient to support the amount of translation that a cell requires. Hence, the translation machinery itself might be best poised to make this assessment. Binding is observed between eIF4A and NAT1 that is strong in the presence of amino acids, and is reduced upon amino acid withdrawal, independently of TORC1 signaling. These epistasis experiments are consistent with NAT1 acting as the upstream mediator of the amino acid signal, binding and inhibiting eIF4A in the presence of amino acids, but not in the absence of amino acids. Hence, NAT1 might play a role in this sensing process (Tsokanos, 2016).

In sum, these data identify the eIF4F complex as an important upstream regulator of TORC1, which acts via TSC2 to inactivate TORC1 upon withdrawal of amino acids (Tsokanos, 2016).

MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila

miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. Previous work has shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. This study examined the assembly of translation initiation factors on the target mRNA under repression. Ago1-RISC was shown to induce dissociation of eIF4A>, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block the assembly of the eIF4F complex during translation initiation (Fukaya, 2014).

MicroRNAs (miRNAs) silence their complementary target mRNAs via formation of the effector ribonucleoprotein complex called RNA-induced silencing complex (RISC). The core component of RISC is a member of the Argonaute (Ago) proteins. In Drosophila, miRNAs are sorted into two functionally distinct Ago proteins, Ago1 and Ago2, according to their structural features and the identity of the 5' end nucleotides. Compared to fly Ago2, fly Ago1 shares more common features with mammalian Ago1-4, making it a suitable model for investigating miRNA-mediated gene silencing in animals. Ago1-RISC mediates translational repression as well as shortening of the poly(A) tail followed by mRNA decay. While deadenylation per se disrupts the closed-loop configuration of mRNA and leads to inhibition of translation initiation, Ago1-RISC can repress translation independently of deadenylation. Such a deadenylation-independent 'pure' translational repression mechanism seems to be widely conserved among species (Fukaya, 2014).

Ago is not the only protein involved in the miRNA-mediated gene silencing pathway. In flies, a P-body protein GW182 specifically interacts with Ago1, but not with Ago2, through the N-terminal glycine/tryptophan (GW) repeats and provides a binding platform for PAN2-PAN3 and CCR4-NOT deadenylase complexes (Braun, 2011, Chekulaeva, 2011). This protein interaction network is conserved in animals including zebrafish, nematodes, and humans. Accordingly, GW182 is essential for shortening of the poly(A) tail by miRNAs. On the other hand, recent studies revealed that miRNA-mediated translational repression occurs in both GW182-dependent and -independent manners. Previous sedimentation analysis on sucrose density gradient suggested that both of the two translational repression mechanisms block recruitment of the ribosomal 43S preinitiation complex to the target mRNA independently of deadenylation (Fukaya, 2014).

In eukaryotes, recruitment of the 43S preinitiation complex is initiated by the formation of eukaryotic translation initiation factor 4F (eIF4F). eIF4F is a multiprotein complex composed of the cap-binding protein eIF4E, which recognizes the 7-methyl guanosine (m7G) structure of the capped mRNA; the scaffold protein eIF4G, which interacts with 40S ribosome-associated eIF3 and bridges the mRNA and the 43S preinitiation complex; and the DEAD-box RNA helicase eIF4A, which plays a pivotal role in translation initiation supposedly through unwinding the secondary structure of the 5' UTR for landing of the 43S complex. In addition, the poly(A)-binding protein PABP stimulates translation initiation through its direct interaction with eIF4G. miRNAs likely block one (or more) of these steps to repress translation initiation. It was recently proposed that, in mammals, preferential recruitment of eIF4AII-one of the two eIF4A paralogs-is required for miRNA-mediated translational repression (Meijer, 2013). This model postulates that eIF4AII acts to inhibit rather than activate translation, unlike its major counterpart eIF4AI. However, the role of eIF4AII in translation remains largely unexplored, as opposed to eIF4AI's well-established function to promote translation. Moreover, invertebrates have only one eIF4A, making this model incompatible in flies. Thus, it still remains unclear how miRNAs repress translation initiation. This is largely due to technical limitations in directly monitoring the assembly of the translation initiation complex specifically on the mRNA targeted by miRNAs (Fukaya, 2014).

By using site-specific UV crosslinking, this study examined the association of translation initiation factors on the target RNA under repression. Fly Ago1-RISC was shown to specifically induce dissociation of eIF4A from the target mRNA without affecting the 5' cap recognition by eIF4E in a manner independent of GW182 or PABP. On the other hand, direct tethering of GW182 to the target mRNA promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block assembly of the eIF4F complex during translation initiation, in addition to their established role in deadenylation and decay of their target mRNAs (Fukaya, 2014).

