TIBS 23 – NOVEMBER 1998 REFLECTIONS

In addition, one can construct curated 5 Smith, T. F. and Waterman, M. S. (1981) J. Mol. 20 Bucher, P., Karplus, K., Moeri, N. and library of PSI-BLAST compatible profiles Biol. 147, 195–197 Hofmann, K. (1996) Comput. Chem. 20, 3–23 6 Karlin, S. and Altschul, S. F. (1990) Proc. Natl. 21 Durbin, R., Eddy, S., Krogh, A. and Mitchison, G. and modify the program in order to Acad. Sci. U. S. A. 87, 2264–2268 (1998) Biological Sequence Analysis. compare a query directly to this library. 7 Karlin, S. and Altschul, S. F. (1993) Proc. Natl. Probabilistic Models of Proteins and Nucleic We hope that future refinements that Acad. Sci. U. S. A. 90, 5873–5877 Acids, Cambridge University Press 8 Dembo, A., Karlin, S. and Zeitouni, O. (1994) 22 Mushegian, A. R. et al. (1997) Proc. Natl. Acad. perhaps incorporate some of these Ann. Prob. 22, 2022–2039 Sci. U. S. A. 94, 5831–5836 ideas will further enhance our ability to 9 Waterman, M. S. and Vingron, M. (1994) Stat. 23 Huynen, M. et al. (1998) J. Mol. Biol. 280, make sense of protein sequences. Sci. 9, 367–381 323–326 10 Pearson, W. R. (1998) J. Mol. Biol. 276, 71–84 24 Aravind, L. et al. Trends Genet. (in press) 11 Pearson, W. R. (1995) Protein Sci. 4, 25 Bult, C. J. et al. (1996) Science 273, Acknowledgements 1145–1160 1058–1073 We thank the developers of PSI- 12 Holm, L. and Sander, C. (1997) Structure 5, 26 Sterky, F., Holmberg, A., Pettersson, B. and BLAST, who include D. J. Lipman, T. L. 165–171 Uhlen, M. (1996) Yeast 12, 1091–1095 13 Brenner, S. E., Chothia, C. and Hubbard, T. J. P. 27 Shuman, S. and Schwer, B. (1995) Mol. Madden, W. Miller, A. A. Schäffer, (1998) Proc. Natl. Acad. Sci. U. S. A. 95, Microbiol. 17, 405–410 J. Zhang and Z. Zhang. We also thank 6073–6078 28 Wootton, J. C. and Federhen, S. (1996) L. Aravind for his collaboration on the 14 Schneider, T. S., Stormo, G. D., Gold, L. and Methods Enzymol. 266, 554–571 application of PSI-BLAST to the detection Ehrenfeucht, A. (1986) J. Mol. Biol. 188, 29 Lupas, A. (1996) Methods Enzymol. 266, 415–431 513–525 of subtle relationships among proteins. 15 Gribskov, M., McLachlin, A. D. and Eisenberg, D. 30 Zhang, Z. et al. (1998) Nucleic Acids Res. 26, (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3986–3990 References 4355–4358 1 Pearson, W. R. and Lipman, D. J. (1988) Proc. 16 Staden, R. (1989) Comput. Appl. Biosci. 4, Natl. Acad. Sci. U. S. A. 85, 2444–2448 53–60 STEPHEN F. ALTSCHUL AND 2 Altschul, S. F. et al. (1990) J. Mol. Biol. 215, 17 Gribskov, M. (1992) Gene 119, 107–111 EUGENE V. KOONIN 403–410 18 Tatusov, R. L., Altschul, S. F. and Koonin, E. V. 3 Altschul, S. F. and Gish, W. (1996) Methods (1994) Proc. Natl. Acad. Sci. U. S. A. 91, Enzymol. 266, 460–480 12091–12095 NCBI, National Library of Medicine, NIH, 4 Altschul, S. F. et al. (1997) Nucleic Acids Res. 19 Yi, T-M. and Lander, E. S. (1994) Protein Sci. 3, Bethesda, MD 20894, USA. 25, 3389–3402 1315–1328 Email: [email protected]

radioactive labelling, cell fractionation, sucrose-gradient centrifugation and poly- Eukaryotic rRNA methylation: the acrylamide-gel electrophoresis, and many features of cellular and viral composition and biosynthesis were being revealed. calm before the Sno storm Sheldon Penman had developed a cell- fractionation procedure that allowed purification of the nucleolar precursors Much excitement has arisen from the Biology, in Cambridge. Robin had been a of rRNA (Ref. 8). When I arrived, Jon discovery that small nucleolar RNA mol- postdoc with Fritz Lipmann and was an Warner was developing this procedure ecules (snoRNAs) function as guides for early leader in the characterization of further for isolation and characteriz- post-synthetic modifications of eukary- the partial reactions of protein synthe- ation of nucleolar preribosomal RNP