- Email: [email protected]
Is the nucleus in need of translation?
Research Update
TRENDS in Cell Biology Vol.11 No.10 October 2001
395
Research News
Is the nucleus in need of translation? Thoru Pederson In prokaryotic cells, protein synthesis commences on nascent messenger RNAs while they are still being synthesized on the DNA template. The eukaryotic nuclear envelope spatially segregates protein synthesis away from the genome and messenger RNA production – this being thought to be a defining feature of the Eukarya. New results suggest that the eukaryotic ‘standard model’ might be an oversimplification. How good is the evidence for translation in the cell nucleus, and, if sound, where do the new findings take us?
In prokaryotes, ribosomes bind to 5′-end entry sites on messenger RNAs while the transcript is still being made, and translation commences concurrently, there being no reason to wait until the mRNA is completed. So long as the rates of translational initiation and elongation don’t exceed the velocity of RNA polymerase along the DNA template, this straightforward process works. In eukaryotes, it has long been thought that the physical separation provided by the nucleus between the genome and at least the great majority of the protein synthetic apparatus has some profound evolutionary meaning. It almost certainly does, and the notion that mRNA and proteins are produced at different sites remains an unsullied general principle of the molecular genetics of eukaryotic cells. However, an exception to this longstanding ‘rule’ has appeared on the scene. A recent paper now provides a plausible case that some protein synthesis occurs in the nucleus of mammalian cells1.
contamination (i.e. ribosomes, tRNAs and other protein synthesis factors). We shall view these early studies in more detail later, but in essence these experiments were neither individually nor collectively persuasive, at least to the great majority of eukaryotic cell biologists and to the molecular biology community. The main difficulty with these early studies was ruling out the possibility that some, almost finished, cytoplasmic translation products picked up label and moved into the nuclei during the experiment, however brief the incubation time. The recent study by Cook and colleagues presents an interesting approach to circumvent this problem1. Rather than using a labeled amino acid, they incubated permeabilized HeLa cells with a biotin-tagged lysyl-tRNAlys. In addition to the biotin-lysyl tRNAlys, the system contained the other 19 amino acids, bovine liver tRNAs and aminoacyltRNA synthetases, GTP, phosphocreatine and creatine phosphokinase. Suboptimal conditions for translation were used so that nascent polypeptides became extended an average of 15 amino acids, minimizing the likelihood that much of the label ends up in completed proteins that can move away from the synthesis sites to other locations (see below). Other experiments established that the protein synthesis observed in this system was sensitive to inhibitors of eukaryotic protein synthesis and did not reflect
the activity of contaminating bacterial ribosomes. The biotin-lysyl-tRNAlys became incorporated into proteasesensitive material to the same extent as 3H-lysine or 35S-methionine, as revealed by avidin selection of biotin-containing polypeptides and analysis of their 3H:35S ratios. Finally, additional controls established that the detection of biotin labeling sites in the permeabilized cells was not compromised by endogenous biotin depots. Nuclear translation sites
The sites of biotin-lysyl-tRNAlys labeling were mainly cytoplasmic, as expected, but there were also distinct foci of incorporation within the nuclei, both scattered about in the nucleoplasm and in the nucleoli (Fig. 1b). Confocal microscopy convincingly revealed intranuclear foci, as did gold immunoelectron microscopy detection of the incorporated biotin-lysine (Fig. 2a). The labeling of the nuclear sites was greatly reduced in the presence of the translation inhibitor cycloheximide (Fig. 1c). As mentioned above, the longstanding problem with such studies is that, however brief the label time, there might nonetheless be some nuclear import of just-completed proteins from the cytoplasm. In the new studies, the average elongation of only ~15 amino acids was achieved not merely by the brevity of the label time, but also by use of a sub-physiological temperature (27.5°C) and a deliberately suboptimal
Designing an experimental approach
Protein synthesis in the nucleus was claimed back in the 1950s and 1960s, in studies in which isolated liver and thymocyte nuclei were found to incorporate radioactive amino acids into high molecular mass material2,3. The authors of these papers presented evidence, of varying cogency, that their isolated nuclei were relatively, or in some cases largely, free of cytoplasmic http://tcb.trends.com
Fig. 1. Biotin-lysyl-tRNAlys incorporation of permeabilized HeLa cells. Cells were allowed to extend nascent peptides labeled with biotin-lysyl-tRNAlys in the presence of 19 amino acids, bovine liver tRNAs and aminoacyl-tRNA synthetases, GTP, phosphocreatine and creatine phosphokinase at 27.5°C. Samples were taken and examined by confocal microscopy either immediately before labeling (a), revealing the endogenous background labeling, especially by mitochondria, or after 10 min of labeling in the absence (b) or presence (c) of 1 mg ml–1 cycloheximide (chx), an inhibitor of eukaryotic protein synthesis. Note the nuclear foci of incorporated label in (b), which is sensitive to the presence of chx (c). Bar, 3 µm. (Images reproduced by permission of the American Association for the Advancement of Science. See Ref. 1 for further details.)