Thus fly Ago1-RISC induces dissociation of eIF4A without affecting the cap recognition by eIF4E. Although it was not possible to detect eIF4G via any of the crosslinking positions spanning from 2 nt to 13 nt downstream of the cap, it was previously shown that noncanonical translation driven by direct tethering of eIF4G to the 5' UTR was fully susceptible to translational repression by Ago1-RISC (Fukaya, 2012). Therefore, it was reasoned that Ago1-RISC directly targets eIF4A rather than eIF4E or eIF4G. In the accompanying paper, Fukao (2014) revealed that human Ago2-RISC specifically induces dissociation of eIF4A-both eIF4AI and eIF4AII-without affecting eIF4E or eIF4G in a cell-free system deriving from HEK293F cells. Thus, eIF4A is likely a target of miRNA action conserved among species. In agreement with this model, miRNA-mediated gene silencing is cancelled by the eIF4A inhibitors silvestrol, hippuristanol, or pateamine A in human cells (Fukaya, 2014).

GW182 is a well-known interactor of miRNA-associated Ago proteins and is a prerequisite for miRNA-mediated deadenylation/decay of target mRNAs. GW182 directly binds to both NOT1 and CAF40/CNOT9, thereby recruiting the CCR4-NOT deadenylase complex to the target mRNA. It has been suggested that the CCR4-NOT complex not only shortens the poly(A) tail but also plays a role in miRNA-mediated translational repression, because direct tethering of the CCR4-NOT complex was capable of inducing translational repression independently of deadenylation. It was originally proposed that, in humans, the CCR4-NOT complex specifically binds to eIF4AII (but not to eIF4AI) to repress translation. However, this model was challenged by recent studies showing that, although the MIFG4 domain of human CNOT1 structurally resembles the middle domain of eIF4G, it does not bind eIF4AI or II but instead partners with the DEAD-box RNA helicase DDX6, which has been implicated in repression of translation initiation and/or translation elongation as well as activation of decapping. Given that miRNAs mediate gene silencing via multiple different pathways, recruitment of DDX6 by GW182 via the CCR4-NOT complex may well play a role in inhibiting protein synthesis from miRNA targets. Indeed, this study observed strong dissociation of both eIF4E and eIF4A by direct tethering of GW182. However, at the physiological stoichiometry between Ago1 and GW182 in S2 cell lysate, eIF4A was specifically dissociated without apparent effect on eIF4E by canonical miRNA targeting, which is in agreement with the result of the reporter assay in S2 cells depleted of each eIF4F component. It is envisioned that, although GW182 is clearly essential for miRNA-mediated deadenylation, the degree of contribution of GW182 to translational repression can vary in different cell types and conditions, depending on the concentrations of GW182 and Ago proteins, as well as their protein interaction networks that are subject to regulation by extracellular signaling. In this regard, direct tethering of GW182 may potentially overestimate its role in miRNA-mediated translational repression (Fukaya, 2014).

How could Ago1-RISC specifically dissociate eIF4A from the initiation complex? Previous studies hade shown that none of GW182, the CCR4-NOT complex, or PABP is required for translational repression by Ago1-RISC (Fukaya, 2012). The current data extend these findings to reveal that Ago1-RISC can induce dissociation of eIF4A independently of GW182 or PABP. It is tempting to speculate that an as-yet-unidentified factor associated with Ago1-RISC, or perhaps Ago1-RISC itself, blocks the interaction between eIF4G and eIF4A (e.g., similarly to Programmed Cell Death 4 [PDCD4] whose tandem MA-3 domains compete with the MA-3 domain of eIF4G to bind the N-terminal domain of eIF4A, thereby displacing eIF4A from the eIF4F initiation complex). Alternatively, Ago1-RISC might directly or indirectly inhibit the ATP-dependent RNA-binding activity of eIF4A, which is tightly regulated by its accessory proteins eIF4B and eIF4H (Abramson, 1988, Richter, 1999). Future studies are warranted to determine how miRNAs block the assembly of the eIF4F translation initiation complex (Fukaya, 2014).

The insulin receptor cellular IRES confers resistance to eIF4A inhibition

Under conditions of stress, such as limited growth factor signaling, translation is inhibited by the action of 4E-BP and PDCD4. These proteins, through inhibition of eIF4E and eIF4A, respectively, impair cap-dependent translation. Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression. This study shows that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress. Previous work has shown that the 5' UTR of the Drosophila insulin-like receptor (dINR) supports cap-independent translation that is resistant to 4E-BP. Using hippuristanol, an eIF4A inhibitor, this study found that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition. In addition, the murine insulin receptor and insulin-like growth factor receptor 5' UTRs support cap-independent translation and have a similar resistance to hippuristanol. This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions (Olson, 2013).

eIF4A controls germline stem cell self-renewal by directly inhibiting BAM function in the Drosophila ovary

