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Translational Control: A Cup Half Full
Current Biology, Vol. 14, R282–R283, April 6, 2004, ©2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.03.025
Translational Control: A Cup Half Full Paul M. Macdonald
Repression of translation of oskar and nanos mRNAs prior to their posterior localization in the egg and embryo is essential for body patterning in Drosophila. The Cup protein is now found to have an important role in repression of both mRNAs, and apparently does so in a manner similar to the action of the Xenopus Maskin protein.
Not all mRNAs are translated equally. Variations in mRNA translation efficiency can enhance the production of proteins required at high levels, or restrict the accumulation of proteins that are necessarily rare. For those proteins that must at different times be either abundant or scarce, the efficiency of translation can be adjusted by a spectrum of regulatory mechanisms collectively termed translational control. Typically, control is negative, and translation of the affected mRNAs is repressed until they are specifically activated. A common feature of such control is intervention at the point of translation initiation, in which a necessary contact between two initiation factors — the capbinding protein eIF4E and eIF4G — is blocked via binding of an inhibitor to eIF4E [1]. The structural basis for the eIF4E–eIF4G interaction is known and, of greater use in characterizing regulatory proteins, a signature eIF4E-binding site motif has been defined [1–3]. For this discussion, the most relevant eIF4E-binding regulatory protein is Maskin, which associates with the 3′′ untranslated regions (UTRs) of specific mRNAs and competes with eIF4G for binding to eIF4E. Maskin does not bind directly to the mRNA, but is recruited there by CPEB, which binds to a regulatory element termed the CPE [4]. Translational control is prominent in early development, and Drosophila provides many examples. The body plan of the fruitfly embryo is laid down in large part by the action of several key localized determinants, each subject to translational control. In particular, posterior body patterning relies on the sequential posterior deployment of first Oskar (Osk) protein in the oocyte, and later Nanos (Nos) protein in the embryo [5,6]. Control here is especially elaborate, in that translation is coordinated with mRNA localization: translation of unlocalized transcripts is repressed, and localization triggers activation. A number of proteins that influence the translation of osk and nos mRNAs have been identified, but any detailed explanation of an actual mechanism has been elusive. Three recent reports [7–9] have now provided some welcome clarity. Each group arrived at a protein known as Cup through biochemical experiments, looking for proteins that interact with factors known to contribute to the Institute for Cellular and Molecular Biology, Section of Molecular Cell and Developmental Biology, The University of Texas at Austin, Texas 78712-0159, USA. Email: [email protected]
Dispatch
control of the osk or nos mRNAs. Although the details of the papers differ — and do so in interesting but not entirely consistent ways — they all agree that Cup contains a conventional eIF4E-binding site and does indeed bind eIF4E. Surprisingly, Nelson et al. [8] also detected an unconventional contact between Cup and eIF4E, between regions of each protein that are distinct from those involved in the conventional interaction. This contact must be taken seriously, as like the conventional interaction, it has the property of blocking the eIF4E–eIF4G interaction. Why the different groups obtained different results is not certain, but it may be significant that the experiment in which the unconventional contact was not detected — affinity chromatography of soluble Cup and immobilized GSTtagged eIF4E — made use of Cup that was synthesized by in vitro translation and therefore provided in a mixture of many proteins, including eIF4E. Independent of the experimental differences between the three studies [7–9], all point to the same type of mechanism. Cup is proposed to act much like Maskin: when recruited to the 3′′ UTR of osk or nos mRNA, Cup binds eIF4E and blocks its interaction with eIF4G, thereby preventing successful initiation of translation. This model provides a solid start for understanding the details of the mechanism, although exactly how an inhibitory interaction with a molecule eIF4E at one end of an mRNA — the 3′′ UTR, where the proteins that recruit Maskin and Cup are known or thought to bind — would prevent the use of any other molecule of eIF4E at the other end of the mRNA (the cap) is not completely clear. Presumably there is very little