0962-8924/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02105-5
396
Research Update
TRENDS in Cell Biology Vol.11 No.10 October 2001
rNTP-dependent increase in biotin-lysine incorporation was not observed. Most intriguing, dual-diameter gold immunoelectron microscopy showed that the sites of biotin-lysine labeling tended to be near sites of Br-UTP incorporation as well as close to the location of the SR class of splicing factors. These are extremely provocative results, indicating that nuclear translation might depend on transcription – reminiscent of the way in which these processes are coupled in prokaryotes. It is of interest to note that, in one of the more convincing early studies, the incorporation of 14C-amino acids in isolated thymocyte nuclei was observed to be significantly reduced if the nuclei were first exposed to the RNA synthesis inhibitor DRB (Ref. 4). Fig. 2. Gold immunoelectron microscopy detection of biotin-lysyl-tRNAlys incorporation. Permeabilized HeLa cells were incubated as described in Fig. 1 and a sample prepared for electron microscopy (EM) after 10 min of labeling with biotin-lysyl-tRNAlys. The dark line from 1 to 7 o’clock is the nuclear envelope. On its right is the nucleus, to its left is the cytoplasm. Note the dark spots indicating foci of intranuclear biotin incorporation (bio-peptide). Bar, 200 nm. (b, c) Dual-diameter gold immuno-EM shows that the sites of biotin-lysine labeling of extending peptides tend to be near sites of RNA synthesis, indicated by Br-UTP incorporation (b), as well as close to the location of the ribosomal protein L7 (c). Bar, 50 nm. (Images reproduced by permission of the American Association for the Advancement of Science. See Ref. 1 for further details.)
concentration of amino acids. In addition, the investigators carried out parallel studies with isolated nuclei, which they estimated to be freed of ∼95% of the cytoplasmic ribosomes initially present in intact cells, and observed approximately the same level of labeling inside the nuclei as in the permeabilized cell experiments. In another key control, the authors observed that a concentration of aurin tricarboxylic acid (ATA) known to block translational initiation did not reduce the nuclear labeling, whereas higher concentrations of ATA that block elongation reduced nuclear labeling by ~90%, further bolstering the case that the nuclear labeling represents incorporation of the ‘proximal precursor’ biotin-lysyltRNAlys into sites that are already engaged in translation. Although most of the earlier studies on protein synthesis in the nucleus probably reflected rapid nuclear import of newly made proteins synthesized on cytoplasmic ribosomes2,3, there was one finding, however, that seemed particularly interesting and quite convincing. In an extensive series of studies by Vincent Allfrey and Alfred Mirsky of protein synthesis in nuclei carefully isolated from thymocytes, their claimed nuclear protein http://tcb.trends.com
synthesis was observed to display a distinctive dependence on sodium ions4. This was, and remains, an intriguing finding since there is evidence that the nucleus sequesters sodium ions to a level about 12-fold higher than the cytoplasm5. The permeabilized cell system of Iborra et al. lends itself nicely to investigating the Na+ versus K+ dependence of the intranuclear biotin-lysine incorporation. Is nuclear translation coupled with transcription?
Cook and colleagues also suggest a link between nuclear translation and transcription1. When the standard biotinlysyl-tRNAlys labeling experiment was performed in the presence of increasing concentrations of ribonucleoside triphosphates, it was found that the amount of nuclear biotin-lysine incorporation increased. Moreover, the rNTP-dependent increase in biotin-lysine incorporation was not observed if the endogenous RNA polymerase II was selectively poisoned with α-amanitin. Also, if the RNA-chain-terminating adenosine analog cordycepin (3′-deoxyadenosine triphosphate) was added, or an essential RNA synthesis nucleotide (CTP) was omitted, the
What might be the function of translation in the nucleus?
