RNA surveillance by nuclear scanning? - Nature

commentary

RNA surveillance by nuclear scanning? Miles F. Wilkinson and Ann-Bin Shyu

There are many quality-control mechanisms that ensure high fidelity of gene expression. One of these is the nonsense-mediated decay (NMD) pathway, which destroys aberrant mRNAs that contain premature termination codons generated as a result of biosynthetic errors or random and programmed gene mutations. Two complexes that initially bind to RNA in the nucleus have been suggested to be involved in NMD in the cytoplasm. Here we propose an alternative model that involves nuclear scanning, on the basis of recent evidence for nuclear translation.

“Forgive me my nonsense as I also forgive the nonsense of those who think they talk sense.” – Robert Frost ammalian cells transcribe thousands of different precursor messenger RNAs (pre-mRNAs), many of which are alternatively spliced to generate in excess of 100,000 different mature mRNAs, each encoding a different protein. This complexity requires several layers of quality control to ensure high fidelity of gene expression. One of these quality-control mechanisms is nonsense-mediated decay (NMD), an RNA surveillance pathway that detects and destroys aberrant mRNAs containing premature termination (nonsense) codons1–3. One class of substrates for this NMD pathway is transcripts from mutated genes containing either nonsense or frameshift mutations that lead to pre-mature translation termination. Another class of substrates is transcripts from normal genes that contain nonsense codons as a result of errors in RNA splicing or transcription. A surprising discovery — originally made more than 15 years ago — was that this NMD response can occur in the nuclear fraction of mammalian cells4–8. This was paradoxical because the signal that elicits NMD is a translation signal, which was thought to be recognized only by the cytoplasmic translation apparatus. Yet another paradoxical aspect of NMD was revealed by studies designed to determine how premature termination codons that trigger mRNA decay are distinguished from normal stop codons that do not elicit NMD. It was found that the NMD response requires ‘a second signal’ in the form of an intron downstream of the stop codon (the first signal)8–10. This nicely explained the selectivity of NMD, as normal stop codons are typically in the terminal 3′ exon of an mRNA (that is, not followed by an intron), whereas premature stop codons are typically in internal exons (that is, followed by one or more introns). As pleasing as this twosignal rule was, it was unexpected, as introns are only present in pre-mRNAs in the nucleus; they are spliced out by the time

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an mRNA encounters the translation apparatus in the cytoplasm. One solution to the unexpected involvement of the nucleus and introns in NMD came from the discovery of an exon–junction protein complex (EJC) left behind after RNA splicing, near exon–exon junctions, in mature mRNAs11. This EJC was found to travel with mature mRNA to the cytoplasm, meaning that it could be recognized as a second signal for NMD by cytoplasmic ribosomes. This solved the problem of how a nuclear entity (an intron) could be involved in an event involving cytoplasmic translation, as it suggested that introns, after splicing, only serve to leave a mark (the EJC) that remains attached to mRNA when it meets the translation apparatus in the cytoplasm. According to this model, NMD is elicited in the cytoplasm when this EJC is recognized by a putative surveillance complex that forms after a ribosome meets a stop codon. To explain the many cases in which NMD can occur in the nuclear fraction of cells, it was further hypothesized that scanning of stop codons and EJCs occurs in a compartment that biochemically cofractionates with the nucleus but is actually in the cytoplasm, probably near the nuclear pore3,7,10. This model can be called a ‘nuclear-history’ model, as it rests on the notion that proteins bound to mRNAs in the nucleus affect the fate of the mRNA after it reaches the cytoplasm12. In this commentary, we propose an alternative explanation for the paradoxical role of the nucleus and introns in the NMD RNA-surveillance pathway. This explanation is a resurrection of the ‘nuclear-scanning’ model originally proposed by Chasin and co-workers6, which has gained new life as a result of the recent evidence that translation can occur in the nucleus of mammalian cells13,14. This nuclear-scanning model posits that mRNAs are scanned for nonsense codons in the nucleus itself rather than in the cytoplasm. Here we update this model to include nuclear molecules recently proposed to be involved in NMD, including the EJC, the cap-binding complex (CBC), and translation initiation factors known to accumulate in the nucleus.

