- Email: [email protected]
Nups, Kaps, NXFs and others: making sense in the Babel of nuclear transport
6
Research Update
TRENDS in Cell Biology Vol.12 No.1 January 2002
Nups, Kaps, NXFs and others: making sense in the Babel of nuclear transport Patrick J. Rogue The spatio-temporal aspects of signal transduction, and the molecular basis of nucleocytoplasmic transport, have attracted much attention recently. How they might be linked was the theme of a recent conference*. Many aspects of nuclear transport were discussed and novel results presented, either in talks or posters, by the approximately 80 participants. (*EMBO workshop on ‘Signal-transductionmediated regulation of nuclear transport’; 11–14 August 2001; Strasbourg, France. Organized by A.N. Malviya.)
The nuclear pore complex (NPC), which represents the passage obligé for access to the nuclear interior, is an obvious site for regulating nuclear transport. And, indeed, it does seem to be modulated by signalling pathways. The NPC has been observed in different morphological states by electron microscopy. Its nuclear basket component appears to be an especially dynamic structure, with a distal ring that opens and closes in response to ATP and micromolar Ca2+. H. Pickersgill (from the laboratories of M. Goldberg and T. Allen, Manchester, UK) reported that the NPC is also sensitive to Ran–GTP, but not Ran–GDP, suggesting that energy is required not only for driving macromolecules against their chemical gradients or overcoming entropic exclusion at the pore entrance, but also for converting the NPC to a favorable conformation. An important prerequisite to fully understanding NPC regulation is to identify and localize the nucleoporins (Nups). H. Pickersgill presented results demonstrating that CAN/Nup214 is localized to the central ring of the NPC. J. Hanover (Bethesda, USA) explained that Nups are often modified by addition of O-linked N-acetylglucosamine catalyzed by O-GlcNAc transferase (OGT). Overexpression of OGT has a dramatic effect on cell metabolism. However, the physiological significance of glycosylation or phosphorylation [1] remains difficult to interpret. F. Melchior (Martinsried, Germany) discussed the role of SUMO modification of the Ran system. She had demonstrated previously that RanGAP1 is conjugated http://tcb.trends.com
by SUMO-1, which targets it to Nup358/RanBP2 on the NPC cytoplasmic filaments. She now presented data showing that Nup358/RanBP2 is itself a SUMO-1 E3 ligase. Because many known SUMO substrates contain a functional nuclear-localization sequence (NLS), this suggests that SUMO conjugation might occur during the translocation process. It could thus be involved in the regulation of the nuclear transport of these substrates. S. Swaminathan and colleagues, from the F. Melchior lab, showed that RanGAP1 is phosphorylated by Cdk1–cyclin B, although this modification does not affect its ability to stimulate the Ran GTPase in vitro. The cytoskeleton shared part of the limelight at the meeting. Much is known about the nucleocytoplasmic phase of transport, but how proteins come close enough to the NPC to engage the import machinery is less well understood. It was long thought that diffusion alone presided over the approach to nuclear pores, but now a picture is emerging of a highly organized process that involves the cytoskeleton, particularly the microtubule network and its motor proteins. Thus, accumulation of p53 in the nucleus in response to DNA damage requires a functional microtubule network, and dynein moves p53 along the network towards the NPC (P. Giannakakou, Atlanta, USA). Especially interesting in the context of the meeting is the observation that signals affecting the normal function of microtubules also regulate p53 signaling. Ca2++ signaling and nuclear transport
Recent results have suggested that cytosolic Ca2+ transients might regulate a novel ATP-dependent import pathway. However, J. Hanover introduced a cautionary note concerning nuclear transport that is not coupled to GTP hydrolysis. In digitonin-permeabilized cells, NLS-dependent protein import in the presence of low Ca2+ concentrations requires GTP, whereas at elevated Ca2+ concentrations it can occur even in the absence of GTP, provided ATP and