otic rRNA1–6. The title of one review, ‘Sno sis. The subject had interested me particles9. In a separate study, Jim’s storm in the nucleolus: new roles for greatly, and my contribution made a group discovered 5.8S rRNA (initially myriad small RNPs’6, abundantly con- mark7, but I had also become interested called 7S rRNA)10. The discovery exem- veys the excitement. A prerequisite for in RNA biosynthesis in animal cells plified Jim’s perceptive eye; he had no- this work was the accurate mapping of through reading Jim Darnell’s work; ticed that 28S rRNA sedimented slightly the numerous modified nucleosides when Jim offered me a postdoctoral pos- more slowly extraction with hot within eukaryotic rRNA. My colleagues ition, I accepted enthusiastically. phenol than after extraction with cold and I had the good fortune to contribute Jon Warner – who was already well phenol. He guessed that a small piece of to this earlier work, especially the map- known for discovering polysomes dur- noncovalently attached RNA might be ping of the RNA methyl groups. Here, I ing his PhD with Alex Rich at MIT – was released by heat treatment, and he and look back on the mapping work, which, closely associated with Jim’s research colleagues sought and characterized in retrospect, was a mini golden era of group. Because of my background in this RNA10. Jim and Jon were both most ‘calm before the Sno storm’. ribosomes, we agreed that I should work interested in regulation, however. One jointly with Jon and Jim. This was an approach to regulation was to observe Albert Einstein College of Medicine, ideal arrangement and was further en- the effects of depriving cells of a nutri- 1967–1969 hanced by the stimulating overall envi- tionally essential amino acid – and In January 1967, I arrived as a post- ronment. Harry Eagle (of Eagle’s thereby slowing down protein synthesis doc in Jim Darnell’s laboratory at the medium) had been influential in attract- to turnover levels – on ribosome for- Albert Einstein College of Medicine, New ing several leading cell biologists to mation. I used valine deprivation as a York. I had completed my PhD, on ribo- ‘Einstein’, and there were excellent model11; withholding this essential amino some-catalyzed peptidyl transfer, under interactions between research groups. A acid led to a reversible slowing down of Robin Monro at the Medical Research wide range of topics was amenable pre-rRNA synthesis and processing, but Council Laboratory for Molecular to the (then) illuminating methods of not to complete cessation. 0968 – 0004/98/$ – See front matter © 1998, Elsevier Science. All rights reserved. PII: S0968-0004(98)01304-8 447 REFLECTIONS TIBS 23 – NOVEMBER 1998

only by float plane (sea plane) from mid-1960s, Fred Sanger and colleagues Watson Lake on the Alaska Highway. I had developed two-dimensional separ- asked for the proofs to be sent to the ation methods for RNA oligonucleo- Watson Lake post office. Upon emerging tides16. Just before I left Cambridge, a from the wilds and collecting the proofs, new PhD student of Sanger, Peter I found that my short description of the Fellner, had started to study methylated newly discovered 7S rRNA had been em- oligonucleotides from enzymatic digests bellished with the words ‘if it is not an of Escherichia coli rRNA. His work was artefact’. I deleted the phrase and published17 shortly before I arrived in mailed the proofs back straightaway, Glasgow. It was a small logical step to but I was too late – the article had gone conceive of applying the methodology to print! It was an embarrassed postdoc to eukaryotic rRNA and pre-rRNA from who arrived back in the lab after his HeLa cells. long holiday. Fortunately, however, the When Salim arrived, we discussed the review was a success from all other points possibility. I calculated the amounts of of view and, to everyone’s pleasure, cells and labelled methionine, and the reprint requests came pouring in. labelling times, that would be needed to produce methyl-labelled rRNA and pre- Glasgow rRNA that had the necessary specific ac-