The EJC platform A remarkable finding was the recent discovery that, after RNA splicing, a protein complex remains bound to the mature mRNA product, always about 20–24 nucleotides upstream of its exon–exon junctions, regardless of sequence11. This EJC was found to comprise several interesting proteins, including the RNA-export factor REF/Aly, the RNA-splicing factor RNPS1, the nuclear-matrix protein SRm160, the nucleo-cytoplasmic shuttling protein Y14, and the oncoprotein DEK11,15–17. More recently, the NMD protein UPF3 and MAGOH, a Y14-interacting protein, were also found to be components of the EJC16,18–20. Because this EJC remains bound to mRNA after RNA splicing, it seemed an ideal candidate for the second signal that triggers NMD. Further support for this idea was the finding that tethering some members of the EJC downstream of a normal stop codon transforms this into a ‘premature’ stop codon that triggers NMD19,21. Evidence for nuclear scanning Models developed to explain how the EJC triggers NMD have proposed that this complex is recognized by the cytoplasmic translation apparatus 15,16,19,21. Although this is a reasonable idea, here we propose that recent data also support a nuclear-scanning model in which recognition of a nonsense codon and the downstream EJC occurs in the nucleus proper (Fig. 1). At least four lines of evidence are consistent with this nuclearscanning model14. First, nonsense codons have been shown to increase the levels of nuclear unspliced mRNA from genes that acquire nonsense codons during normal lymphocyte development (Ig-κ, Ig-µ and T-cell receptor (TCR)-β), as well as unspliced mRNA from the minute virus of mice22–24. In the case of Ig-µ and TCR-β, it was shown by fluorescent in situ hybridization analysis that nonsense codons cause the accumulation of unspliced mRNA at or very near the site of transcription in the nucleus proper24. This surprising stimulatory effect of nonsense codons on nuclear precursor mRNA levels suggests that they affect RNA splicing,

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commentary Normal transcript

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Figure 1 Model for nuclear nonsense-mediated decay (NMD). During pre-mRNA splicing in the nucleus, several proteins, including UPF3, are deposited on CBC-bound mRNA 20–24 nucleotides upstream of exon–exon junctions to form the exon junction complex (EJC). a, During translation of normal transcripts, the EJC is remodelled and CBC is replaced by 4E, which promotes mRNA export to the cytoplasm. b, For transcripts containing nonsense codons (right), the ribosome pauses at the premature termination codon and is then converted into a surveillance complex, which

nuclear RNA stability, or both, in the nucleus. Second, several laboratories have observed that nonsense and frameshift mutations are associated with increased levels of alternatively spliced transcripts that have skipped the offending mutation, suggesting that nonsense codons modulate RNA splicing25,26. Although it is clear in some cases that nonsense mutations alter RNA splicing merely because they disrupt splicing enhancers25,26, recently it was demonstrated that a translation-like mechanism acting independently of splicing–enhancer disruption is responsible for upregulation of an alternatively spliced TCR transcript in

: Tanslation stop codon : Exon : Intron

has been hypothesized to be the 40S ribosomal subunit bound to factors such as the NMD protein UPF1. Collision of this surveillance complex with the EJC leads to nuclear mRNA decay. A surveillance complex also forms on normal transcripts but there is no EJC downstream and hence NMD is not elicited. PABP2, nuclear poly(A)-binding protein; PABP, cytoplasmic poly(A)-binding protein; TAP, mRNA nuclear export mediator; 4E, translation initiation factor eIF-4E.

response to nonsense and frameshift mutations27. Third, some of the basic ingredients for translation are in the nucleus, including charged transfer RNAs, and the translation initiation factors eIF-2α, eIF-3, eIF-4E (4E), eIF-4G (4G), and the elongation factor EF1 (refs 13,14,28–32). Finally, some studies suggest that a modest but significant fraction (~10%) of total cellular translation occurs in the nucleus. The evidence for this originally came from amino-acid incorporation studies performed some 30 years ago33. But this evidence for nuclear translation was largely ignored, as it was considered to be the result of contamination of

