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© 1999 Nature America Inc. • http://genetics.nature.com news & views © 1999 Nature America Inc. • http://genetics.nature.com different (adjacent) genes, in contrast with Neurospora crassa, where a single gene encodes a product with both activites6. Last year Peer Bork and colleagues published a report7 showing that interacting proteins could be identified simply on the basis of gene order, at least in Bacteria and Archaea. They searched genomes for evidence of conserved gene order as a clue to interaction, identifying 301 proteins in the process, of which more than threequarters were found to have been reported to interact by direct experiment. They suggested that the temporal coordination of synthesis is advantageous for protein-protein interactions, and critical to interdependent co-translational folding8. Ultimately, the new findings of Marcotte et al. are quite similar to those of Bork and colleagues, although in the latter case, the genes are adjacent instead of fused. Clearly the thermodynamic advantage of the tether cannot be the only issue here. Perhaps the explanation has to do with differences in bacterial and eukaryotic assembly processes9, although some ‘Rosetta Stones’ are provided by bacterial genomes. One is left wondering why some genes sit next to each other in some bacteria but not in others. Even the gyrase A and B genes, while widely separated in E. coli, are adjacent in many other bacteria. Clearly, genomes are in constant foment, and gene order is continually reshuffled11. The evolutionary advantages of recombination are widely accepted. So there may be two opposing forces at play here: one that shuffles the genome, and another, that preserves gene associations for functional reasons. This is not to say that these new genomic approaches aren’t both useful and exciting. It seems certain that they will generate valuable data. The fact that all the data presented by Marcotte et al. are freely available on the internet will probably set off a rush of activity (their web site at www.doe-mbi.ucla.edu has already hosted more than 1,500 visits). Where to look first? I think that most of the significant connections involving known genes will turn out to be of the sort where the functional relationships were already apparent from ordinary homology searches—with occasional help from gene-order considerations. The most useful ‘connections’ will be those where the function of only one of the separated components (equivalent to half of the fused ‘pair’) is known; in these cases a genuine new finding—a clue to the function of a previously ‘anonymous’ gene—is possible (Fig. 2). Even if no functions have been ascribed to either component, the very existence of the fused gene product implies some kind of connection between two separated genes in the organism under scrutiny, which should get at least some biologists grooving. 1. 2. 3. Doolittle, R.F. Nature 392, 339–342 (1998). Marcotte, E.M. et al. Science 285, 751–753 (1999). Corpet, F., Gouzy, J. & Kahn, D. Nucleic Acids Res. 26, 323–326 (1998). 4. Pellegrini, M., Marcotte, E.M., Thompson, M.J., Eisenberg, D. & Yeates, T.O. Proc. Natl Acad. Sci. USA 96, 4285–4288 (1999). 5. Gaertner, F.H., Ericson, M.C. & DeMoss, J.A. J. Biol. Chem. 245, 595–600 (1970). 6. DeMoss, J. Biochem. Biophys. Res. Comm. 18, 850–857 (1965). 7. Dandekar, T., Snel, B., Huynen, M. & Bork, P. Trends Biochem. Sci. 23, 324–328 (1998). 8. Thanaraj, T.A. & Argos, P. Protein. Sci. 5, 1594–1612 (1996). 9. Netzer, W.J. & Hartl, F.U. Nature 388, 343–349 (1997). 10. Lawrence, J.G. & Roth, J.R. Genetics 143, 1843–1860 (1996). 