free eIF4E available, and the pseudo-unimolecular interaction between different proteins bound to the same mRNA predominates over bimolecular reactions between proteins bound to different mRNAs. Nelson et al. [8] address the role of Cup in control of nos mRNA. Here it is Smaug, a protein known to mediate repression of nos translation [10–12], that recruits Cup. cup mutants are defective in Smaugmediated repression, and the function of Cup in binding to eIF4E is clearly established through use of mutants in which both eIF4E interactions are disrupted. This demonstration of control at the level of translational initiation adds further complexity to regulation of nos mRNA, as a significant fraction of nos mRNA is associated with polysomes under conditions when no Nos protein accumulates [13]. Thus, there appear to be overlapping or redundant means of preventing the accumulation of Nos protein away from the posterior pole of the Drosophila embryo. Nakamura et al. [7] isolated Cup in a complex with Me31B, a protein required for repression of osk mRNA translation [14]. The unique feature of this study is the evidence that Cup binds directly to Bruno (Bru), a translational repressor that binds to regulatory sequences in the osk mRNA 3′′ UTR [15]. The authors propose that this interaction underlies the role of Bru. However, this model fails to account for the fact that loss of Cup activity has a much greater effect on osk translation than
Current Biology R283
does preventing Bru from binding to osk mRNA. This discrepancy can most simply be explained by one of two basic models. In one, Cup plays a role in osk translational repression that is independent of its association with osk mRNA. For example, free Cup that is not bound to mRNAs could act as a non-specific translational repressor, much like the inhibitory eIF4E-BPs [16], although some feature of the osk mRNA or its translation would have to be unusually susceptible to this repression. Alternatively, the recruitment of Cup to osk mRNA might be a partially redundant process, one in which more than one protein can act as the bridging factor. The paper from Wilhelm et al. [9] includes evidence that would be consistent with the latter model. Wilhelm et al. [9] identified Cup as a component of a large protein complex implicated in the localization of osk mRNA [17]. In keeping with that genealogy, cup mutants prove to be defective in the later stages of osk mRNA localization. This result stands in sharp contrast to the work of Nakamura et al. [7], who found no such defect. Here the availability of multiple cup alleles, usually a boon to genetic analysis, proves to be a hindrance (for now) to comparison and possible resolution of this conflict, as none of the three groups used the same set of alleles. Nevertheless, an explanation of the localization defect of the cup mutant is provided, as the posterior localization of Barentsz, a protein required for osk mRNA localization [18], is disrupted. This work is particularly notable because it breaks a long-standing pattern, in which mutants defective in osk mRNA localization also fail to translate osk mRNA. Thus, the cup mutants will be an important tool for understanding how localization and translation are coordinated, and Cup itself may play a central role in the process. Indeed, Wilhelm et al. [9] provide a detailed and Cupcentric proposal for how a series of interactions could be formed and then broken to orchestrate both localization and translation. History suggests that any simple model of osk or nos regulation will be at best incomplete, but the basic role of Cup, as suggested by the more extensive analysis of Maskin, appears likely to be correct. Additional complexities, suggested by differences in the results in the three papers [7–9], are likely to be revealed, but even a pessimist would conclude that the recent discoveries represent a decisive step forward. A final lesson to be learned from the papers is of more general interest. With the discovery of a second protein that appears to follow the Maskin prototype in its function, it seems reasonable to expect that there will be still other proteins with the same function. Because Maskin and Cup are not strikingly similar in primary structure, identifying such proteins may not be simple. Nevertheless, this class of proteins could prove to be widely used to regulate translation, a type of control now recognized to be used in many cell types. References 1. Gingras, A.C., Raught, B., and Sonenberg, N. (1999). eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68, 913-963. 2. Mader, S., Lee, H., Pause, A., and Sonenberg, N. (1995). The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4Ebinding proteins. Mol. Cell Biol. 15, 4990-4997.