Stem cell self-renewal is controlled by concerted actions of extrinsic niche signals and intrinsic factors in a variety of systems. Drosophila ovarian germline stem cells (GSCs) have been one of the most productive systems for identifying the factors controlling self-renewal. The differentiation factor BAM is necessary and sufficient for GSC differentiation, but it still remains expressed in GSCs at low levels. However, it is unclear how its function is repressed in GSCs to maintain self-renewal. This study reports the identification of the translation initiation factor eIF4A for its essential role in self-renewal by directly inactivating BAM function. eIF4A can physically interact with BAM in Drosophila S2 cells and yeast cells. eIF4A exhibits dosage-specific interactions with bam in the regulation of GSC differentiation. It is required intrinsically for controlling GSC self-renewal and proliferation but not survival. In addition, it is required for maintaining E-cadherin expression but not BMP signaling activity. Furthermore, BAM and BGCN together repress translation of E-cadherin through its 3' UTR in S2 cells. Therefore, it is proposed that BAM functions as a translation repressor by interfering with translation initiation and eIF4A maintains self-renewal by inhibiting BAM function and promoting E-cadherin expression (Shen, 2009).

This study has revealed the biochemical function of the BAM/BGCN complex as a translational repressor. eIF4A in the regulation of GSC self-renewal to be a direct antagonist of BAM function in the Drosophila ovary. A model is proposed explaining how GSC self-renewal is controlled by concerted actions of intrinsic factors and the extrinsic BMP signal. BMP signaling directly represses bam expression, yet leaves low levels of BAM protein expression in the GSC. eIF4A and other unidentified germline factors in the GSC can effectively dismantle BAM/BGCN's repression of GSC maintenance factors, including E-cadherin, through physical interactions, leading to high expression of maintenance factors in the GSC. In the cystoblast (CB), high levels of BAM along with BGCN can keep eIF4A proteins out of the active pool and thus effectively repress GSC maintenance factors, promoting CB differentiation. Therefore, this study has significantly advanced current understanding of how GSC self-renewal and differentiation are regulated by translation factors (Shen, 2009).

bam and bgcn genetically require each other's function to control CB differentiation. Although they are expressed at low levels in GSCs, they have an important role in regulating GSC competition. However, their biochemical functions remained unclear until this study. This study showed that BAM specifically interacts with BGCN, but not other RNA-binding proteins VASA, Rm62, and Me31B, to form a protein complex. In addition, BAM and BGCN are shown to act together; BAM or BGCN alone are not capable of suppressing the expression of the reporter containing the shg 3' UTR. Furthermore, BAM and BGCN do not affect the stability of the reporter mRNA, further supporting that they regulate mRNA translation but not stability. To reveal the role of BGCN in the function of the BAM/BCGN complex, this study showed that direct tethering of BAM to the 3' UTR of the target mRNA can bypass the requirement of BGCN and sufficiently suppress the expression of the reporter. Based on the fact that BGCN contains a putative DEXH RNA binding domain, it is proposed that BGCN helps bring BAM to its target mRNAs to repress their translation. Therefore, this study has revealed the biochemical functions of BAM and BGCN (Shen, 2009).

Previous genetic study showed that BAM and BGCN negatively regulate E-cadherin expression in GSCs to control GSC competition, but the underlying molecular mechanism remains defined. This study showed that in Drosophila S2 cells BAM and BGCN could repress E-cadherin expression through its 3' UTR at the translational level. Along with previous observation that BAM and BGCN negatively regulate E-cadherin expression in GSCs in vivo, it is proposed that BAM and BGCN likely repress E-cadherin expression in GSCs at the translational level. In the future, it will be important to show if BAM and BGCN directly bind to the shg 3' UTR to repress E-cadherin expression in the GSC (Shen, 2009).

eIF4A, an RNA helicase, is one component of the translation initiation complex eIF4F, which is required for loading the small 40S ribosome subunit onto the target mRNA to initiate its translation. The helicase activity of eIF4A itself is weak but is enhanced upon binding to eIF4G, another component of eIF4F. Such helicase activity is important to remove the secondary structure of the 5' UTR, facilitating the ribosome scanning along mRNA to find the initiation codon ATG. To reveal how BAM and BGCN confer translation repression, the yeast 2-hybrid screen was used to identify eIF4A as a BAM interacting protein. Then, two pieces of genetic of evidence were provided supporting the idea that eIF4A and bam function together to control the balance between GSC self-renewal and differentiation. First, one copy of the mutations in eIF4A can dramatically promote germ cell differentiation in the hypomorphic bam^Z/bam^δ86 transheterozygous ovaries. However, a mutation in eIF4A cannot suppress the tumorous phenotype of the bam^?86 homozygous ovaries (no bam function at all), suggesting that the reduction of eIF4A dosage helps enhance the remaining BAM function. Second, overexpression of eIF4A can enhance the differentiation defect in the bam^?86 heterozygote. These genetic results support the antagonizing relationship between bam and eIF4A (Shen, 2009).