11. Siefert, J.L. et al. J. Mol. Evol. 45, 467–472 (1997). Baby, don’t stop! Alexander S. Mankin1 & Susan W. Liebman2 1Center for Pharmaceutical Biotechnology and 2Department of Biological Sciences, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, Illinois 60607, USA. e-mail: [email protected] and [email protected] A large number of human genetic diseases result from mutations that cause the premature termination of the synthesis of the protein encoded by the mutant gene1. A study by Elisabeth Barton-Davis and colleagues2, exploring antibiotic effect on a mouse model of Duchenne muscular dystrophy (DMD) and published in a recent issue of the Journal of Clinical Investigation, suggests that gentamicin may provide a readily accessible treatment for disease caused by mutant stop codons. The stop (nonsense) codons UAA, UAG and UGA signal the termination of protein synthesis. While the anticodons of aminoacyl transfer RNAs (tRNAs) recognize sense codons, leading to the incorporation of a specific amino acid, there are normally no tRNAs with anticodons that precisely match any of the three nonsense codons. Rather, these codons are recognized by proteins that promote the release of the completed polypeptide chain 8 (Fig. 1a). When a nonsense codon in a structural gene results from mutation, the protein product is incomplete (or truncated; Fig. 1b) and the messenger RNA (mRNA) often rapidly degraded3. Start making sense The synthesis of complete protein from such a mutant gene can be partially restored by unlinked mutations that affect the translational apparatus. For example, tRNAs that have been mutated so that their anticodon can recognize a stop codon will bind to the stop codon in competition with release factors, thereby preventing premature chain termination some fraction of the time. Alternatively, aminoglycoside antibiotics allow normal tRNAs to recognize ‘incorrect’ codons, including stop codons4 (Fig. 1c). Aminoglycosides interact with the highly conserved decoding centre of the ribosomal RNA (rRNA). This centre normally fac- ilitates accurate codon-anticodon pairing, but when bound with a drug, RNA conformation is altered and accuracy reduced. Depending upon the dose, these drugs may inhibit protein synthesis. An ideal treatment of genetic disease would be to replace or supplement the mutant gene with a wild-type copy. An alternative approach to treating human genetic diseases caused by premature stop codons would be to engineer mutant human tRNA genes capable of decoding stop codons5. A less ideal, but accessible treatment is the use of aminoglycosides6, as suggested in 1985 by Burke and Mogg. Using cultured mammalian cells, they showed that the aminoglycoside antibiotics paromomycin and G-418 could partially restore the synthesis of a full-size protein from a mutant gene with a premature UAG mutation. Later, G-418 and gentamicin (another aminoglycoside) were shown to restore the expression of the cysnature genetics • volume 23 • september 1999 © 1999 Nature America Inc. • http://genetics.nature.com wild type a start codon AUG mRNA CAA b stop codon UAG AUG mutant premature stop codon UAA c UAG news & views mutant in the presence of gentamycin AUG UAA UAG protein product dystrophin nascent peptide aminoacyl tRNA glutamine RF tRNA © 1999 Nature America Inc. • http://genetics.nature.com ribosome RF CAA UAA UAA dystrophin mRNA gentamicin Trumping termination. a, Normal dystrophin mRNA encodes the complete dystrophin protein. The ‘correct’ tRNA binds to the dystrophin CAA sense codon. b, A mutant mRNA with a premature stop codon and the truncated polypeptide it encodes. Release factor (RF) proteins bind to the premature UAA stop codon within the mutant dystrophin mRNA. c, Gentamicin occasionally allows the incorporation of an amino acid at the internal stop codon of mutant dystrophin mRNA. The truncated protein is still made, but in some cases a full-length protein results from read-through and has an amino acid substitution at the site corresponding to the premature stop codon. tic fibrosis transmembrane conductance regulator (CFTR) protein in a cell line carrying a nonsense mutation in CFTR (refs 7,8). As gentamicin is currently used to treat bacterial infections in cystic fibrosis (CF) patients and about 10% of all CF patients carry a nonsense codon in CFTR, it should be possible to determine the clinical efficacy of gentamicin in suppressing CFTR nonsense mutations. Going live The work of Barton-Davis et al.2 represents an important step towards the use of aminoglycosides in the treatment of genetic diseases; it brings drug-mediated bypass of premature stop codons from cell culture to the organismal level. DMD is caused by inactivation of the human dystrophin gene—which is effected by a premature stop codon in 5–15% of DMD patients. The mdx mouse, an