3. Marcotrigiano, J., Gingras, A.C., Sonenberg, N., and Burley, S.K. (1999). Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. Mol. Cell 3, 707-716. 4. Mendez, R., and Richter, J.D. (2001). Translational control by CPEB: a means to the end. Nat. Rev. Mol. Cell Biol. 2, 521-529. 5. Johnstone, O., and Lasko, P. (2001). Translational regulation and RNA localization in Drosophila oocytes and embryos. Annu. Rev. Genet. 35, 365-406. 6. Palacios, I.M., and St. Johnston, D. (2001). Getting the message across: the intracellular localization of mRNAs in higher eukaryotes. Annu. Rev. Cell Dev. Biol. 17, 569-614. 7. Nakamura, A., Sato, K., and Hanyu-Nakamura, K. (2004). Drosophila Cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis. Dev. Cell 6, 69-78. 8. Nelson, M.R., Leidal, A.M., and Smibert, C.A. (2004). Drosophila Cup is an eIF4E-binding protein that functions in Smaug-mediated translational repression. EMBO J. 23, 150-159. 9. Wilhelm, J.E., Hilton, M., Amos, Q., and Henzel, W.J. (2003). Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz. J. Cell Biol. 163, 1197-1204. 10. Smibert, C.A., Wilson, J.E., Kerr, K., and Macdonald, P.M. (1996). smaug protein represses translation of unlocalized nanos mRNA in the Drosophila embryo. Genes Dev. 10, 2600-2609. 11. Smibert, C.A., Lie, Y.S., Shillinglaw, W., Henzel, W.J., and Macdonald, P.M. (1999). Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro. RNA 5, 1535-1547. 12. Dahanukar, A., Walker, J.A., and Wharton, R.P. (1999). Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila. Mol. Cell 4, 209-218. 13. Clark, I.E., Wyckoff, D., and Gavis, E.R. (2000). Synthesis of the posterior determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism. Curr. Biol. 10, 1311-1314. 14. Nakamura, A., Amikura, R., Hanyu, K., and Kobayashi, S. (2001). Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development 128, 3233-3242. 15. Kim-Ha, J., Kerr, K., and Macdonald, P.M. (1995). Translational regulation of oskar mRNA by bruno, an ovarian RNA-binding protein, is essential. Cell 81, 403-412. 16. Haghighat, A., Mader, S., Pause, A., and Sonenberg, N. (1995). Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J. 14, 5701-5709. 17. Wilhelm, J.E., Mansfield, J., Hom-Booher, N., Wang, S., Turck, C.W., Hazelrigg, T., and Vale, R.D. (2000). Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes. J. Cell Biol. 148, 427-440. 18. van Eeden, F.J., Palacios, I.M., Petronczki, M., Weston, M.J., and St Johnston, D. (2001). Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole. J. Cell Biol. 154, 511-523.
Translational Control: A Cup Half Full Paul M. Macdonald
Repression of translation of oskar and nanos mRNAs prior to their posterior localization in the egg and embryo is essential for body patterning in Drosophila. The Cup protein is now found to have an important role in repression of both mRNAs, and apparently does so in a manner similar to the action of the Xenopus Maskin protein.
Not all mRNAs are translated equally. Variations in mRNA translation efficiency can enhance the production of proteins required at high levels, or restrict the accumulation of proteins that are necessarily rare. For those proteins that must at different times be either abundant or scarce, the efficiency of translation can be adjusted by a spectrum of regulatory mechanisms collectively termed translational control. Typically, control is negative, and translation of the affected mRNAs is repressed until they are specifically activated. A common feature of such control is intervention at the point of translation initiation, in which a necessary contact between two initiation factors — the capbinding protein eIF4E and eIF4G — is blocked via binding of an inhibitor to eIF4E [1]. The structural basis for the eIF4E–eIF4G interaction is known and, of greater use in characterizing regulatory proteins, a signature eIF4E-binding site motif has been defined [1–3]. For this discussion, the most relevant eIF4E-binding regulatory protein is Maskin, which associates with the 3′′ untranslated regions (UTRs) of specific mRNAs and competes with eIF4G for binding to eIF4E. Maskin does not bind directly to the mRNA, but is recruited there by CPEB, which binds to a regulatory element termed the CPE [4]. Translational control is prominent in early development, and Drosophila provides many examples. The body plan of the fruitfly embryo is laid down in large part by the action of several key localized determinants, each subject to translational control. In particular, posterior body patterning relies on the sequential posterior deployment of first Oskar (Osk) protein in the oocyte, and later Nanos (Nos) protein in the embryo [5,6]. Control here is especially elaborate, in that translation is coordinated with mRNA localization: translation of unlocalized transcripts is repressed, and localization triggers activation. A number of proteins that influence the translation of osk and nos mRNAs have been identified, but any detailed explanation of an actual mechanism has been elusive. Three recent reports [7–9] have now provided some welcome clarity. Each group arrived at a protein known as Cup through biochemical experiments, looking for proteins that interact with factors known to contribute to the Institute for Cellular and Molecular Biology, Section of Molecular Cell and Developmental Biology, The University of Texas at Austin, Texas 78712-0159, USA. Email: [email protected]