calmodulin are added. But calmodulin is not a true import factor, and what one might be seeing is a change in the conformation of the NPC induced by calmodulin in the presence of ATP. Similarly, in digitonin-permeabilized cells, export of Rev requires the addition ATP, not GTP. This unusual dependence on ATP does not derive from intrinsic requirements of the export pathway but from the tethering of Rev to the nucleolus, with a subsequent ATP-dependent release step. J. Hanover also reported that calreticulin can serve as an export factor for Rev (Fig. 1). Nuclear export is relatively insensitive to Ca2+, and calreticulin is thus a rather unlikely export factor – in fact, Ca2+ signals might only contribute to the process by facilitating the release of calreticulin from the endoplasmic reticulum (ER). It is now well accepted that intranuclear Ca2+ signals can be generated by the liberation of Ca2+ from a pool in the lumen of the nuclear envelope (NE) through activation of secondmessenger-regulated release channels located on the inner leaflet. At least two such release channels have been characterized: inositol (1,4,5)trisphosphate receptors (IP3Rs) and ryanodine receptors. Nuclear IP3Rs have been observed to cluster in patches in the vicinity of the NPCs: presumably, they induce high Ca2+ microdomains that could modulate the conformation of NPCs directly. The NE is also endowed with Ca2+-uptake systems, including a Ca2+ATPase on the outer NE leaflet (NCA) and inositol (1,3,4,5)-tetrakisphosphate receptors reported on the inner leaflet [2], that replenish the NE Ca2+ pool and thus indirectly influence the transport of macromolecules. Indeed, A.N. Malviya and C. Gensburger (Strasbourg, France) presented results from in vivo studies showing how the Ca2+ pumping activity of NCA is modulated by phosphorylation by protein kinase A (PKA), with subsequent changes in the diffusion of macromolecules into the nucleus. Treatment of cells in culture with db-cAMP triggers Ca2+ accumulation in
0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02204-8
Research Update
TRENDS in Cell Biology Vol.12 No.1 January 2002
7
Ca 2
Signal
B A
Ca2+/CM
Ca2+
+
Ca2+/CM B A NES
IP3
B
(3)
NE
S
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Ca2+/CM B A NES
NES
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(2)
/C
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Ca2+/CM
NE
Crm1
S
NES NLS
Other NFAT kinase
Ca2+ Ca2+/CM B A NES
Casein kinase1
NLS
NLS B A
CM TRENDS in Cell Biology
Fig. 1. Signal-regulated nuclear transport: nuclear transport appears to be a highly regulated process, with many potential levels of control. For example, calcium signals (1) modulate the conformation of the nuclear pore complex (NPC) in an ATP-dependent fashion, acting either directly on contractile components of the NPC (actin, myosin) or through nuclear calmodulin. Furthermore, Ca2+ in the NE lumen modulates diffusion of macromolecules through the NPC, possibly by means of gp210, an NE protein that interacts with the NPC and presents an intraluminal EF hand motif; (2) act, indirectly, at the level of transport complexes: calreticulin (Crt), which is apparently released from the ER under stimulated conditions, can serve as an export factor for both Rev and the glucocorticoid receptor (GR) in a process that involves formation of a trimeric complex with RanGTP; (3) act at the level of protein cargos themselves, by regulating the accessibility of NLSs and NESs to the nuclear transport machinery. Thus, phospho- and dephospho-NFATs differ in their kinetics of nuclear import and export. Calcineurin regulates both the nuclear import (it dephosphorylates the NFAT regulatory region, unmasking two NLSs and sending it into the nucleus) and export (it competes with Crm1 for binding to NFAT). Abbreviations: A, B, catalytic and regulatory Ca2+-binding subunits of calcineurin, respectively; Ca2+/CM, calcium/calmodulin; ER, endoplasmic reticulum; NE, nuclear envelope; NES, nuclear-export sequence; NLS, nuclear-localization sequence.