In the spring of 1969, I took up a tivity for T1-RNase digestion followed by lectureship in Biochemistry at the two-dimensional electrophoresis (finger- University of Glasgow. Martin Smellie, printing). Fingerprinting, however, was Figure 1 who had been my PhD external exam- new and daunting ground to me, and we Salim displays his RNA methyl finger- iner, was a professor there and had en- did not have the equipment. Fortunately, prints in the Glasgow lab. couraged me to apply. The head and Bob Williamson, who was then working founder of the department was Professor nearby at the Beatson Institute for Cancer Mike Vaughan, a postdoc in the lab, J. N. Davidson, whose small book The Research, had the necessary expertise had obtained very different results when Nucleic Acids I had read many years pre- and equipment, which he had been using studying methionine deprivation. He viously as an undergraduate. At first I to study 5S rRNA. He offered to collabo- was interested in the observation that was in awe of ‘JND’, but I soon perceived rate, and our first results showed im- eukaryotic rRNA appeared to be more that he considered me an asset, and he mediately that we were in business. 18S heavily methylated than prokaryotic used his influence to offer me two re- and 28S rRNA yielded different methyl rRNA, and to exhibit a preponderance of search students during my first year. fingerprints and, as predicted, the 45S 2Ј-0-methylation and only a minority of The department was in the process of fingerprint contained 18S and 28S spots. base methylation. Greenberg and installing good cell-culture facilities, The results18 were only a first glimpse Penman12 had shown that (most) rRNA through a grant from the Wellcome of what would eventually unfold, but methylation in HeLa cells occurs rapidly Trust, so I was able to continue using the they were sufficiently promising to on pre-rRNA, apparently on nascent cell labelling and fractionation methods justify the installation of a high-voltage- chains. Mike and his colleagues asked I had learned at Einstein. electrophoresis facility in the basement whether pre-rRNA methylation is essen- My first student, Jim Shepherd (who of the Biochemistry Department. They tial for ribosome formation. Exploiting is Professor of Pathological Bio- also helped me to obtain independent the fact that methionine is the source of chemistry at Glasgow Royal Infirmary), grant money. methyl groups for nucleic acid methyl- worked on a peptide-mapping project, Salim was amply fulfilling JND’s assur- ation, and that it is nutritionally essen- studying ribosomal proteins. We were ance that he was an excellent student, tial in animal cells, they showed that, seeking to extend Jon Warner’s ribosome- and we made an effective pair. Early on, I during methionine deprivation, pre-rRNA assembly studies9 by labelling ribosomal was interested in establishing the quali- methylation is largely suppressed and proteins with [35S]amino acids and then tative and quantitative relationships be- rRNA maturation is completely inhibited. displaying tryptic peptides in two- tween methylation of rRNA and pre- They inferred that pre-rRNA methylation dimensional fingerprints. The work met rRNA, whereas Salim was interested in is essential for ribosome maturation13. with some early success15, but the num- sequencing the methylated oligonu- I mentioned these findings in a short ber of labelled peptides was too large cleotides, using [14C]methyl-labelled review article14. A member of the edito- for full characterization, and two-dimen- and 