Dispatch
control of the osk or nos mRNAs. Although the details of the papers differ — and do so in interesting but not entirely consistent ways — they all agree that Cup contains a conventional eIF4E-binding site and does indeed bind eIF4E. Surprisingly, Nelson et al. [8] also detected an unconventional contact between Cup and eIF4E, between regions of each protein that are distinct from those involved in the conventional interaction. This contact must be taken seriously, as like the conventional interaction, it has the property of blocking the eIF4E–eIF4G interaction. Why the different groups obtained different results is not certain, but it may be significant that the experiment in which the unconventional contact was not detected — affinity chromatography of soluble Cup and immobilized GSTtagged eIF4E — made use of Cup that was synthesized by in vitro translation and therefore provided in a mixture of many proteins, including eIF4E. Independent of the experimental differences between the three studies [7–9], all point to the same type of mechanism. Cup is proposed to act much like Maskin: when recruited to the 3′′ UTR of osk or nos mRNA, Cup binds eIF4E and blocks its interaction with eIF4G, thereby preventing successful initiation of translation. This model provides a solid start for understanding the details of the mechanism, although exactly how an inhibitory interaction with a molecule eIF4E at one end of an mRNA — the 3′′ UTR, where the proteins that recruit Maskin and Cup are known or thought to bind — would prevent the use of any other molecule of eIF4E at the other end of the mRNA (the cap) is not completely clear. Presumably there is very little free eIF4E available, and the pseudo-unimolecular interaction between different proteins bound to the same mRNA predominates over bimolecular reactions between proteins bound to different mRNAs. Nelson et al. [8] address the role of Cup in control of nos mRNA. Here it is Smaug, a protein known to mediate repression of nos translation [10–12], that recruits Cup. cup mutants are defective in Smaugmediated repression, and the function of Cup in binding to eIF4E is clearly established through use of mutants in which both eIF4E interactions are disrupted. This demonstration of control at the level of translational initiation adds further complexity to regulation of nos mRNA, as a significant fraction of nos mRNA is associated with polysomes under conditions when no Nos protein accumulates [13]. Thus, there appear to be overlapping or redundant means of preventing the accumulation of Nos protein away from the posterior pole of the Drosophila embryo. Nakamura et al. [7] isolated Cup in a complex with Me31B, a protein required for repression of osk mRNA translation [14]. The unique feature of this study is the evidence that Cup binds directly to Bruno (Bru), a translational repressor that binds to regulatory sequences in the osk mRNA 3′′ UTR [15]. The authors propose that this interaction underlies the role of Bru. However, this model fails to account for the fact that loss of Cup activity has a much greater effect on osk translation than
Current Biology R283
does preventing Bru from binding to osk mRNA. This discrepancy can most simply be explained by one of two basic models. In one, Cup plays a role in osk translational repression that is independent of its association with osk mRNA. For example, free Cup that is not bound to mRNAs could act as a non-specific translational repressor, much like the inhibitory eIF4E-BPs [16], although some feature of the osk mRNA or its translation would have to be unusually susceptible to this repression. Alternatively, the recruitment of Cup to osk mRNA might be a partially redundant process, one in which more than one protein can act as the bridging factor. The paper from Wilhelm et al. [9] includes evidence that would be consistent with the latter model. Wilhelm et al. [9] identified Cup as a component of a large protein complex implicated in the localization of osk mRNA [17]. In keeping with that genealogy, cup mutants prove to be defective in the later stages of osk mRNA localization. This result stands in sharp contrast to the work of