Among their findings, Iborra et al. emphasize the provocative presence of numerous translation factors in the nucleus (see also Ref. 6). But presence does not necessarily signify function, and the roles of various translation factors that are present in the nucleus still remain unclear. It is conceivable, as Iborra et al. suggest, that nuclear translation functions in nonsensemediated decay (NMD), a quality-control mechanism in which messenger RNAs are surveyed for the presence of nonsense codons. The tRNA-assisted ribosome is the best, and admittedly the only, mRNA codon surveyor we know. Because NMD appears to occur within the nucleus or to be intimately connected to nuclear events, the results of Iborra et al. will expectedly stir the interest of those in the NMD field. Although the new results do not directly bear on nonsensemediated mRNA decay, at least the NMD field can now proceed from the stronger epistemological position of envisioning ribosome-based scanning as a plausible nuclear process. If there is indeed translation in the nucleus, what are the protein products, where do they go and what do they do? Iborra et al. estimate that the nuclear translation they observe constitutes ~15% of the total protein synthesis of the cell. Their data indicate that much of the observed labeling is in high molecular mass proteins. Are these translation products used to build the nucleus and
Research Update
TRENDS in Cell Biology Vol.11 No.10 October 2001
support nuclear functions? Notwithstanding a possible nucleuslocalized NMD pathway, are there nucleus-restricted mRNAs that are translationally competent but nuclear export incompetent? And what about the necessary nuclear ribosomes? Are they the most recently assembled ones, just out of the nucleolus and taking one fleeting stab at translation before they exit the nucleus? And since there is no evidence for polysomes in the nucleus, is the initiation rate of the reported nuclear translation low? A low initiation rate is expected for NMD scanning, contrasting with the higher initiation rates of cytoplasmic mRNAs (thus generating polysomes), where the biological aim is protein production, not quality control. And what about the labeling Iborra et al. see in the nucleolus itself, the very place ribosomes are being assembled? Is there mRNA in the nucleolus? There is some evidence that there might be7,8. Are these assembling nucleolar ribosomes functional? Are the nucleolar sites of labeling observed by Iborra et al. in the granular component of the nucleolus, where finished ribosomes are present? Is nucleolar translation coupled to ribosomal RNA transcription? Are any of the mRNAs that are translated in the nucleus or nucleolus ones that encode secreted or membrane proteins? In such
cases, is there an involvement of the signal recognition particle (SRP)? Is this why the SRP appears itself to be assembled in the nucleolus6? These numerous questions, and others, are now rendered intellectually viable and scientifically tractable by the new results. Concluding remarks
The nucleus of most eukaryotic somatic cells is essentially and literally enveloped by a major protein synthesis system – the endoplasmic reticulum. Cytological studies in the 19th century and all through the 20th, together with evolutionary considerations, teach us that the eukaryotic nucleus and cytoplasm are separate worlds. The findings of Iborra et al. bring about a moment of reflection. Perhaps the nucleus and protein synthesis are more anciently, intimately and still today connected than their apparent cytological segregation implies. We are reminded of how little we know for sure. The American newspaper writer, H.L. Mencken, put it thus: ‘Penetrating so many secrets, we cease to believe in the unknowable. But there it sits, nevertheless, calmly licking its chops.’ Acknowledgements
I thank Mahlon Hoagland, Arthur Pardee and Joel Richter for their helpful
397