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the nuclear fraction with cytoplasmic ribosomes. But when several groups demonstrated that NMD occurs in the nuclear fraction of cells4–8, the possibility of nuclear translation was considered to be worth revisiting. The first data that began to erode the cytoplasmic-translation dogma were short-pulse experiments performed in the slime-mold Dictyostelium discoideum which suggested that nuclear polyribosomes (or polysomes) exist34. More recently, a series of amino-acid incorporation localization studies performed in several different permeabilized cell lines provided evidence that nuclear translation also occurs in mammalian cells13. E145

commentary Rather than being uniform throughout the nucleus, amino-acid incorporation was localized in specific spots that also had high concentrations of transcription factors, RNA splicing factors, and translation factors. Intriguingly, indirect evidence from both mammalian cells and D. discoideum indicated that nuclear translation is coupled with transcription, suggesting the possibility that these two events occur in the same part of the nucleus13,34. Advantages of nuclear scanning A nuclear-surveillance mechanism that scans and destroys aberrant mRNAs would seem to have some advantages over cytoplasmic scanning. Decay of bad mRNAs immediately after (or during) transcription is a more efficient way of preventing truncated, potentially deleterious proteins from being made than degrading them in the cytoplasm. This may be particularly important for genes that normally acquire premature termination codons during normal development, such as TCR and Ig genes. If truncated TCR and Ig proteins from nonsensecodon-bearing genes were expressed, they could severely inhibit normal immune responses as a result of dominant-negative effects2. Another class of transcripts that could benefit from a nuclear NMD mechanism would be transcripts that are so rapidly turned over in the cytoplasm (for example, those from early-responsegenes) that it is unlikely a cytoplasmic NMD mechanism could significantly decrease their expression if they acquired a nonsense codon by mutation. We suggest that a nuclear-scanning mechanism would recognize such mRNAs as being aberrant and rapidly degrade them before they enter the cytoplasm. It is important that such highly unstable mRNAs are efficiently surveyed for mistakes, as they encode many potent regulators of cell growth, including proto-oncoproteins, cytokines and transcription factors35. Nuclear scanning makes possible more layers of protection from deleterious proteins than could be provided by cytoplasmic scanning. For one, because the likely purpose of nuclear scanning is to proofread mRNAs for mistakes and not to generate proteins, perhaps most proteins generated by nuclear translation are targeted for decay. This nuclear proteolytic pathway would serve to protect the cell from truncated, potentially toxic proteins generated from messages with nonsense codons. It is also possible that nuclear scanning does not involve translation but instead is only a codon-scanning proofreading process — in this case, the potentially deleterious effects of truncated proteins encoded by nonsense-codon-bearing mRNAs would be completely eliminated. E146

Pioneer round of translation Recent evidence from two groups suggested that a unique complex of proteins that binds to the 5′ cap of mRNAs mediates an initial (‘pioneer’) round of translation that can detect nonsense codons36,37. This capbinding complex (CBC) is composed of two proteins, CBP20 and CBP80, that bind as a heterodimer to newly transcribed premRNAs and promotes their splicing. CBCbound mRNAs can also be translated, shown by in vitro translation experiments in Saccharomyces cerevisiae in which CBC was shown to recruit the central scaffold translation initiation factor 4G (ref. 36). Binding of CBC to 4G has also recently been demonstrated in mammalian cells32. The CBC may also recruit other initiation factors, as its CBP80 subunit has a motif (MIF4F) called a NIC domain that may bind to eIF-3 and eIF-4A (refs 32,38). The evidence that CBC-bound mRNAs are the substrates for NMD comes from the finding that an anti-CBP80 antibody immunoprecipitates a cell fraction in which NMD occurs and that this antibody also coimmunoprecipitates the NMD factors UPF2 and UPF3 (ref. 37). These observations collectively led to a model in which CBC-bound mRNAs are scanned for nonsense codons during a first round of translation and then the CBC is displaced from the mRNA 5′ cap by the initiation factor 4E, which then initiates multiple rounds of bulk translation36,37. This displacement model is consistent with the finding that 4E competes with CBC for binding to 4G (ref. 36). Where are CBC–mRNAs scanned? Current models posit that both the pioneer round of CBC-mediated translation and the CBC-to-4E ‘handoff ’ occurs in the cytoplasm32,36,37,39,40. This is supported by the finding that CBC is exported from the nucleus while bound to Balbiani ring transcripts in the larval salivary glands of Chironomus tentans40 and that CBC promotes the export of mRNAs from Xenopus laevis oocytes39. But these situations in which CBC is exported to the cytoplasm may be special cases. C. tentans Balbiani ring transcripts are unique, highly transcribed mRNAs with enormous exons about 100 times larger than mammalian exons. Xenopus oocytes are very specialized cells that differ in many ways from somatic cells, including having unique translational regulatory mechanisms. So it is unclear how general a cytoplasmic CBC-to-4E exchange is, especially in mammalian cells in which 4E accumulates at high levels in the nucleus14,29. Importantly, the nuclear accumulation of 4E is probably not just a passive event, as a specific shuttling factor 4E-T that mediates its import, has been identified30. Moreover, 4E is not randomly localized in the nucleus but instead accumulates