animal model of DMD, carries a mutant UAA stop codon in place of a glutamine CAA codon in its dystrophin gene. BartonDavis and colleagues found that injecting mdx mice with high doses of gentamicin for two weeks restored the amount of dystrophin in muscles to up to 20% of the normal level. Furthermore, post-mortem examination of these mice following the treatment period revealed a decrease in some symptoms of the disease. nature genetics • volume 23 • september 1999 Most aminoglycosides are highly active against bacterial ribosomes, but do not affect the cytoplasmic ribosomes in human cells. The sensitivity of eukaryotic ribosomes to some aminoglycosides, such as gentamicin, G-418, paromomycin, hygromycin and a few others, has been viewed as an unwanted side effect associated with these antibacterial drugs. But it is precisely that ‘side’ effect that opens the possibility of using these drugs for the treatment of human genetic diseases. So far, the clinical application of aminoglycosides has been limited to their use as antibacterials; no effort has been made to optimize their ability to cause translation errors in eukaryotic cells. This leaves hope that their capacity to ‘bypass’ stop-codons could be improved by screening combinatorial chemical libraries or rational drug design—especially as the structures of several aminoglycosides complexed with their target sites have been solved9. At the moment, aminoglycosides are the only drugs known to affect translational fidelity in eukaryotes. However, alterations in several different rRNA regions (both in small and large ribosomal subunits), ribosomal proteins and translation factors can affect accuracy of translation. So, in addition to the decoding centre, other ribosomal sites and translation-elongation and termination factors could be viewed as potential targets for drugs that would allow the cell to synthesize a full-length protein from a gene with a mutant stop codon. On the downside, such approaches are expected to be effective only against a small fraction of deleterious mutations. The effects of some genetic alterations, such as deletions, will not be ameliorated by drugs affecting translational accuracy. It is also not clear if any missense or frameshift mutants could be suppressed, but this remains a possibility. Furthermore, not all nonsense mutations are likely to be countered by this approach. This is because competition between release factors and aminoacyl tRNA depends on the composition of sequence flanking the stop codon10. In model studies with eukaryotic cells, bypass of stop codons only within specific ‘contexts’ could be stimulated by aminoglycoside antibiotics11,12. Other factors may further reduce the spectrum of genetic diseases amenable to aminoglycoside treatment. The activity of the protein containing an altered amino acid in place of the premature stop, the amount of the protein required to carry out its cellular function and its stability will clearly impose additional limitations. Finally, the increased frequency of translation errors will result in the production of aberrant proteins, which, together with the other known side 9 news & views © 1999 Nature America Inc. • http://genetics.nature.com effects of gentamicin (and yet unknown consequences of the long-term use of the drug), will make it tricky to find effective treatment regimens. Nevertheless, as there is no effective treatment for most genetic disorders, this readily accessible approach is exciting. More than ten years elapsed before the idea of treating genetic diseases with aminoglycosides6 was tested in an animal model2. Hopefully, it will take less time to extrapolate this approach to the point where it can actually help patients. 1. 2. 3. 4. 5. Atkinson, J. & Martin, R. Nucleic Acids Res. 22, 1327–1334 (1994). Barton-Davis, E.R., Cordier, L., Shoturma, D.I., Leland, S.E. & Sweeney, H.L. J. Clin. Invest. 104, 375–381 (1999). Culbertson, M.R. Trends Genet. 15, 74–80 (1999). Davies, J., Gilbert, W. & Gorini, L. Proc. Natl Acad. Sci. USA 51, 883–890 (1964). Temple, G.F., Dozy, A.M., Roy, K.L. & Kan, Y.W. Nature 296, 537–540 (1982). Burke, J.F. & Mogg, A.E. Nucleic Acids Res. 13, 6265–6272 (1985). 7. Bedwell, D.M. et al. Nature Med. 3, 1280–1284 (1997). 