Nakamura et al. [7], who found no such defect. Here the availability of multiple cup alleles, usually a boon to genetic analysis, proves to be a hindrance (for now) to comparison and possible resolution of this conflict, as none of the three groups used the same set of alleles. Nevertheless, an explanation of the localization defect of the cup mutant is provided, as the posterior localization of Barentsz, a protein required for osk mRNA localization [18], is disrupted. This work is particularly notable because it breaks a long-standing pattern, in which mutants defective in osk mRNA localization also fail to translate osk mRNA. Thus, the cup mutants will be an important tool for understanding how localization and translation are coordinated, and Cup itself may play a central role in the process. Indeed, Wilhelm et al. [9] provide a detailed and Cupcentric proposal for how a series of interactions could be formed and then broken to orchestrate both localization and translation. History suggests that any simple model of osk or nos regulation will be at best incomplete, but the basic role of Cup, as suggested by the more extensive analysis of Maskin, appears likely to be correct. Additional complexities, suggested by differences in the results in the three papers [7–9], are likely to be revealed, but even a pessimist would conclude that the recent discoveries represent a decisive step forward. A final lesson to be learned from the papers is of more general interest. With the discovery of a second protein that appears to follow the Maskin prototype in its function, it seems reasonable to expect that there will be still other proteins with the same function. Because Maskin and Cup are not strikingly similar in primary structure, identifying such proteins may not be simple. Nevertheless, this class of proteins could prove to be widely used to regulate translation, a type of control now recognized to be used in many cell types. References 1. Gingras, A.C., Raught, B., and Sonenberg, N. (1999). eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68, 913-963. 2. Mader, S., Lee, H., Pause, A., and Sonenberg, N. (1995). The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4Ebinding proteins. Mol. Cell Biol. 15, 4990-4997.
3. Marcotrigiano, J., Gingras, A.C., Sonenberg, N., and Burley, S.K. (1999). Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. Mol. Cell 3, 707-716. 4. Mendez, R., and Richter, J.D. (2001). Translational control by CPEB: a means to the end. Nat. Rev. Mol. Cell Biol. 2, 521-529. 5. Johnstone, O., and Lasko, P. (2001). Translational regulation and RNA localization in Drosophila oocytes and embryos. Annu. Rev. Genet. 35, 365-406. 6. Palacios, I.M., and St. Johnston, D. (2001). Getting the message across: the intracellular localization of mRNAs in higher eukaryotes. Annu. Rev. Cell Dev. Biol. 17, 569-614. 7. Nakamura, A., Sato, K., and Hanyu-Nakamura, K. (2004). Drosophila Cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis. Dev. Cell 6, 69-78. 8. Nelson, M.R., Leidal, A.M., and Smibert, C.A. (2004). Drosophila Cup is an eIF4E-binding protein that functions in Smaug-mediated translational repression. EMBO J. 23, 150-159. 9. Wilhelm, J.E., Hilton, M., Amos, Q., and Henzel, W.J. (2003). Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz. J. Cell Biol. 163, 1197-1204. 10. Smibert, C.A., Wilson, J.E., Kerr, K., and Macdonald, P.M. (1996). smaug protein represses translation of unlocalized nanos mRNA in the Drosophila embryo. Genes Dev. 10, 2600-2609. 11. Smibert, C.A., Lie, Y.S., Shillinglaw, W., Henzel, W.J., and Macdonald, P.M. (1999). Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro. RNA 5, 1535-1547. 12. Dahanukar, A., Walker, J.A., and Wharton, R.P. (1999). Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila. Mol. Cell 4, 209-218. 13. Clark, I.E., Wyckoff, D., and Gavis, E.R. (2000). Synthesis of the posterior determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism. Curr. Biol. 10, 1311-1314. 14. Nakamura, A., Amikura, R., Hanyu, K., and Kobayashi, S. (2001). Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development 128, 3233-3242. 15. Kim-Ha, J., Kerr, K., and Macdonald, P.M. (1995). Translational regulation of oskar mRNA by bruno, an ovarian RNA-binding protein, is essential. Cell 81, 403-412. 16. Haghighat, A., Mader, S., Pause, A., and Sonenberg, N. (1995). Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J. 14, 5701-5709. 17. Wilhelm, J.E., Mansfield, J., Hom-Booher, N., Wang, S., Turck, C.W., Hazelrigg, T., and Vale, R.D. (2000). Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes. J. Cell Biol. 148, 427-440. 18. van Eeden, F.J., Palacios, I.M., Petronczki, M., Weston, M.J., and St Johnston, D. (2001). Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole. J. Cell Biol. 154, 511-523.