in a nuclear speckled pattern that is reminiscent of (if not identical to) the pattern observed for 4G and several RNA splicing factors14,29,31,32. Why would 4E travel to specific sites in the nucleus if it is exchanged for CBC in the cytoplasm? Furthermore, why is the central scaffold initiation factor 4G, which binds to both CBC and 4E, also found at specific sites in the mammalian nucleus31,32? Here we hypothesize, to explain this dilemma, that CBC-mediated translation and the exchange of CBC-for-4E can occur in the mammalian nucleus (Fig. 1). This would explain the available data, including the fact that CBC-bound β-globin mRNA is degraded in the nuclear fraction of cells4,5,37. It would also explain why 4G interacts with CBC in the nuclear fraction of mammalian cells and why 4G is stably associated with capped mRNA while it is spliced by nuclear extracts in vitro32. Furthermore, nuclear translation followed by a CBC-to-4E nuclear exchange would be a more efficient way to rapidly initiate bulk translation in the cytoplasm. Priming mRNAs with 4E before their export to the cytoplasm would allow them to engage in translation as soon as they emerged from the nuclear pore into the cytoplasm. In addition, we speculate that 4E could promote export of mRNAs from the nucleus, as its binding to mRNAs may act as a second signal that together with EJC remodelling permits a subset of mRNAs to be exported to the cytoplasm (Fig. 1). Future directions The discovery that unique protein complexes bind both to the 5′ cap and exon–exon junctions of mRNAs while they are in the nucleus raises many questions for future research. For example, how does CBC binding to the 5′ cap of an mRNA direct only one initial round of translation36,37? One possibility is that CBC accomplishes this simply because it cannot direct RNA-circle formation (the circularization of mRNAs as a result of factors at the 5′ and 3′ ends interacting with each other), which has been hypothesized to be essential for multiple rounds of translation41. In contrast, the 4E-containing 5′-cap-binding complex that replaces CBC is known to direct RNA-circle formation because it can bind to poly (A)-binding protein, which interacts with the 3′ end of most mRNAs41 (Fig. 1). Another possibility is that 4E curtails CBC-mediated nuclear translation by pushing CBC off mRNA. To address this, it will be important to determine where the CBC-to-4E exchange occurs. Is it only in the cytoplasm or can it occur in the nucleus, and if the latter, is it at the site of trancription or at the nuclear pore? Another question is why are CBCbound mRNAs the substrates for NMD?

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commentary Does CBC preferentially recruit the appropriate factors for NMD? Or is CBC only a passive participant in NMD that binds to nascent mRNAs and so happens to be around when mRNAs are scanned for mistakes during the first round of translation? There are also many unanswered questions about the EJC. For example, what do the individual components of the EJC do? How do RNPS1 and Y14 elicit NMD? How does REF/Aly drive mRNA export and does its presence in the EJC explain why removing introns from mammalian genes (that is, in cDNAs) dramatically decreases the amount of mRNA they express in the cytoplasm12? How do other members of the EJC identified to date, such as SRm160 and DEK, function? Why is there a dynamic exchange of individual EJC proteins during the transit of an mRNA from the nucleus to the cytoplasm11,15–17? Finally, how does reading frame dictate the fate of the EJC? All published reports on the EJC have used substrate mRNAs without normal openreading frames. If mRNAs are normally scanned in the nucleus for reading frame, as we have suggested here, it will be interesting to see how this alters the binding or composition of the EJC. The nuclear-scanning model presented here is not mutually exclusive with cytoplasmic-scanning models. Clearly, some nonsense-codon-containing mRNAs are degraded in the cytoplasmic fraction rather than in the nuclear fraction of mammalian cells1,3. It will be important to determine whether this cytoplasmic NMD is fundamentally distinct from nuclear NMD and whether the site of the CBC-to-4E exchange dictates which pathway is taken. The nuclear-scanning model requires that one have faith in the idea that mRNAs are scanned for nonsense codons in the nucleus proper. To definitively prove whether this is so will require new approaches, such as blocking nuclear mRNA export and assessing whether NMD still occurs, or approaches involving nuclear injection into Xenopus laevis oocytes or in vitro decay systems using mammalian nuclear extracts. An important issue that remains unresolved is whether all the factors essential for NMD and translation are in the nucleus. It is clear that UPF3 and UPF3B are correctly localized for nuclear NMD, as they are nucleo-cytoplasmic shuttling proteins that primarily accumulate in the nucleus of mammalian cells19,21,42. In