32P-labelled rRNA separately or in rial staff of Nature had visited Einstein to sional fractionation of intact ribosomal combination, and a variety of enzymatic commission a review on rRNA and ribo- proteins – as developed by Wittmann’s and chemical degradation procedures. some biosynthesis. Jim and Jon both group – took over in such work soon One problem was to obtain sufficient had other writing commitments, so the afterwards. yields of all of the methyl-labelled nu- offer came to me. It was a great opportu- Initially, I was reluctant to take an- cleolar pre-rRNA species, including the nity for a fairly junior scientist, and I other student during my first year, but quantitatively minor 41S intermediate, worked hard to produce an informative JND did not accept ‘no’ for an answer. for fingerprinting. Weinberg and account of the state of the art. There He assured me that the student, Salim Penman had shown19 that these species was a small glitch in publication. That (Fig. 1), who was from Pakistan, was ex- accumulated during infection of HeLa summer (1968), I had planned a moun- cellent. I therefore accepted JND’s offer cells with poliovirus. I therefore ar- taineering trip to a remote area in north- and began to think out a project that ranged to revisit Einstein and work in ern Canada. The area was accessible had been at the back of my mind. In the Don Summers’ lab to do the necessary 448 TIBS 23 – NOVEMBER 1998 REFLECTIONS polio experiments, and bring the RNA While working on the problem of the I was also considering the possibility back to Glasgow for fingerprinting20. The absolute molar yields of the methylated that I could begin to localize the methyl poliovirus strain was an attenuated one oligos, we had established a friendly re- groups by analyzing the RNA recovered and, as I recall, we did not need to han- lationship with Rudi Planta’s group in after hybridization to specific segments dle it again in Glasgow. Nevertheless, Amsterdam, who were doing very simi- of DNA. Don had electrophoresis tanks JND and Martin Smellie rightly insisted lar work on yeast rRNA. They showed that I would be able to use for finger- that, for safety, all members of the de- that there were fewer methyl groups in printing the recovered rRNA segments. I partment should receive polio vaccine; I yeast rRNA than in vertebrates26, but think both Don and Ron were surprised had the congenial duty of administering that qualitative aspects (the many early that I should wish to pursue this seem- sugar lumps treated with vaccine to the ribose methylations and fewer late base ingly idiosyncratic (but intuitive) line of entire staff as they filed past! methylations) were highly similar in research rather than work on transcrip- Another problem concerned the ab- yeast and vertebrates. We collaborated tion. However, I was, and remain, very solute molar yields of the methylated in the characterization of an oligonu- grateful that they agreed to my pursuing oligos. Previously published data that cleotide that contains a conserved, it. I made a start by determining which related indirectly to this question were hypermodified nucleoside in 18S rRNA. methylated oligos were recovered in confusing. The solution came fortu- The nucleoside, m1acp3y, had been RNA that hybridized to the left, and itously when Salim and I visited identified earlier27 and is remarkable be- which were recovered in RNA that hy- Cambridge to learn some further separ- cause it incorporates label (in separate bridized to the right, of the unique ation methods from George Brownlee. biosynthetic reactions) from the methyl EcoRI sites in the 18S and 28S genes29. I George had been a student of Fred group and then C1 of methionine28. also subcloned rDNA into smaller pieces, Sanger when I was with Robin Monro, All of this work