The antagonizing genetic relationship between bam and eIF4A suggests that eIF4A favors GSC maintenance over differentiation. The genetic analysis of the marked eIF4A mutant GSC clones shows that eIF4A is indeed required in GSCs for their self-renewal and division. To uncover the genetic mechanism underlying the function of eIF4A in maintaining GSCs, it was also shown that the marked eIF4A mutant GSC has normal BMP signaling activities in comparison with its neighboring wild-type GSC based on expression results from 2 BMP responses genes, bam and Dad, but has significantly reduced E-cadherin expression in comparison with its neighboring wild-type GSC. These genetic and cell biological results demonstrate that eIF4A controls GSC maintenance at least partly by maintaining E-cadherin expression. In mammalian cells, overexpression of translation initiation factors, such as eIF4A, 4G, and 4E, is implicated in different kinds of cancer due to their ability to increase cell proliferation. In the Drosophila imaginal disc, the block in cell proliferation caused by mutations in eIF4A can be bypassed by E2F overexpression, indicating that eIF4A regulates cell cycle progression and consequently cell proliferation. In this study, it was shown that eIF4A is also required for controlling GSC division. Therefore, it is proposed that eIF4A controls GSC proliferation by regulating cell cycle progression like in Drosophila imaginal tissues (Shen, 2009).

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components, proteins were isolated that associate in early embryos with Vasa (Vas) and Tudor (Tud), two known polar granule associated molecules. Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) were identified as components of both Vas and Tud complexes and their localization to polar granules was confirmed by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. The recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly (Thomson, 2008).

Little is understood of the molecular events that link the assembly of germ plasm to the formation of germ cells. There is a strong correlation between polar granule formation and germ cell formation, yet their functional relationship is still unclear. In an attempt to understand polar granule formation and function this study set out to isolate polar granule components with a biochemical approach; proteins common to both Tud and Vas complexes were isolated. These complexes were isolated by cross-linking proteins from early embryonic extracts followed by anti-Tud or anti-Vas immunoprecipitation; proteins found in both complexes were then immunolocalized using EM. Using this method it was confirmed that Aub is a polar granule component and three new polar granule components were identified: ME31B, TER94 and eIF4A. Through genetic interaction analysis in transheterozygous embryos it was shown that decreasing the levels of Vas or Tud along with either Aub, ME31B, Ter94 or eIF4A reduces germ cell number. This approach both identified novel polar granule components and implicated novel processes in germ cell formation (Thomson, 2008).

The presence of Aub, ME31B and eIF4A in polar granules supports the hypothesis that polar granules and P bodies are structurally, and perhaps functionally, related. Recovery of CUP, an ME31B-interacting protein, in the Tud complex further supports this. Similar parallels have recently been found for the mouse chromatoid body, an electron dense structure in the male germ line with similarities to Drosophila polar granules and nurse cell nuage. RNAs in P bodies are stored in a translationally quiescent state and can later be either degraded or translationally activated in response to physiological cues. Translational repression in P bodies occurs at the level of mRNA recruitment to the ribosome, and through miRNA silencing pathways. The polar granule components that were identified suggest their involvement in both types of post-transcriptional regulation. Aub has been implicated in processing of germ line specific piRNAs. Findings that Aub associates with polar granules implicates piRNAs in germ cell formation, as has a previous study. In contrast, Vas and eIF4A have been closely linked with translational regulation and are not known to participate in miRNA silencing pathways (Thomson, 2008).

The RNA-rich nature of early polar granules supports the idea that specific germ line-specific mRNAs are stored in polar granules in a translationally repressed state. Subsequently, these RNAs are translated and their function may be required for germ cell formation and further development. How could general translational repression mediated by polar granules be overcome? Conceivably Vas could be a key factor. Vas, a highly conserved polar granule component with homologues in other species involved in germ line formation, binds directly to eIF5B. Disrupting the eIF5B-Vas interaction abrogates germ cell formation, presumably due to the loss of the ability of Vas to initiate translation of yet unidentified mRNAs. Thus, Vas may act as a germ line specific mRNA translation derepression factor. Other tissue specific factors could adapt a P body to a specific function or cell line. Identification of mRNAs that localize in polar granules and are dependent on Vas for their translation will no doubt provide more insight into this mechanism (Thomson, 2008).

Ultrastructural analysis of proteins found in the Vas and Tud containing complexes revealed that polar granules were often in close proximity or in contact with ER. Supporting such a link, Ter94 and ME31B were present in both Tud and Vas complexes, and are enriched in polar granules. Further work is required to elucidate what proportion of polar granules associate with ER, and whether this association is stage dependent. The presence of Ter94, an ER exit site marker, with Vas, ME31B, Aub and eIF4A in the same structure suggests that ER exit sites directly associate with the translational machinery with both activating and repressing factors. Polar granules may form at ER exit sites, which could provide a mechanism for the localization and assembly of mRNPs required for the translational regulation of their constituent mRNAs. There is evidence that P bodies associate with ER exit sites. In the Drosophila ovary, Trailer Hitch (TRAL) associates with ER exit sites and associates with P body components such as ME31B and CUP. The C. elegans homologue of TRAL, Car-1, associates with DCAP-1, a P-body marker, and car-1 mutations affect ER assembly. A single TRAL peptide was recovered in one immunoprecipitation with Tud, perhaps lending additional support to an association between polar granules and ER exit sites (Thomson, 2008).