contrast, UPF1 is predominantly cytoplasmic at steady state but it could also participate in nuclear NMD, as it was recently also shown to shuttle between the cytoplasm and the nucleus (H. Dietz, personal communication). UPF2 is also cytoplasmic at steady state but is poised to enter the nucleus, as it accumulates near the nuclear envelope and has classical nuclear-localization signals21,42,43. It has been shown that many translation initiation factors are in the nucleus3,14,28–32 but it has not yet been determined whether eRF1 and eRF3, the release factors that actually recognize stop codons, are in the nucleus. If one accepts the possibility of nuclear scanning, then an important future issue to resolve is that of its substrate specificity. If fully spliced (mature) transcripts are its only substrates, then how are the intronbearing (precursor) mRNAs that are so prevalent in the nucleus excluded from the process? Alternatively, if intron-containing mRNAs are among its substrates, how are the multiple stop codons in their introns interpreted? One possibility is that these stop codons help direct splice-site recognition, as their recognition by nuclear ribosomes may serve to direct the splicesome to cleave at appropriate splice sites near the stop codons and ignore the many cryptic splice sites that are typically scattered throughout the rest of mammalian mRNAs. Another possibility is that introns are excluded from being scanned as a result of their being looped out by an exon definition mechanism, thereby restricting nuclear ribosomes to scanning coding regions for disruptions in reading frame by nonsense codons. Clearly, the notion of nuclear RNA surveillance has provided us with a whole host of fascinating implications but has also left us with many unsolved riddles. Miles Wilkinson is in the Department of Immunology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA. Ann-Bin Shyu is in the Department of Biochemistry and Molecular Biology, The University of Texas–Houston Medical School, Houston, Texas 77030, USA. e-mail: [email protected]