comments on a first draft. The author’s work is supported by grant GM-21595 from the US National Institutes of Health, which requires him to state, according to the agency’s rules, that the views expressed in this article are not those of the US government. (Did you really think they were?) References 1 Iborra, F.J. et al. (2001) Coupled transcription and translation within nuclei of mammalian cells. Science 293, 1139–1142 2 Goldstein, L. (1970) On the question of protein synthesis by cell nuclei. Adv. Cell Biol. 1, 187–210 3 Pederson, T. (1976) Cellular aspects of histone synthesis. Protein Synthesis (Vol. 2) (McConkey E.H., ed.), pp. 69–123, Marcel Dekker 4 Allfrey, V.G. et al. (1957). Protein synthesis in isolated cell nuclei. J. Gen. Physiol. 40, 451–490 5 Lagendorf, H. et al. (1961). Kationenverteilung in Zellkern und Cytoplasma der Rattenleber. Biochem. Z. 335, 273–284 6 Pederson, T. and Politz, J.C. (2000) The nucleolus and the four ribonucleoproteins of translation. J. Cell Biol. 148, 1091–1095 7 Harris, H. (1974) Nucleus and Cytoplasm (3rd edn), Oxford 8 Pederson, T. (1998) The plurifunctional nucleolus. Nucleic Acids Res. 26, 3871–3876
Thoru Pederson Dept of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 377 Plantation Street, Worcester, MA 01605, USA. e-mail: [email protected]
Meeting Report
Ubiquitin – more than just a signal for protein degradation Cezary Wójcik Research related to the ubiquitin- and proteasome-dependent proteindegradation pathway is growing exponentially. Top scientists in this field met recently in the green mountains of Vermont. Among the numerous topics discussed at this meeting* were ubiquitinlike proteins, ubiquitin-interacting proteins, non-proteolytic roles of ubiquitin and the regulation and function of proteasomes. (*FASEB Summer Conference: Ubiquitin and Intracellular Protein Degradation; Saxtons River, Vermont, USA; 23–28 June 2001. Organized by George DeMartino and Daniel Finley.) http://tcb.trends.com
The ubiquitin–proteasome pathway (UPP) of protein degradation is responsible for the degradation of most cell proteins, not only regulatory, abnormal and short-lived proteins, but also those that serve structural roles and are long lived1. Since the discovery of ubiquitin in the late 1970s, ubiquitin research has expanded into a formidable field impacting all areas of cell biology2. In a typical mammalian cell, 30% of the total protein pool is degraded within 10 min of its synthesis. This can be calculated to correspond to three substrates being degraded by each proteasome per minute
(Jon Yewdell, Bethesda, USA). In Arabidopsis thaliana, 4.6% of the proteome participates in the UPP (Richard D. Vierstra, Madison, USA). Ticket to ride
Ubiquitin, a small polypeptide of 76 amino acids, can be covalently attached to other proteins, forming polyubiquitin chains, using the E1–E2–E3 cascade of enzymes (Fig. 1). Polyubiquitin Lys48-linked chains are usually attached to internal lysines of the substrates, targeting them for degradation by the 26S proteasome. However, several proteins, such as MyoD,
0962-8924/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02084-0
TRENDS in Cell Biology Vol.11 No.10 October 2001
395
Research News
Is the nucleus in need of translation? Thoru Pederson In prokaryotic cells, protein synthesis commences on nascent messenger RNAs while they are still being synthesized on the DNA template. The eukaryotic nuclear envelope spatially segregates protein synthesis away from the genome and messenger RNA production – this being thought to be a defining feature of the Eukarya. New results suggest that the eukaryotic ‘standard model’ might be an oversimplification. How good is the evidence for translation in the cell nucleus, and, if sound, where do the new findings take us?
In prokaryotes, ribosomes bind to 5′-end entry sites on messenger RNAs while the transcript is still being made, and translation commences concurrently, there being no reason to wait until the mRNA is completed. So long as the rates of translational initiation and elongation don’t exceed the velocity of RNA polymerase along the DNA template, this straightforward process works. In eukaryotes, it has long been thought that the physical separation provided by the nucleus between the genome and at least the great majority of the protein synthetic apparatus has some profound evolutionary meaning. It almost certainly does, and the notion that mRNA and proteins are produced at different sites remains an unsullied general principle of the molecular genetics of eukaryotic cells. However, an exception to this longstanding ‘rule’ has appeared on the scene. A recent paper now provides a plausible case that some protein synthesis occurs in the nucleus of mammalian cells1.