Repeated attempts to biochemically isolate polar granules were made over 30 years ago, before the advent of modern analytical techniques that allow the identification of very small amounts of protein. From this work a major polar granule component of approximately 95 kDa was identified. The nature of this protein was not determined although Ter94 has approximately the same molecular mass, as does PIWI, a 97-kDa likely polar granule component that has eluded the currently used screens. PIWI associates with Vas, a polar granule component, as well as with components of the miRNA machinery. PIWI RNA and protein are enriched in germ plasm and piwi mutants have defects in germ cell formation. The screen also did not identify Osk, which was shown by a yeast two-hybrid screen to bind directly to Vas. This may be because these proteins were not present in high enough abundance for detection. Alternatively, since the reactive ends of the cross-linkers that were used specifically cross-link cysteine residues, they would not stabilize a particular protein-protein interaction unless a pair of cysteine residues is within the range of the cross-linker. The work demonstrates that a molecular approach can be a powerful complement to genetics, and that purification schemes based on two independent reagents can reduce signal-to-noise problems that are inherent in co-immunoprecipitation experiments. Molecular approaches such as this one also have the capacity to identify proteins involved in a developmental process that are encoded by genes with multiple functions, or required for cellular viability, that will therefore elude phenotype-based genetic screens (Thomson, 2008).

Identification of the Drosophila eIF4A gene as a target of the DREF transcription factor

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. A genetic screening was carried out, taking advantage of the rough eye phenotype of transgenic flies that express full-length DREF in the eye imaginal discs, and the eukaryotic initiation factor 4A (eIF4A) gene was identified as a dominant suppressor of the DREF-induced rough eye phenotype. The eIF4A gene was found to carry three DRE sequences, DRE1 (-40 to -47), DRE2 (-48 to -55), and DRE3 (-267 to -274) in its promoter region, these all being important for the eIF4A gene promoter activity in cultured Drosophila Kc cells and in living flies. Knockdown of DREF in Drosophila S2 cells decreased the eIF4A mRNA level and the eIF4A gene promoter activity. Furthermore, specific binding of DREF to genomic regions containing DRE sequences was demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. Band mobility shift assays using Kc cell nuclear extracts revealed that DREF could bind to DRE1 and DRE3 sequences in the eIF4A gene promoter in vitro, but not to the DRE2 sequence. The results suggest that the eIF4A gene is under the control of the DREF pathway and DREF is therefore involved in the regulation of protein synthesis (Ida, 2007).

A novel function of Drosophila eIF4A as a negative regulator of Dpp/BMP signalling that mediates SMAD degradation

Signalling by the TGF-beta superfamily member and BMP orthologue Decapentaplegic (Dpp) is crucial for multiple developmental programmes and has to be tightly regulated.This study demonstrated that the Drosophila Dpp pathway is negatively regulated by eukaryotic translation initiation factor 4A (eIF4A), which mediates activation-dependent degradation of the Dpp signalling components Mad and Medea. eIF4A mutants exhibit increased Dpp signalling and accumulation of Mad and phospho-Mad. Overexpression of eIF4A decreases Dpp signalling and causes loss of Mad and phospho-Mad. Furthermore, eIF4A physically associates with Mad and Medea, and promotes their degradation following activation of Dpp signalling in a translation-independent manner. Finally, this study shows that eIF4A acts synergistically with, but independently of, the ubiquitin ligase DSmurf, indicating that a dual system controls SMAD degradation. Thus, in addition to being an obligatory component of the cap-dependent translation initiation complex, eIF4A has a novel function as a specific inhibitor of Dpp signalling that mediates the degradation of SMAD homologues (Li, 2006).

A genetic screen for maternal-effect suppressors of decapentaplegic identifies the eukaryotic translation initiation factor 4A in Drosophila

The Dpp signaling pathway is essential for many developmental processes in Drosophila and its activity is tightly regulated. To identify additional regulators of Dpp signaling, a genetic screen was conducted for maternal-effect suppressors of dpp haplo-insufficiency. Approximately 7000 EMS-mutagenized genomes were screened, and seven independent dominant suppressors of dpp, Su(dpp), were recovered as second-site mutations that resulted in viable flies in trans-heterozygous with dpp^H46, a dpp null allele. Most of the Su(dpp) mutants exhibited increased cell numbers of the amnioserosa, a cell type specified by the Dpp pathway, suggesting that these mutations may augment Dpp signaling activity. This study reports the unexpected identification of one of the Su(dpp) mutations as an allele of the eukaryotic translation initiation factor 4A (eIF4A). Su(dpp)^YE9 maps to eIF4A and this allele is associated with a substitution, arginine 321 to histidine, at a well-conserved amino acid and behaves genetically as a dominant-negative mutation. This result provides an intriguing link between a component of the translation machinery and Dpp signaling (Li, 2005).