was done before we which would allow subsequent re-itera- and he was by this time a staff member. had any means for determining the com- tion of this general approach. Lastly, I He mentioned a technique for character- plete sequences of the rRNA molecules learned the intricacies of the Maxam izing oligo-A tracts in RNA by digesting or the whereabouts of the methyl and Gilbert sequencing method from the RNA using combined T1 RNase groups in the sequence. The methylated Ron Peterson, a postdoc who was working (which cleaves after G residues) and oligos were unplaced pieces in a linear with Don Brown. These beginnings pancreatic RNase (which cleaves after U puzzle. By this time, the E. coli 16S rRNA paved the way to the subsequent map- and C residues). It was known that these sequence was nearing completion in ping of all but a few of the methyl groups. enzymes would not cleave after 2Ј-O- Strasbourg, but its assembly from the methylated nucleotides. I realized that necessary partial digestions and over- Glasgow to Liverpool several rRNA oligos containing such 2Ј- laps had been a daunting task. We did When I returned to Glasgow in au- O-methyls could be purified by elec- not seriously contemplate applying tumn 1978, I had a new student, Lucinda trophoresis from digests of 32P-labelled such methods to human 18S or 28S Hall. I was also able to arrange for Salim, rRNA and, therefore, quantified. rRNA. However, a revolution in method- who had been on the faculty at Application of this method successively ology was about to occur. Islamabad for a few years, to a to 32P-labelled and then [14C]methyl- year on sabbatical with me. Lucinda set labelled rRNA gave the required stoi- Carnegie sabbatical up the Maxam and Gilbert sequencing chiometries and, hence, for the first I had been following the Carnegie methodology and applied it to the diffi- time, accurate values for the total num- Embryology group’s work on rRNA cult, GC rich, internal transcribed bers of methyl groups21,22 – about 115 genes in Xenopus laevis with great inter- spacer region of Xenopus rDNA (Ref. 30). per human ribosome. est. In 1969, I had paid a brief visit. Salim acquired the new skills and se- In 1972, Salim was awarded his PhD Later, in 1977–1978, I spent a sabbatical quenced the 18S gene. Meanwhile, I con- (Fig. 2); sadly, JND passed away shortly year at their lab. I was particularly fortu- tinued to map the 18S and 28S rRNA before this. In 1974, we published a com- nate for two reasons: (1) Carnegie was methyl groups to regions defined by prehensive paper on this phase of the one of the leading centres at the begin- smaller segments of rDNA. This work work23. Shah Khan had by that time ning of the cloning revolution; and (2) was technically demanding because the joined the group, and we showed that there had been considerable competi- amounts of radioactivity recovered in rRNA-methylation patterns are highly tion for places there. [14C]methyl experiments were quite low, conserved among vertebrates24. We When I arrived, the large-scale struc- and I often had to wait several weeks for were also directing our thoughts to the tural characterization of ribosomal gene good autoradiographs. I specialized in recognition processes that generate the organization was essentially complete. this technique but also familiarized my- many methyl groups present in different Interest had moved to DNA sequencing self with DNA sequencing. local sequence environments. One pos- (by the Maxam and Gilbert method) By the time Salim had finished the 18S sibility was that some recurrent feature and, especially, to transcription. Don rDNA sequence, I had mapped the rRNA of secondary or tertiary structure was Brown’s group were by that time con- methyl groups to within tracts of a few recognized, perhaps on the rRNA sur- centrating on 5S RNA genes, while Ron hundred nucleotides. In most instances, face. To start to address this problem, Reeder’s group were working on the we had sufficient sequence data from