the NE lumen (visualized by fluo-3AM) along with accumulation of 10-kDa dextran into the nucleoplasm. Whether modulation of Ca2+ levels in the NE can also influence NLS-dependent import remains uncertain. In his talk, O. Petersen (Liverpool, UK) focused on the function of perinuclear mitochondria in shielding the nucleoplasm from invasions by ‘unwanted’ cytosolic Ca2+ waves. http://tcb.trends.com
The export of mRNA
The issue of how mRNA gets out of the nucleus is of course very important. A major step in our understanding occurred with the identification of Tap/NXF1 as the crucial export factor. E. Izaurralde (Heidelberg, Germany) and B. Cullen (Durham, USA) discussed structure–function relationships for Tap/NXF1, which shows a conserved
modular organization, with separate RNA-binding and nuclear-export domains. A controversial issue concerns the Tap/NXF1 binding partner, p15/NXT1, which was described by C. Dargemont (Paris, France) as also binding to Crm1. Using a novel in vitro RNA export assay, she analyzed its role in the export of U1 snRNA, tRNA, mRNA and in the Crm1-dependent export of NES-containing proteins [3]. It appears that p15/NXT1 is not only involved in Tap-mediated mRNA export, but also in the Crm1- and Ran–GTP-dependent Rev export pathway, where it stimulates the dissociation of the export complex in the cytoplasm. However, neither E. Izaurralde nor B. Cullen find an interaction between p15/NXT1 and Crm1 or Ran, and Rev function is unaffected if p15/NXT1 expression is knocked-out in tissue-culture cells using RNA
8
Research Update
interference. The basis for the observed differences remains unclear. NXF proteins define an evolutionarily conserved family of putative mRNAexport receptors, distinct from karyopherins (Kaps). hNXF5, for instance, is expressed mainly in the brain, and interruption of the nxf5 gene by a chromosomal inversion is associated with a form of male mental retardation [4]. A. Hatton (from the P. Silver laboratory, Boston, USA) described how stress signals differentially regulate the export of mRNPs through coordinated phosphorylation and arginine methylation of hnRNP proteins. And in his lecture, G. Dreyfuss (Philadelphia, USA) discussed a key regulatory process that couples splicing with nonsense-mediated decay (NMD), a cytoplasmic qualitycontrol system that degrades aberrant transcripts. Spliceosomes deposit on mRNAs a specific set of proteins in a sequence-independent but positionspecific manner, forming multicomponent complexes ~20 nucleotides upstream of the exon–exon junctions (EEJ complexes). G. Dreyfuss presented exciting new data concerning Y14 and its interactors Upf3 and Magoh [5], showing how these EEJ complex components remain associated with the mRNA in the cytoplasm and thus communicate positional information that is essential for the NMD system. This suggests a model whereby, during translation, Y14/Magoh/Upf3 complexes are sequentially removed. In the vast majority of transcripts, the legitimate termination codon is found on the last exon – by the time the ribosome reaches there, all Y14/Magoh/Upf3 complexes will have gone. But, if the ribosome encounters a premature termination codon, it cannot distinguish it from the wild-type stop codon and disassembles just the same, leaving Y14/Magoh/Upf3 complexes on the mRNA. Upf3 will then recruit mRNA degradation factors, initiating the NMD response. Regulating nuclear transport at freight level
Masking targeting sequences by phosphorylation or peptide binding is a common mechanism for regulating the accessibility of cargos to the transport machinery. In her lecture, however, A. Rao (Boston, USA) emphasized that, in the case of heavily phosphorylated proteins such as NFAT, progressive phosphorylation http://tcb.trends.com
TRENDS in Cell Biology Vol.12 No.1 January 2002
does not function solely to occlude localization sequences but also confers cooperativity as it translates into functional changes. Thus, dephosphorylation by calcineurin of the so-called ‘gate-keeper’ region in NFAT unfolds the protein sufficiently such that the other regulatory domain phosphoserines (13 in all) can also be dephosphorylated, and their accessibility increases cooperatively with the number of sites already dephosphorylated. Mathematical modeling (C. Salazar, Berlin, Germany) suggests a threshold effect, compatible with this process, whereby a small change in the Ca2+ signal can induce complete NFAT activation. Remarkably, casein kinase 1, the major NFAT kinase for the gate-keeper region, shows a pattern of processive phosphorylation also in accord with this model. E. Carafoli (Padova, Italy) discussed another pathway by which calcineurin controls transcription, involving nuclear import of calcineurin itself [6]. Concluding remarks