Identification and characterization of the expression of the translation initiation factor 4A (eIF4A) from Drosophila melanogaster

The initiation factor 4A (eIF4A) was identified in a two-dimensional protein database of Drosophila wing imaginal discs. eIF4A, a member of the DEAD-box family of RNA helicases, forms the active eIF4F complex that in the presence of eIF4B and eIF4H unwinds the secondary structure of the 5'-UTR of mRNAs during translational initiation. Two-dimensional gel electrophoresis and microsequencing allowed purification of eIF4A and the generation specific polyclonal antibodies. A combination of immunoblotting and labelling with [(35)S]methionine + [(35)S]cysteine revealed the existence of a single eIF4A isoform encoded by a previously reported gene that maps to chromosome 2L at 26A7-9. Expression of this gene yields two mRNA species, generated by alternative splicing in the 3'-untranslated region. The two mRNAs contain the same open reading frame and produce the identical eIF4A protein. No expression was detected of the eIF4A-related gene CG7483. EIF4A protein expression was detected in the wing imaginal discs of several Drosophila species, and in haltere, leg 1, leg 2, leg 3, and eye-antenna imaginal discs of D. melanogaster. Examination of eIF4A in tumor suppressor mutants showed significantly increased (> 50%) expression in the wing imaginal discs of these larvae. Ubiquitous expression of eIF4A mRNA and protein was observed during Drosophila embryogenesis. Yeast two-hybrid analysis demonstrated the in vivo interaction of Drosophila eIF4G with the N-terminal third of eIF4A (Hernandez, 2004).

Drosophila PTEN regulates cell growth and proliferation through PI3K-dependent and -independent pathways

Pten, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, Pten minus cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of Pten results in opposite phenotypes. The loss-of-function phenotypes of Pten are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that Pten acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of Pten stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, Pten does not play a major role in cell survival during Drosophila development. These results provide a potential explanation for the high frequency of PTEN mutation in human cancer (Gao, 2000).

The observation that clones of Pten mutant cells have a proliferative advantage over their wild-type twin spots indicates that Pten controls progression through the cell division cycle. To determine which phase(s) of the cell cycle Pten regulates, FACS analysis was used to examine the DNA content of dissociated wing imaginal disc cells containing clones of dPTEN mutant cells. Loss of Pten results in a decrease in the percentage of cells in the G1 phase of the cell cycle and a relative increase in the S and G2 population. Clonal overexpression of Pten has a complementary effect, causing a slight decrease in the number of S-phase cells. FACS analysis also reveals complementary changes in cell size in response to Pten levels: loss of Pten causes an increase in average cell size, while Pten overexpression decreases average cell size. Similar effects were observed throughout all phases of the cell cycle and at multiple developmental stages. The cell size and cell cycle FACS profile of Pten mutant cells is remarkably similar to that of cells overexpressing Drosophila PI3K. In both cases, the percentage of cells in G1 is reduced, indicating an acceleration of this phase of the cell cycle. Since overexpression of PI3K does not increase proliferation rates, this shortened G1 appears to be balanced by a commensurate lengthening in the duration of S and/or G2 phases. A similar phenomenon has been described for cells whose G1 phase is accelerated by overexpression of Cyclin E, dMyc, or activated Ras. In contrast, the rapid proliferation rate of Pten mutant cells indicates that S and G2 phases do not lengthen in response to the abbreviated G1 and thus suggest that Pten regulates multiple phases of the cell cycle (Gao, 2000).

Genetic interactions between Pten and PI3K, a component of the insulin signaling pathway in Drosophila, were examined. Overexpression of the PI3K catalytic subunit, Dp110, results in increased wing size, while overexpression of a dominant negative Dp110 construct (PI3KDN) results in the opposite phenotype. These phenotypes are due to changes in cell size. Coexpression of Pten with PI3KDN further reduces wing size. In addition, overexpression of Pten suppresses the increased wing size resulting from PI3K overexpression. To further examine the genetic interaction between Pten and PI3K, Pten mutant cells were examined in a genetic background of overexpressing PI3K or PI3KDN. When PI3K is overexpressed, Pten mutant cells are indistinguishable in cell size from their nonmutant siblings, suggesting that overexpression of PI3K results in increased PIP3 levels and increased signaling that cannot be further activated by removing Pten. Conversely, overexpression of PI3KDN partially suppresses the increased cell size of Pten mutant cells. Thus, PTEN and PI3K antagonize each other in regulating cell size (Gao, 2000).