5. Takeshita, K., Forget, B. G., Scarpa, A. & Benz, E. J. Blood 64, 13–22 (1984). 6. Urlaub, G., Mitchell, P. J., Ciudad, C. J. & Chasin, L. A. Mol. Cell. Biol. 9, 2868–2880 (1989). 7. Cheng, J. & Maquat, L. E. Mol. Cell. Biol. 13, 1892–1902 (1993). 8. Carter, M. S., Li, S. & Wilkinson, M. F. EMBO J. 15, 5965–5975 (1996). 9. Cheng, J., Belgrader, P., Zhou, X. & Maquat, L. E. Mol. Cell. Biol. 14, 6317–6325 (1994). 10. Thermann, R. et al. EMBO J. 17, 3484–3494 (1998). 11. Le Hir, H., Moore, M. J. & Maquat, L. E. Genes Dev. 14, 1098–1108 (2000). 12. Shyu, A. B. & Wilkinson, M. F. Cell 102, 135–138 (2000). 13. Iborra, F. J., Jackson, D. A. & Cook, P. R. Science 293, 1139–1142 (2001). 14. Wilkinson, M. F. & Shyu, A. B. Bioessays 23, 775–787 (2001). 15. Kim, V. N. et al. EMBO J. 20, 2062–2068 (2001). 16. Kim, V. N., Kataoka, N. & Dreyfuss, G. Science 293, 1832–1836 (2001). 17. Le Hir, H., Izaurralde, E., Maquat, L. E. & Moore, M. J. EMBO J. 19, 6860–6869 (2000). 18. Le Hir, H., Gatfield, D., Izaurralde, E. & Moore, M. J. EMBO J. 20, 4987–4997 (2001). 19. Lykke-Andersen, J., Shu, M. D. & Steitz, J. A. Science 293, 1836–1839 (2001). 20. Kataoka, N., Diem, M. D., Kim, V. N., Yong, J. & Dreyfuss, G. EMBO J. 20, 6424–6433 (2001). 21. Lykke-Andersen, J., Shu, M. D. & Steitz, J. A. Cell 103, 1121–1131 (2000). 22. Gersappe, A. & Pintel, D. J. Mol. Cell. Biol. 19, 1640–1650 (1999). 23. Lozano, F., Maertzdorf, B., Pannell, R. & Milstein, C. EMBO J. 13, 4617–4622 (1994). 24. Muhlemann, O. et al. Mol. Cell 8, 33–43 (2001). 25. Maquat, L. E. Am. J. Hum. Genet. 59, 279–286 (1996). 26. Valentine, C. R. Mut. Res. 411, 87–117 (1998). 27. Wang, J., Hamilton, J. I., Carter, M. S., Li, S. & Wilkinson, M. F. Science (in the press). 28. Lund, E. & Dahlberg, J. E. Science 282, 2082–2085 (1998). 29. Dostie, J., Lejbkowicz, F. & Sonenberg, N. J. Cell Biol. 148, 239–245 (2000). 30. Dostie, J., Ferraiuolo, M., Pause, A., Adam, S. A. & Sonenberg, N. EMBO J. 19, 3142–3156 (2000). 31. Etchison, D. & Etchison, J. R. J. Virol. 61, 2702–2710 (1987). 32. McKendrick, L., Thompson, E., Ferreira, J., Morley, S. J. & Lewis, J. D. Mol. Cell. Biol. 21, 3632–3641 (2001). 33. Goidl, J. & Allen, W. R. Trends Biochem. Sci. 3, N225–N228 (1978). 34. Mangiarotti, G. Biochemistry 38, 3996–4000 (1999). 35. Chen, A. C.-Y. & Shyu, A.-B. Trends Biochem. Sci. 20, 465–470 (1995). 36. Fortes, P. et al. Mol. Cell 6, 191–196 (2000). 37. Ishigaki, Y., Li, X., Serin, G. & Maquat, L. E. Cell 106, 607–617 (2001). 38. Aravind, L. & Koonin, E. V. Genome Res 10, 1172–1184 (2000). 39. Ohno, M., Segref, A., Bachi, A., Wilm, M. & Mattaj, I. W. Cell 101, 187–198 (2000). 40. Visa, N., Izaurralde, E., Ferreira, J., Daneholt, B. & Mattaj, I. W. J. Cell Biol. 133, 5–14 (1996). 41. Wells, S. E., Hillner, P. E., Vale, R. D. & Sachs, A. B. Mol. Cell 2, 135–140 (1998). 42. Serin, G., Gersappe, A., Black, J. D., Aronoff, R. & Maquat, L. E. Mol. Cell. Biol. 21, 209–223 (2001). 43. Mendell, J. T., Medghalchi, S. M., Lake, R. G., Noensie, E. N. & Dietz, H. C. Mol. Cell. Biol. 20, 8944–8957 (2000).

1. Hentze, M. W. & Kulozik, A. E. Cell 96, 307–310 (1999). 2. Li, S. & Wilkinson, M. F. Immunity 8, 135–141 (1998). 3. Maquat, L. E. in Translational Control of Gene Expression (eds Hershey, J. W. B., Mathews, M. B. & Sonenberg, N.) 849–868 (Cold Spring Harbor Laboratory Press, 2000). 4. Humphries, R. K., Ley, T. J., Anagnou, N. P., Baur, A. W. & Nienhuis, A. W. Blood 64, 23–32 (1984).

ACKNOWLEDGEMENTS We thank T. Cooper, G. Cote, M. Goode, M. Moore, and N. Sonenberg, and members of the Wilkinson and Shyu laboratories for helpful comments. Special thanks go to L. Bankey for the artwork. Some of the work discussed here was supported by NIH grants (M.F.W. and A.-B.S.) and NSF grant (M.F.W.).

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