Amongst the other topics, U. Greber (Zurich, Switzerland) described how viruses manipulate the transport machinery, escaping checkpoints and ‘highjacking’ molecular motors: in essence, these large cargoes import themselves. Also, J.M. Lehn (Strasbourg, France), 1987 Nobel prizewinner in Chemistry, lectured on the promises of ‘bio-organic supramolecular chemistry’. All in all, the regulation of such a tremendous and vital process as nuclear transport is surely complex. One goal is to understand how it is fine tuned by signaling pathways. Yet, despite much progress, it is still unclear in what way this is achieved. Nonetheless, this EMBO
workshop reflected the dynamism of current research. Certainly, those involved in the field are more fortunate than the apocryphal Alexander The Great, who cried because there were ‘no more worlds to conquer’. Acknowledgements
Owing to limitations on space, but certainly not for lack of interest, only certain presentations are highlighted in this article. References 1 Kehlenbach, R.H. and Gerace, L. (2000) Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro. J. Biol. Chem. 275, 17848–17856 2 Humbert, J.P. et al. (1996) Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear Ca2+ signaling by inositol 1,4,5-trisphosphate. Discrete distribution of inositol phosphate receptors to inner and outer nuclear membranes. J. Biol. Chem. 271, 478–485 3 Ossareh-Nazari, B. et al. (2000) RanGTP-binding protein NXT1 facilitates nuclear export of different classes of RNA in vitro. Mol. Cell. Biol. 20, 4562–4571 4 Jun, L. et al. (2001) NXF5, a novel member of the nuclear RNA export family, is lost in a male patient with a syndromic form of mental retardation. Curr. Biol. 11, 1381–1391 5 Kataoka, N. et al. (2001) Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon–exon junction complex. EMBO J. 20, 6424–6433 6 Li, L. et al. (2000) Calcineurin controls the transcription of Na+/Ca2+ exchanger isoforms in developing cerebellar neurons. J. Biol. Chem. 275, 20903–29010
Patrick J. Rogue Centre de Neurochimie, 5 rue Blaise Pascal, Strasbourg cedex, 67084, France. e-mail: [email protected]
Trends in Cell Biology editorial policy Articles in Trends in Cell Biology are generally commissioned by the editorial team, but proposals and suggestions are welcome and are particularly encouraged for the Opinion section. Prospective authors should send a short synopsis (~200 words) with key references to the Editor at [email protected]. Full guidelines on manuscript preparation will be supplied if the proposal is accepted. Please note that completed articles should not be submitted without prior consultation and that all major review articles are peer reviewed and publication cannot be guaranteed.
Research Update
TRENDS in Cell Biology Vol.12 No.1 January 2002
Nups, Kaps, NXFs and others: making sense in the Babel of nuclear transport Patrick J. Rogue The spatio-temporal aspects of signal transduction, and the molecular basis of nucleocytoplasmic transport, have attracted much attention recently. How they might be linked was the theme of a recent conference*. Many aspects of nuclear transport were discussed and novel results presented, either in talks or posters, by the approximately 80 participants. (*EMBO workshop on ‘Signal-transductionmediated regulation of nuclear transport’; 11–14 August 2001; Strasbourg, France. Organized by A.N. Malviya.)
The nuclear pore complex (NPC), which represents the passage obligé for access to the nuclear interior, is an obvious site for regulating nuclear transport. And, indeed, it does seem to be modulated by signalling pathways. The NPC has been observed in different morphological states by electron microscopy. Its nuclear basket component appears to be an especially dynamic structure, with a distal ring that opens and closes in response to ATP and micromolar Ca2+. H. Pickersgill (from the laboratories of M. Goldberg and T. Allen, Manchester, UK) reported that the NPC is also sensitive to Ran–GTP, but not Ran–GDP, suggesting that energy is required not only for driving macromolecules against their chemical gradients or overcoming entropic exclusion at the pore entrance, but also for converting the NPC to a favorable conformation. An important prerequisite to fully understanding NPC regulation is to identify and localize the nucleoporins (Nups). H. Pickersgill presented results demonstrating that CAN/Nup214 is localized to the central ring of the NPC. J. Hanover (Bethesda, USA) explained that Nups are often modified by addition of O-linked N-acetylglucosamine catalyzed by O-GlcNAc transferase (OGT). Overexpression of OGT has a dramatic effect on cell metabolism. However, the physiological significance of glycosylation or phosphorylation [1] remains difficult to interpret. F. Melchior (Martinsried, Germany) discussed the role of SUMO modification of the Ran system. She had demonstrated previously that RanGAP1 is conjugated http://tcb.trends.com