Certain combinations of loss-of-function alleles of the Drosophila Insulin-like receptor (inr) result in flies with a decreased cell size. This provides an opportunity to examine the genetic epistasis between Pten and inr. The increased cell size of Pten mutant cells can not be reversed in inr mutant animals, and thus loss of Pten function is epistatic to (acts downstream from) mutations in inr (Gao, 2000).

Previous studies have shown that loss of PTEN function promotes cell survival in mammals through activation of Akt. In addition, PTEN acts through Akt in metabolic and longevity control in C. elegans. Dakt1, a Drosophila homolog of Akt, has been suggested to play a role in cell survival in embryogenesis (Staveley, 1998) and cell size control (Verdu, 1999). A hypomorphic allele of Dakt1 has been identified in the large-scale gene disruption project carried out by the Berkeley Drosophila Genome Project. This allele is semilethal, and homozygous survivors show reduced body size and cell size, consistent with a role of Dakt1 in growth control. To examine whether Pten controls cell size through regulating Akt activity, Pten mutant clones were generated in Dakt1 mutant animals. Dakt1 mutation completely suppressed the increase of cell size associated with the Pten mutation. This result provides strong in vivo evidence that Dakt1 functions downstream of (or in parallel to) Pten in the control of cell size. Taken together, these genetic interactions suggest that the role of Pten in opposing signaling through the PI3K/Akt pathway is conserved between flies and vertebrates (Gao, 2000).

eif4A is an ATP-dependent RNA helicase that is an essential component of the eif4F translation initiation complex. Previous studies have identified an allelic series of eif4A mutants that affects larval growth, DNA replication, and cell proliferation (Galloni, 1999). Since the defect in DNA synthesis in eif4A mutants can be bypassed by overexpressing the E2F transcription factor, it has been suggested that eif4A preferentially regulates a specific set of cell-cycle regulatory genes (Galloni, 1999). To examine whether the proliferative advantage of Pten mutant cells requires the same set of cell-cycle regulatory genes as those controlled by eif4A, Pten;eif4A double-mutant clones were examined. A hypomorphic allele and a stronger allele of eif4A were used. The weaker allele confers proliferation disadvantage to the cells, resulting in mutant clones that are smaller than the twin spots without affecting cell size (Galloni, 1999). Hypomorphilc eif4A can partially suppress the overproliferation of Pten mutant cells, but the increased cell size of Pten mutant cells is not suppressed. The stronger eif4A allele completely suppresses the proliferation of Pten mutant cells. Pten;eif4A double-mutant clones are undetectable, as has been observed in the eif4A strong single mutant (Galloni, 1999). It is suggested that modulation of translation initiation is an important aspect of Pten function in regulating cell proliferation (Gao, 2000).

It is proposed that Pten regulates cell proliferation by multiple mechanisms, both PI3K-dependent and -independent. One potential PI3K-independent mechanism is suggested by the domain in Pten related to tensin, an actin filament capping protein that localizes to focal adhesions. Overexpression of tensin can suppress anchorage-independent proliferation of Ras-transformed 3T3 cells, and therefore this domain may provide a growth-regulatory function in mammalian PTEN as well. Moreover, in addition to its role as a lipid phosphatase, PTEN also possesses a dual-specificity protein phosphatase activity. PTEN has been shown to bind and dephosphorylate the focal adhesion kinase FAK and to down-regulate the formation of focal adhesions. Such cell contacts play a critical role in regulating proliferation in Drosophila, and the gene products of several Drosophila tumor suppressors such as expanded, fat, and l(2) discs large all localize to adherens or septate junctions. The results for Pten are thus consistent with a model in which PTEN suppresses cell growth and G1/S progression by down-regulating the PI3K/Akt pathway and inhibiting the G2/M transition through an alternative mechanism, perhaps involving regulation of the cytoarchitecture. The ability to regulate both growth and cell division may explain why PTEN is such a common target in advanced tumors. This model is also consistent with the different mutant phenotypes between a null Pten allele and an allele that carries a point mutation (Huang, 1999). While the point mutation changes an invariant amino acid within the phosphatase active site and is likely to inactivate the lipid phosphatase activity, the other domains of Pten are still intact. Characterization of Pten mutants that are specifically defective in cell growth or proliferation may shed further light on its role in the control of overall growth (Gao, 2000).