contamination (i.e. ribosomes, tRNAs and other protein synthesis factors). We shall view these early studies in more detail later, but in essence these experiments were neither individually nor collectively persuasive, at least to the great majority of eukaryotic cell biologists and to the molecular biology community. The main difficulty with these early studies was ruling out the possibility that some, almost finished, cytoplasmic translation products picked up label and moved into the nuclei during the experiment, however brief the incubation time. The recent study by Cook and colleagues presents an interesting approach to circumvent this problem1. Rather than using a labeled amino acid, they incubated permeabilized HeLa cells with a biotin-tagged lysyl-tRNAlys. In addition to the biotin-lysyl tRNAlys, the system contained the other 19 amino acids, bovine liver tRNAs and aminoacyltRNA synthetases, GTP, phosphocreatine and creatine phosphokinase. Suboptimal conditions for translation were used so that nascent polypeptides became extended an average of 15 amino acids, minimizing the likelihood that much of the label ends up in completed proteins that can move away from the synthesis sites to other locations (see below). Other experiments established that the protein synthesis observed in this system was sensitive to inhibitors of eukaryotic protein synthesis and did not reflect
the activity of contaminating bacterial ribosomes. The biotin-lysyl-tRNAlys became incorporated into proteasesensitive material to the same extent as 3H-lysine or 35S-methionine, as revealed by avidin selection of biotin-containing polypeptides and analysis of their 3H:35S ratios. Finally, additional controls established that the detection of biotin labeling sites in the permeabilized cells was not compromised by endogenous biotin depots. Nuclear translation sites
The sites of biotin-lysyl-tRNAlys labeling were mainly cytoplasmic, as expected, but there were also distinct foci of incorporation within the nuclei, both scattered about in the nucleoplasm and in the nucleoli (Fig. 1b). Confocal microscopy convincingly revealed intranuclear foci, as did gold immunoelectron microscopy detection of the incorporated biotin-lysine (Fig. 2a). The labeling of the nuclear sites was greatly reduced in the presence of the translation inhibitor cycloheximide (Fig. 1c). As mentioned above, the longstanding problem with such studies is that, however brief the label time, there might nonetheless be some nuclear import of just-completed proteins from the cytoplasm. In the new studies, the average elongation of only ~15 amino acids was achieved not merely by the brevity of the label time, but also by use of a sub-physiological temperature (27.5°C) and a deliberately suboptimal
Designing an experimental approach
Protein synthesis in the nucleus was claimed back in the 1950s and 1960s, in studies in which isolated liver and thymocyte nuclei were found to incorporate radioactive amino acids into high molecular mass material2,3. The authors of these papers presented evidence, of varying cogency, that their isolated nuclei were relatively, or in some cases largely, free of cytoplasmic http://tcb.trends.com
Fig. 1. Biotin-lysyl-tRNAlys incorporation of permeabilized HeLa cells. Cells were allowed to extend nascent peptides labeled with biotin-lysyl-tRNAlys in the presence of 19 amino acids, bovine liver tRNAs and aminoacyl-tRNA synthetases, GTP, phosphocreatine and creatine phosphokinase at 27.5°C. Samples were taken and examined by confocal microscopy either immediately before labeling (a), revealing the endogenous background labeling, especially by mitochondria, or after 10 min of labeling in the absence (b) or presence (c) of 1 mg ml–1 cycloheximide (chx), an inhibitor of eukaryotic protein synthesis. Note the nuclear foci of incorporated label in (b), which is sensitive to the presence of chx (c). Bar, 3 µm. (Images reproduced by permission of the American Association for the Advancement of Science. See Ref. 1 for further details.)
0962-8924/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02105-5
396
Research Update
TRENDS in Cell Biology Vol.11 No.10 October 2001
rNTP-dependent increase in biotin-lysine incorporation was not observed. Most intriguing, dual-diameter gold immunoelectron microscopy showed that the sites of biotin-lysine labeling tended to be near sites of Br-UTP incorporation as well as close to the location of the SR class of splicing factors. These are extremely provocative results, indicating that nuclear translation might depend on transcription – reminiscent of the way in which these processes are coupled in prokaryotes. It is of interest to note that, in one of the more convincing early studies, the incorporation of 14C-amino acids in isolated thymocyte nuclei was observed to be significantly reduced if the nuclei were first exposed to the RNA synthesis inhibitor DRB (Ref. 4). Fig. 2. Gold immunoelectron microscopy detection of biotin-lysyl-tRNAlys incorporation. Permeabilized HeLa cells were incubated as described in Fig. 1 and a sample prepared for electron microscopy (EM) after 10 min of labeling with biotin-lysyl-tRNAlys. The dark line from 1 to 7 o’clock is the nuclear envelope. On its right is the nucleus, to its left is the cytoplasm. Note the dark spots indicating foci of intranuclear biotin incorporation (bio-peptide). Bar, 200 nm. (b, c) Dual-diameter gold immuno-EM shows that the sites of biotin-lysine labeling of extending peptides tend to be near sites of RNA synthesis, indicated by Br-UTP incorporation (b), as well as close to the location of the ribosomal protein L7 (c). Bar, 50 nm. (Images reproduced by permission of the American Association for the Advancement of Science. See Ref. 1 for further details.)