Shah performed S1-nuclease digests on genes for 18S and 28S rRNA (rDNA); this the earlier work on the methylated oli- methyl-labelled rRNA (Ref. 25). Contrary led to their ground-breaking work on gos23 to identify a unique sequence in to our expectations, a wide range of sen- transcription by RNA polymerases III the relevant tract of rDNA defined by the sitivites to S1 nuclease was evident; this and I, respectively. hybridization experiments. We could implied that some methylation sites The possibility of using rDNA to purify therefore pinpoint the RNA methyl were exposed but that others were buried specific segments of rRNA for further groups in the complete sequence derived in the three-dimensional structure. analysis had been at the back of my mind. from rDNA. This work was published in 449 REFLECTIONS TIBS 23 – NOVEMBER 1998

References (a) (b) 3 5 1 Kiss-Laszlo, Z. et al. (1996) Cell 85, 1077–1088 4 6 2 Nicoloso, M., Qu, L-H., Michot, B. and Bachellerie, J-P. (1996) J. Mol. Biol. 260, 178–195 1 2 3 Ni, J., Tien, L. and Fournier, M. J. (1997) Cell 89, 565–573 4 Ganot, P., Bortolin, M-L. and Kiss, T. (1997) Cell 89, 799–809 5 Bachellerie, J-P. and Cavaillé, J. J. (1997) Trends Biochem. Sci. 22, 257–261 Figure 2 6 Smith, C. and Steitz, J. A. (1997) Cell 89, 669–672 (a) Salim’s leaving party (1973). 7 Maden, B. E. H., Traut, R. R. and Monro, R. E. (b) Key to (a). 1, Salim; 2, Jim (1968) J. Mol. Biol. 35, 333–345 Shepherd; 3, Bob Williamson; 4, Ted 8 Penman, S., Smith, I. and Holtzman, E. (1966) Maden; 5, John Goddard; 6, Nigel Science 154, 786 Fraser. 9 Warner, J. R. and Soeiro, R. (1967) Proc. Natl. Acad. Sci. U. S. A. 58, 1984–1990 10 Pene, J. J., Knight, E. and Darnell, J. E. (1968) J. Mol. Biol. 17, 117–130 198131. (A single correction to the DNA recognition processes that underlie 11 Maden, B. E. H., Vaughan, M. H., Warner, J. R. and sequence was made later32.) what had seemed to be a bewildering di- Darnell, J. E. (1969) J. Mol. Biol. 45, 265–275 12 Greenberg, M. and Penman, S. (1966) J. Mol. In 1983, I became a professor at the versity of modification sites. Moreover, Biol. 21, 527–535 University of Liverpool. At this time, the discovery of the involvement of 13 Vaughan, M. H., Soeiro, R., Warner, J. R. and rRNA sequences, usually derived from snoRNAs, particularly the finding that Darnell, J. E. (1967) Proc. Natl. Acad. Sci. U. S. A. 58, 1527–1534 the genes, were accumulating rapidly in many snoRNAs in higher organisms are 14 Maden, B. E. H. (1968) Nature 219, 685–689 2 the literature, and consensus models for derived from introns , has linked rRNA 15 Shepherd, J. and Maden, B. E. H. (1972) Nature rRNA secondary structures were emerg- modification to a much wider field of 236, 211–214 ing. Knowledge of the methyl-group molecular cell biology. 16 Sanger, F., Brownlee, G. G. and Barrell, B. (1965) J. Mol. Biol. 14, 373–398 locations was becoming increasingly The rRNA methylation work de- 17 Fellner, P. and Sanger, F. (1968) Nature 219, important in this structural context. scribed here paralleled general ad- 236–238 I worried that locating the 18S methyl vances in our understanding of ribo- 18 Salim, M., Williamson, R. and Maden, B. E. H. groups had required a large amount of some structure and biosynthesis, as (1970) FEBS Lett. 12, 109–113 19 Weinberg, R. A. and Penman, S. (1970) J. Mol. evidence, which had taken many years well as of modification of other RNA Biol. 47, 169–178 of research to accumulate, and that it molecules – tRNA, snRNA and eukary- 20 Maden, B. E. H., Salim, M. and Summers, D. F. had not been possible or appropriate to otic mRNA. In 1994, an EMBO