by SUMO-1, which targets it to Nup358/RanBP2 on the NPC cytoplasmic filaments. She now presented data showing that Nup358/RanBP2 is itself a SUMO-1 E3 ligase. Because many known SUMO substrates contain a functional nuclear-localization sequence (NLS), this suggests that SUMO conjugation might occur during the translocation process. It could thus be involved in the regulation of the nuclear transport of these substrates. S. Swaminathan and colleagues, from the F. Melchior lab, showed that RanGAP1 is phosphorylated by Cdk1–cyclin B, although this modification does not affect its ability to stimulate the Ran GTPase in vitro. The cytoskeleton shared part of the limelight at the meeting. Much is known about the nucleocytoplasmic phase of transport, but how proteins come close enough to the NPC to engage the import machinery is less well understood. It was long thought that diffusion alone presided over the approach to nuclear pores, but now a picture is emerging of a highly organized process that involves the cytoskeleton, particularly the microtubule network and its motor proteins. Thus, accumulation of p53 in the nucleus in response to DNA damage requires a functional microtubule network, and dynein moves p53 along the network towards the NPC (P. Giannakakou, Atlanta, USA). Especially interesting in the context of the meeting is the observation that signals affecting the normal function of microtubules also regulate p53 signaling. Ca2++ signaling and nuclear transport
Recent results have suggested that cytosolic Ca2+ transients might regulate a novel ATP-dependent import pathway. However, J. Hanover introduced a cautionary note concerning nuclear transport that is not coupled to GTP hydrolysis. In digitonin-permeabilized cells, NLS-dependent protein import in the presence of low Ca2+ concentrations requires GTP, whereas at elevated Ca2+ concentrations it can occur even in the absence of GTP, provided ATP and
calmodulin are added. But calmodulin is not a true import factor, and what one might be seeing is a change in the conformation of the NPC induced by calmodulin in the presence of ATP. Similarly, in digitonin-permeabilized cells, export of Rev requires the addition ATP, not GTP. This unusual dependence on ATP does not derive from intrinsic requirements of the export pathway but from the tethering of Rev to the nucleolus, with a subsequent ATP-dependent release step. J. Hanover also reported that calreticulin can serve as an export factor for Rev (Fig. 1). Nuclear export is relatively insensitive to Ca2+, and calreticulin is thus a rather unlikely export factor – in fact, Ca2+ signals might only contribute to the process by facilitating the release of calreticulin from the endoplasmic reticulum (ER). It is now well accepted that intranuclear Ca2+ signals can be generated by the liberation of Ca2+ from a pool in the lumen of the nuclear envelope (NE) through activation of secondmessenger-regulated release channels located on the inner leaflet. At least two such release channels have been characterized: inositol (1,4,5)trisphosphate receptors (IP3Rs) and ryanodine receptors. Nuclear IP3Rs have been observed to cluster in patches in the vicinity of the NPCs: presumably, they induce high Ca2+ microdomains that could modulate the conformation of NPCs directly. The NE is also endowed with Ca2+-uptake systems, including a Ca2+ATPase on the outer NE leaflet (NCA) and inositol (1,3,4,5)-tetrakisphosphate receptors reported on the inner leaflet [2], that replenish the NE Ca2+ pool and thus indirectly influence the transport of macromolecules. Indeed, A.N. Malviya and C. Gensburger (Strasbourg, France) presented results from in vivo studies showing how the Ca2+ pumping activity of NCA is modulated by phosphorylation by protein kinase A (PKA), with subsequent changes in the diffusion of macromolecules into the nucleus. Treatment of cells in culture with db-cAMP triggers Ca2+ accumulation in
0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02204-8
Research Update
TRENDS in Cell Biology Vol.12 No.1 January 2002
7
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B A
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+