concentration of amino acids. In addition, the investigators carried out parallel studies with isolated nuclei, which they estimated to be freed of ∼95% of the cytoplasmic ribosomes initially present in intact cells, and observed approximately the same level of labeling inside the nuclei as in the permeabilized cell experiments. In another key control, the authors observed that a concentration of aurin tricarboxylic acid (ATA) known to block translational initiation did not reduce the nuclear labeling, whereas higher concentrations of ATA that block elongation reduced nuclear labeling by ~90%, further bolstering the case that the nuclear labeling represents incorporation of the ‘proximal precursor’ biotin-lysyltRNAlys into sites that are already engaged in translation. Although most of the earlier studies on protein synthesis in the nucleus probably reflected rapid nuclear import of newly made proteins synthesized on cytoplasmic ribosomes2,3, there was one finding, however, that seemed particularly interesting and quite convincing. In an extensive series of studies by Vincent Allfrey and Alfred Mirsky of protein synthesis in nuclei carefully isolated from thymocytes, their claimed nuclear protein http://tcb.trends.com
synthesis was observed to display a distinctive dependence on sodium ions4. This was, and remains, an intriguing finding since there is evidence that the nucleus sequesters sodium ions to a level about 12-fold higher than the cytoplasm5. The permeabilized cell system of Iborra et al. lends itself nicely to investigating the Na+ versus K+ dependence of the intranuclear biotin-lysine incorporation. Is nuclear translation coupled with transcription?
Cook and colleagues also suggest a link between nuclear translation and transcription1. When the standard biotinlysyl-tRNAlys labeling experiment was performed in the presence of increasing concentrations of ribonucleoside triphosphates, it was found that the amount of nuclear biotin-lysine incorporation increased. Moreover, the rNTP-dependent increase in biotin-lysine incorporation was not observed if the endogenous RNA polymerase II was selectively poisoned with α-amanitin. Also, if the RNA-chain-terminating adenosine analog cordycepin (3′-deoxyadenosine triphosphate) was added, or an essential RNA synthesis nucleotide (CTP) was omitted, the
What might be the function of translation in the nucleus?
Among their findings, Iborra et al. emphasize the provocative presence of numerous translation factors in the nucleus (see also Ref. 6). But presence does not necessarily signify function, and the roles of various translation factors that are present in the nucleus still remain unclear. It is conceivable, as Iborra et al. suggest, that nuclear translation functions in nonsensemediated decay (NMD), a quality-control mechanism in which messenger RNAs are surveyed for the presence of nonsense codons. The tRNA-assisted ribosome is the best, and admittedly the only, mRNA codon surveyor we know. Because NMD appears to occur within the nucleus or to be intimately connected to nuclear events, the results of Iborra et al. will expectedly stir the interest of those in the NMD field. Although the new results do not directly bear on nonsensemediated mRNA decay, at least the NMD field can now proceed from the stronger epistemological position of envisioning ribosome-based scanning as a plausible nuclear process. If there is indeed translation in the nucleus, what are the protein products, where do they go and what do they do? Iborra et al. estimate that the nuclear translation they observe constitutes ~15% of the total protein synthesis of the cell. Their data indicate that much of the observed labeling is in high molecular mass proteins. Are these translation products used to build the nucleus and
Research Update
TRENDS in Cell Biology Vol.11 No.10 October 2001
support nuclear functions? Notwithstanding a possible nucleuslocalized NMD pathway, are there nucleus-restricted mRNAs that are translationally competent but nuclear export incompetent? And what about the necessary nuclear ribosomes? Are they the most recently assembled ones, just out of the nucleolus and taking one fleeting stab at translation before they exit the nucleus? And since there is no evidence for polysomes in the nucleus, is the initiation rate of the reported nuclear translation low? A low initiation rate is expected for NMD scanning, contrasting with the higher initiation rates of cytoplasmic mRNAs (thus generating polysomes), where the biological aim is protein production, not quality control. And what about the labeling Iborra et al. see in the nucleolus itself, the very place ribosomes are being assembled? Is there mRNA in the nucleolus? There is some evidence that there might be7,8. Are these assembling nucleolar ribosomes functional? Are the nucleolar sites of labeling observed by Iborra et al. in the granular component of the nucleolus, where finished ribosomes are present? Is nucleolar translation coupled to ribosomal RNA transcription? Are any of the mRNAs that are translated in the nucleus or nucleolus ones that encode secreted or membrane proteins? In such