8. Howard, M., Frizzell, R.A. & Bedwell, D.M. Nature Med. 2, 467–469 (1996). 9. Fourmy, D., Recht, M.I., Blanchard, S.C. & Puglisi, J.D. Science 274, 1367–1371 (1996). 10. Mottagui-Tabar, S., Tuite, M.F. & Isaksson, L.A. Eur. J. Biochem. 257, 249–254 (1998). 11. Singh, A., Ursic, D. & Davies, J. Nature 277, 146–148 (1979). 12. Palmer, E., Wilhelm, J.M. & Sherman, F. Nature 277, 148–150 (1979). 6. © 1999 Nature America Inc. • http://genetics.nature.com gadzooks! Marcy E. MacDonald Molecular Neurogenetics Unit, Massachusetts General Hospital, 13th Street, Charlestown, Massachusetts 02129, USA. e-mail: [email protected] The ubiquitin-proteasome pathway of protein degradation is essential for eukaryotes and involves cellular machinery as elaborate as that required by protein synthesis. Critical molecular steps, however, are emerging from the discovery of mutations that cause problems ranging from cancer to hypertension to fat facets1. On page 47 of this issue, Kazumasa Saigoh and colleagues2 broaden the spectrum by discovering that a mutation in the ubi- Ub quitin carboxy-terminal hydrolase gene (Uchl1) causes gracile axonal dystrophy (gad) in mice. The gad mouse is a simple genetic model with features reminiscent of inherited neurodegenerative disease in human3. It develops subtle sensory ataxia, progressing to motor ataxia. Later, axon terminals of the dorsal root ganglia degenerate, followed by neurons that comprise the gracile nucleus of the medulla oblongata Ub Ub 1 poly-ubiquitin Ub ubiquitin monomer 2 E1 3 E2 The ubiquitin-proteasome system. A stepwise model of the ubiquitin-proteasome pathway1 depicting (step 1) release of ubiquitin from pro-ubiquitin during biosynthesis (steps 2–5) conjugation of ubiquitin to (a) substrate(s) and (steps 6,7) degradation of the polyubiquitinated-substrate by the 26S proteasome complex (19S, 20S, 19S). Conjugation involves activation of ubiquitin by ubiquitin-activating enzyme E1 (step 2), transfer of activated-ubiquitin to a specific E2 ubiquitin-conjugating enzyme (UBC; step 3), transfer of activated ubiquitin from E2 to a substratespecific E3 ubiquitin-ligase (step 4) and formation of a substrate-E3 complex and biosynthesis of the polyubiquitin chain (step 5). Degradation includes binding of the polyubiquitinated-substrate to the 19S complex (step 6), proteolysis to short peptides by the 20S complex, and release of recycled ubiquitin (step 7) from ‘end’ proteolytic products. 10 4 E3 E1 Ub E2 Ub E3 Ub s Ub 5 Ub Ub Ub s Ub Ub E3 Ub s 19S 6 20S 7 Ub 19S Ub peptides Ub recycled ubiquitin region4. The brain of the gad mutant contains ‘spheroid bodies’ and dots of ubiquitin-conjugated protein in ‘dying’ terminals and axons5, reminiscent of the often beautiful, varied dots and swirls of abnormal ubiquitin-conjugated protein found in the post-mortem brains of people with inherited neurodegenerative diseases. The dots seem to hold a clue to the pathogenic process1,6–8, but the discovery of unrelated genes which, when mutated, cause more than a dozen of these disorders has failed, in each instance, to directly link disease mechanism to defects in the cellular machinery that degrades proteins. The identity of the gad gene defect, therefore, comes as a welcome surprise. Saigoh et al. homed in on the gad locus on chromosome 5 by following co-inheritance of the gad phenotype with polymorphic DNA markers in a classic genetic linkagebased cloning strategy. Their reward is an in-frame deletion in Uchl1 that results in a truncated form of ubiquitin hydrolase, a thiol protease that participates in ubiquitin biosythesis and in protein degradation. This discovery raises new issues. As usual, the molecular consequence of the mutation as mode of action needs to be delineated. A simple loss of UCH-L1 activity is the most parsimonious explanation. But, as the authors point out, mRNA is synthesized from the mutant Uchl1 allele at normal levels, which makes it hard to reject a mechanism involving the predicted truncated UCH-L1 product. The larger issue, which remains a key puzzle for cloned genes whose mutation causes most neurodegenerative disease, is to discover why the gad genetic defect affects neurons nature genetics • volume 23 • september 1999