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NE
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NES NLS
Other NFAT kinase
Ca2+ Ca2+/CM B A NES
Casein kinase1
NLS
NLS B A
CM TRENDS in Cell Biology
Fig. 1. Signal-regulated nuclear transport: nuclear transport appears to be a highly regulated process, with many potential levels of control. For example, calcium signals (1) modulate the conformation of the nuclear pore complex (NPC) in an ATP-dependent fashion, acting either directly on contractile components of the NPC (actin, myosin) or through nuclear calmodulin. Furthermore, Ca2+ in the NE lumen modulates diffusion of macromolecules through the NPC, possibly by means of gp210, an NE protein that interacts with the NPC and presents an intraluminal EF hand motif; (2) act, indirectly, at the level of transport complexes: calreticulin (Crt), which is apparently released from the ER under stimulated conditions, can serve as an export factor for both Rev and the glucocorticoid receptor (GR) in a process that involves formation of a trimeric complex with RanGTP; (3) act at the level of protein cargos themselves, by regulating the accessibility of NLSs and NESs to the nuclear transport machinery. Thus, phospho- and dephospho-NFATs differ in their kinetics of nuclear import and export. Calcineurin regulates both the nuclear import (it dephosphorylates the NFAT regulatory region, unmasking two NLSs and sending it into the nucleus) and export (it competes with Crm1 for binding to NFAT). Abbreviations: A, B, catalytic and regulatory Ca2+-binding subunits of calcineurin, respectively; Ca2+/CM, calcium/calmodulin; ER, endoplasmic reticulum; NE, nuclear envelope; NES, nuclear-export sequence; NLS, nuclear-localization sequence.
the NE lumen (visualized by fluo-3AM) along with accumulation of 10-kDa dextran into the nucleoplasm. Whether modulation of Ca2+ levels in the NE can also influence NLS-dependent import remains uncertain. In his talk, O. Petersen (Liverpool, UK) focused on the function of perinuclear mitochondria in shielding the nucleoplasm from invasions by ‘unwanted’ cytosolic Ca2+ waves. http://tcb.trends.com
The export of mRNA
The issue of how mRNA gets out of the nucleus is of course very important. A major step in our understanding occurred with the identification of Tap/NXF1 as the crucial export factor. E. Izaurralde (Heidelberg, Germany) and B. Cullen (Durham, USA) discussed structure–function relationships for Tap/NXF1, which shows a conserved
modular organization, with separate RNA-binding and nuclear-export domains. A controversial issue concerns the Tap/NXF1 binding partner, p15/NXT1, which was described by C. Dargemont (Paris, France) as also binding to Crm1. Using a novel in vitro RNA export assay, she analyzed its role in the export of U1 snRNA, tRNA, mRNA and in the Crm1-dependent export of NES-containing proteins [3]. It appears that p15/NXT1 is not only involved in Tap-mediated mRNA export, but also in the Crm1- and Ran–GTP-dependent Rev export pathway, where it stimulates the dissociation of the export complex in the cytoplasm. However, neither E. Izaurralde nor B. Cullen find an interaction between p15/NXT1 and Crm1 or Ran, and Rev function is unaffected if p15/NXT1 expression is knocked-out in tissue-culture cells using RNA
8
Research Update
interference. The basis for the observed differences remains unclear. NXF proteins define an evolutionarily conserved family of putative mRNAexport receptors, distinct from karyopherins (Kaps). hNXF5, for instance, is expressed mainly in the brain, and interruption of the nxf5 gene by a chromosomal inversion is associated with a form of male mental retardation [4]. A. Hatton (from the P. Silver laboratory, Boston, USA) described how stress signals differentially regulate the export of mRNPs through coordinated phosphorylation and arginine methylation of hnRNP proteins. And in his lecture, G. Dreyfuss (Philadelphia, USA) discussed a key regulatory process that couples splicing with nonsense-mediated decay (NMD), a cytoplasmic qualitycontrol system that degrades aberrant transcripts. Spliceosomes deposit on mRNAs a specific set of proteins in a sequence-independent but positionspecific manner, forming multicomponent complexes ~20 nucleotides upstream of the exon–exon junctions (EEJ complexes). G. Dreyfuss presented exciting new data concerning Y14 and its interactors Upf3 and Magoh [5], showing how these EEJ complex components remain associated with the mRNA in the cytoplasm and thus communicate positional information that is essential for the NMD system. This suggests a model whereby, during translation, Y14/Magoh/Upf3 complexes are sequentially removed. In the vast majority of transcripts, the legitimate termination codon is found on the last exon – by the time the ribosome reaches there, all Y14/Magoh/Upf3 complexes will have gone. But, if the ribosome encounters a premature termination codon, it cannot distinguish it from the wild-type stop codon and disassembles just the same, leaving Y14/Magoh/Upf3 complexes on the mRNA. Upf3 will then recruit mRNA degradation factors, initiating the NMD response. Regulating nuclear transport at freight level