cases, is there an involvement of the signal recognition particle (SRP)? Is this why the SRP appears itself to be assembled in the nucleolus6? These numerous questions, and others, are now rendered intellectually viable and scientifically tractable by the new results. Concluding remarks
The nucleus of most eukaryotic somatic cells is essentially and literally enveloped by a major protein synthesis system – the endoplasmic reticulum. Cytological studies in the 19th century and all through the 20th, together with evolutionary considerations, teach us that the eukaryotic nucleus and cytoplasm are separate worlds. The findings of Iborra et al. bring about a moment of reflection. Perhaps the nucleus and protein synthesis are more anciently, intimately and still today connected than their apparent cytological segregation implies. We are reminded of how little we know for sure. The American newspaper writer, H.L. Mencken, put it thus: ‘Penetrating so many secrets, we cease to believe in the unknowable. But there it sits, nevertheless, calmly licking its chops.’ Acknowledgements
I thank Mahlon Hoagland, Arthur Pardee and Joel Richter for their helpful
397
comments on a first draft. The author’s work is supported by grant GM-21595 from the US National Institutes of Health, which requires him to state, according to the agency’s rules, that the views expressed in this article are not those of the US government. (Did you really think they were?) References 1 Iborra, F.J. et al. (2001) Coupled transcription and translation within nuclei of mammalian cells. Science 293, 1139–1142 2 Goldstein, L. (1970) On the question of protein synthesis by cell nuclei. Adv. Cell Biol. 1, 187–210 3 Pederson, T. (1976) Cellular aspects of histone synthesis. Protein Synthesis (Vol. 2) (McConkey E.H., ed.), pp. 69–123, Marcel Dekker 4 Allfrey, V.G. et al. (1957). Protein synthesis in isolated cell nuclei. J. Gen. Physiol. 40, 451–490 5 Lagendorf, H. et al. (1961). Kationenverteilung in Zellkern und Cytoplasma der Rattenleber. Biochem. Z. 335, 273–284 6 Pederson, T. and Politz, J.C. (2000) The nucleolus and the four ribonucleoproteins of translation. J. Cell Biol. 148, 1091–1095 7 Harris, H. (1974) Nucleus and Cytoplasm (3rd edn), Oxford 8 Pederson, T. (1998) The plurifunctional nucleolus. Nucleic Acids Res. 26, 3871–3876
Thoru Pederson Dept of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 377 Plantation Street, Worcester, MA 01605, USA. e-mail: [email protected]
Meeting Report
Ubiquitin – more than just a signal for protein degradation Cezary Wójcik Research related to the ubiquitin- and proteasome-dependent proteindegradation pathway is growing exponentially. Top scientists in this field met recently in the green mountains of Vermont. Among the numerous topics discussed at this meeting* were ubiquitinlike proteins, ubiquitin-interacting proteins, non-proteolytic roles of ubiquitin and the regulation and function of proteasomes. (*FASEB Summer Conference: Ubiquitin and Intracellular Protein Degradation; Saxtons River, Vermont, USA; 23–28 June 2001. Organized by George DeMartino and Daniel Finley.) http://tcb.trends.com
The ubiquitin–proteasome pathway (UPP) of protein degradation is responsible for the degradation of most cell proteins, not only regulatory, abnormal and short-lived proteins, but also those that serve structural roles and are long lived1. Since the discovery of ubiquitin in the late 1970s, ubiquitin research has expanded into a formidable field impacting all areas of cell biology2. In a typical mammalian cell, 30% of the total protein pool is degraded within 10 min of its synthesis. This can be calculated to correspond to three substrates being degraded by each proteasome per minute
(Jon Yewdell, Bethesda, USA). In Arabidopsis thaliana, 4.6% of the proteome participates in the UPP (Richard D. Vierstra, Madison, USA). Ticket to ride
Ubiquitin, a small polypeptide of 76 amino acids, can be covalently attached to other proteins, forming polyubiquitin chains, using the E1–E2–E3 cascade of enzymes (Fig. 1). Polyubiquitin Lys48-linked chains are usually attached to internal lysines of the substrates, targeting them for degradation by the 26S proteasome. However, several proteins, such as MyoD,
0962-8924/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02084-0