Search PubMed for articles about Drosophila EIF4A

Demetriades, C., Doumpas, N. and Teleman, A. A. (2014). Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2. Cell 156: 786-799. PubMed ID: 24529380

Fukaya, T., Iwakawa, H. O. and Tomari, Y. (2014). MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila. Mol Cell 56: 67-78. PubMed ID: 25280104

Galloni, M., and Edgar, B. A. (1999). Cell-autonomous and non-autonomous growth-defective mutants of Drosophila melanogaster. Development 126: 2365-2375. PubMed Citation: 10225996

Gao, X., Neufeld, T. P. Pan, D. (2000). Drosophila PTEN regulates cell growth and proliferation through PI3K-dependent and -independent pathways. Dev. Biol. 221: 404-418. PubMed Citation: 10790335

Hernandez, G., Lalioti, V., Vandekerckhove, J., Sierra, J. M. and Santaren, J. F. (2004). Identification and characterization of the expression of the translation initiation factor 4A (eIF4A) from Drosophila melanogaster. Proteomics 4(2): 316-326. PubMed ID: 14760701

Ida, H., Yoshida, H., Nakamura, K. and Yamaguchi, M. (2007). Identification of the Drosophila eIF4A gene as a target of the DREF transcription factor. Exp Cell Res 313(20): 4208-4220. PubMed ID: 17888422

Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T. P. and Guan, K. L. (2008). Regulation of TORC1 by Rag GTPases in nutrient response. Nat Cell Biol 10: 935-945. PubMed ID: 18604198

Lee, A. S., Kranzusch, P. J. and Cate, J. H. (2015). eIF3 targets cell-proliferation messenger RNAs for translational activation or repression. Nature 522(7554): 111-114. PubMed ID: 25849773

Lee, A. S., Kranzusch, P. J., Doudna, J. A. and Cate, J. H. (2016). eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation. Nature 536(7614): 96-99. PubMed ID: 27462815

Li, J., Li, W. X. and Gelbart, W. M. (2005). A genetic screen for maternal-effect suppressors of decapentaplegic identifies the eukaryotic translation initiation factor 4A in Drosophila. Genetics 171(4): 1629-1641. PubMed ID: 15972466

Li, J. and Li, W. X. (2006). A novel function of Drosophila eIF4A as a negative regulator of Dpp/BMP signalling that mediates SMAD degradation. Nat Cell Biol 8(12): 1407-1414. PubMed ID: 17115029

Olson, C. M., Donovan, M. R., Spellberg, M. J. and Marr, M. T. (2013). The insulin receptor cellular IRES confers resistance to eIF4A inhibition. Elife 2: e00542. PubMed ID: 23878722

Rode, S., Ohm, H., Anhauser, L., Wagner, M., Rosing, M., Deng, X., Sin, O., Leidel, S. A., Storkebaum, E., Rentmeister, A., Zhu, S. and Rumpf, S. (2018). Differential requirement for translation initiation factor pathways during ecdysone-dependent neuronal remodeling in Drosophila. Cell Rep 24(9): 2287-2299. PubMed ID: 30157424

Sancak, Y., Peterson, T. R., Shaul, Y. D., Lindquist, R. A., Thoreen, C. C., Bar-Peled, L. and Sabatini, D. M. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320: 1496-1501. PubMed ID: 18497260

Sen, N. D., Zhou, F., Harris, M. S., Ingolia, N. T. and Hinnebusch, A. G. (2016). eIF4B stimulates translation of long mRNAs with structured 5' UTRs and low closed-loop potential but weak dependence on eIF4G. Proc Natl Acad Sci U S A 113(38): 10464-10472. PubMed ID: 27601676

Shen, R., Weng C., Yu J. and Xie T. (2009). eIF4A controls germline stem cell self-renewal by directly inhibiting BAM function in the Drosophila ovary. (2009) Proc. Natl. Acad. Sci. U.S.A. 106: 11623-11628. PubMed ID: 19556547

Sokabe, M. and Fraser, C. S. (2017). A helicase-independent activity of eIF4A in promoting mRNA recruitment to the human ribosome. Proc Natl Acad Sci U S A 114(24): 6304-6309. PubMed ID: 28559306

Staveley, B. E., Ruel, L., Jin, J., Stambolic, V., Mastronardi, F. G., Heitzler, P., Woodgett, J. R. and Manoukian, A. S. (1998). Genetic analysis of protein kinase B (AKT) in Drosophila. Curr Biol 8(10): 599-602. PubMed ID: 9601646

Thomson, T., Liu, N., Arkov, A., Lehmann, R. and Lasko, P. (2008). Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins. Mech. Dev. 125(9-10): 865-73. PubMed Citation: 18590813

Tsokanos, F. F., Albert, M. A., Demetriades, C., Spirohn, K., Boutros, M. and Teleman, A. A. (2016). eIF4A inactivates TORC1 in response to amino acid starvation. EMBO J 35(10):1058-76. PubMed ID: 26988032

Verdu, J., Buratovich, M. A., Wilder, E. L. and Birnbaum, M. J. (1999). Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB. Nat Cell Biol 1(8): 500-506. PubMed ID: 10587646

Yourik, P., Aitken, C. E., Zhou, F., Gupta, N., Hinnebusch, A. G. and Lorsch, J. R. (2017). Yeast eIF4A enhances recruitment of mRNAs regardless of their structural complexity. Elife 6. PubMed ID: 29192585

date revised: 2 September 2019

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