Masking targeting sequences by phosphorylation or peptide binding is a common mechanism for regulating the accessibility of cargos to the transport machinery. In her lecture, however, A. Rao (Boston, USA) emphasized that, in the case of heavily phosphorylated proteins such as NFAT, progressive phosphorylation http://tcb.trends.com
TRENDS in Cell Biology Vol.12 No.1 January 2002
does not function solely to occlude localization sequences but also confers cooperativity as it translates into functional changes. Thus, dephosphorylation by calcineurin of the so-called ‘gate-keeper’ region in NFAT unfolds the protein sufficiently such that the other regulatory domain phosphoserines (13 in all) can also be dephosphorylated, and their accessibility increases cooperatively with the number of sites already dephosphorylated. Mathematical modeling (C. Salazar, Berlin, Germany) suggests a threshold effect, compatible with this process, whereby a small change in the Ca2+ signal can induce complete NFAT activation. Remarkably, casein kinase 1, the major NFAT kinase for the gate-keeper region, shows a pattern of processive phosphorylation also in accord with this model. E. Carafoli (Padova, Italy) discussed another pathway by which calcineurin controls transcription, involving nuclear import of calcineurin itself [6]. Concluding remarks
Amongst the other topics, U. Greber (Zurich, Switzerland) described how viruses manipulate the transport machinery, escaping checkpoints and ‘highjacking’ molecular motors: in essence, these large cargoes import themselves. Also, J.M. Lehn (Strasbourg, France), 1987 Nobel prizewinner in Chemistry, lectured on the promises of ‘bio-organic supramolecular chemistry’. All in all, the regulation of such a tremendous and vital process as nuclear transport is surely complex. One goal is to understand how it is fine tuned by signaling pathways. Yet, despite much progress, it is still unclear in what way this is achieved. Nonetheless, this EMBO
workshop reflected the dynamism of current research. Certainly, those involved in the field are more fortunate than the apocryphal Alexander The Great, who cried because there were ‘no more worlds to conquer’. Acknowledgements
Owing to limitations on space, but certainly not for lack of interest, only certain presentations are highlighted in this article. References 1 Kehlenbach, R.H. and Gerace, L. (2000) Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro. J. Biol. Chem. 275, 17848–17856 2 Humbert, J.P. et al. (1996) Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear Ca2+ signaling by inositol 1,4,5-trisphosphate. Discrete distribution of inositol phosphate receptors to inner and outer nuclear membranes. J. Biol. Chem. 271, 478–485 3 Ossareh-Nazari, B. et al. (2000) RanGTP-binding protein NXT1 facilitates nuclear export of different classes of RNA in vitro. Mol. Cell. Biol. 20, 4562–4571 4 Jun, L. et al. (2001) NXF5, a novel member of the nuclear RNA export family, is lost in a male patient with a syndromic form of mental retardation. Curr. Biol. 11, 1381–1391 5 Kataoka, N. et al. (2001) Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon–exon junction complex. EMBO J. 20, 6424–6433 6 Li, L. et al. (2000) Calcineurin controls the transcription of Na+/Ca2+ exchanger isoforms in developing cerebellar neurons. J. Biol. Chem. 275, 20903–29010
Patrick J. Rogue Centre de Neurochimie, 5 rue Blaise Pascal, Strasbourg cedex, 67084, France. e-mail: [email protected]
Trends in Cell Biology editorial policy Articles in Trends in Cell Biology are generally commissioned by the editorial team, but proposals and suggestions are welcome and are particularly encouraged for the Opinion section. Prospective authors should send a short synopsis (~200 words) with key references to the Editor at [email protected]. Full guidelines on manuscript preparation will be supplied if the proposal is accepted. Please note that completed articles should not be submitted without prior consultation and that all major review articles are peer reviewed and publication cannot be guaranteed.