workshop (1972) Nat. New Biol. 237, 5–9 21 Maden, B. E. H. and Forbes, J. (1972) FEBS give that evidence in detail in our Nature on RNA modification and editing, Lett. 28, 289–292 31 paper . I rectified this in 1986, by writ- planned by Henri Grosjean with help 22 Maden, B. E. H., Lees, C. D. and Salim, M. ing a paper that incorporated all the from Glenn Björk, James McCloskey and (1972) FEBS Lett. 28, 293–296 data from our own lab, as well as all rel- myself, was held in Aussois and was 23 Maden, B. E. H. and Salim, M. (1974) J. Mol. 32 Biol. 88, 133–164 evant published data from other labs . deemed highly successful in bringing 24 Khan, M. S. N., Salim, M. and Maden, B. E. H. Then followed the even-more daunting together many different workers and (1978) Biochem. J. 169, 531–542 task of assembling the 28S data (we points of view. The proceedings were 25 Khan, M. S. N. and Maden, B. E. H. (1978) Eur. Biochimie J. Biochem. 84, 241–250 had some unassigned methyl lo- published in (Vols 76 and 77), 26 Klootwijk, J. and Planta, R. J. (1973) Eur. J. 33 cations), and this was published in 1988 . and a comprehensive book, Modification Biochem. 39, 325–333 A review of our work and that of others, and Editing of RNA, which was con- 27 Saponara, A. G. and Enger, M. D. (1974) including the problem of locating ceived at the workshop, has recently Biochim. Biophys. Acta 349, 61–77 39 28 Brand, R. C., Klootwijk, J., Planta, R. J. and pseudouridines, on which only limited been published . Maden, B. E. H. (1978) Biochem. J. 169, 71–77 progress had been made at that time, 29 Maden, B. E. H. and Reeder, R. H. (1979) was published in 199034. Later, both we Note Nucleic Acids Res. 6, 817–830 and Jim Ofengand’s group developed The early conclusion that pre-rRNA 30 Hall, L. M. C. and Maden, B. E. H. (1980) Nucleic Acids Res. 8, 5993–6005 the reverse-transcriptase methodology, methylation is essential for ribosome 31 Salim, M. and Maden, B. E. H. (1981) Nature which facilitated location of pseudo- maturation13 requires, and is receiving, 291, 205–208 uridines35 and 2Ј-O-methyl groups36. further work. In general, ribosome matu- 32 Maden, B. E. H. (1986) J. Mol. Biol. 189, 681–699 33 Maden, B. E. H. (1988) J. Mol. Biol. 201, 289–314 A puzzling aspect of these modifi- ration is not abolished in the single- 34 Maden, B. E. H. (1990) Progr. Nucleic Acid Res. cations was that only one research group, methyl-group knockouts that are Mol. Biol. 39, 241–303 Douane Eichler and colleagues, reported now available. The current, rapidly 35 Ofengand, J. and Bakin, A. (1997) J. Mol. Biol. any success in obtaining site-specific ri- accumulating evidence is beyond 266, 246–268 36 Maden, B. E. H. et al. (1995) Biochimie 77, 22–29 bose methylation in vitro – and this was the scope of this article, but is reviewed 37 Segal, D. M. and Eichler, D. C. (1991) J. Biol. confined to a triplet of methyl groups in elsewhere1,2,5. Chem. 266, 24385–24389 28S rRNA (Ref. 37). Denis Lafontaine and 38 Lafontaine, D. et al. (1994) J. Mol. Biol. 241, 38 Acknowledgements 492–497 colleagues isolated the gene that en- 39 Grosjean, H. and Benne, R., eds (1998) codes the enzyme that modifies the two I thank all mentors and collaborators Modification and Editing of RNA, ASM Press base-methylated dimethyladenosines mentioned in the text, and John Forbes, Ј near the 3 end of the 18S rRNA. Mary Robertson, Elaine Mayers and B. EDWARD H. MADEN The finding that snoRNAs direct Karen Pugh for technical support. The modification (ribose methylation and work has been supported at various School of Biological Sciences, University of pseudouridylation) of the target nucleo- stages by the CRC, the MRC and the Liverpool, Life Sciences Building, Crown sides1–4 has clarified immensely the Wellcome Trust. Street, Liverpool, UK L69 7ZB. 450