oxidative stress shortens telomeres
TRANSCRIPT
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Telomeres were established cytogenetically in the firsthalf of the 20th century as functional chromosome‘caps’, and it is now a quarter of a century since theywere characterized in molecular terms as stretches ofrepetitive DNA with high G–C strand asymmetry.Starting from basic principles of semiconservativereplication of DNA, it was hypothesized thattelomeres should face an ‘end-replication problem’;that is, they should shorten with each round of DNAreplication owing to the loss of the most distal primerfor lagging-strand synthesis [1,2]. Standardpolymerases are unable to fill the resulting terminalgap, resulting in a severe restriction for eukaryoticgrowth in the absence of a compensatory mechanism.In fact, an enzyme was identified in Tetrahymenaextracts that could elongate telomere 3′ ends and thus compensate for the end-replication problem [3].This enzyme (telomerase) was shown to be aribonucleoprotein, carrying a small RNA with asequence that can act as a template for the elongationof the G-rich DNA terminus [4].
Still, telomeres attracted little attention from thewider community until the early 1990s. This changedabruptly after it was experimentally demonstratedthat telomeres in normal human fibroblastsshortened progressively during culture in vitro [5].For the first time, a clear mechanism had beenidentified for a biological clock that could measuremitotic time and account for the phenomenon of cellreplicative senescence [6]. In cell replicativesenescence, under standard culture conditions, cellscease division after a more-or-less-fixed number ofcell divisions (commonly termed the ‘Hayflick limit’)that is a characteristic of the particular cell strain.The timing of senescence depends primarily on thereplicative history of the cells (the number of elapsedcell divisions) and much less on the passage ofchronological time.
Since the early 1990s, the hypothesis thattelomere loss might serve as a mitotic clock has been
strengthened by extensive correlative evidenceindicating the presence of telomerase activity or analternative mechanism of telomere lengthening inimmortal, but not mortal, cell strains. It was finallyconfirmed by the successful immortalization ofnormally mortal (and telomerase-negative) humancell types by forced production of hTERT, the catalyticsubunit of human telomerase [7].
In its most simplistic form, the resulting telomerehypothesis of senescence is summarized in Fig. 1a.The essential features of the hypothesis are:(1) shortening of telomeres to some threshold valuetriggers senescence; and (2) telomeres shorten with aconstant rate so that telomere length is a faithfulindicator of replicative history. Many questionsremained to be answered about how short telomeresactivate senescence and whether the mechanism istriggered when a single telomere or a certain subgroupof or the average of all telomeres reached thethreshold. Despite this, the telomere hypothesis hasdominated our understanding of cell senescence inrecent years. On this basis, telomere length is widelyused to obtain estimates of the replicative history ofcells. However, such a simple ‘mitotic clock’hypothesiscannot easily accommodate the marked intrinsicheterogeneity of replicative lifespan that is seenamong the individual cells that make up cell cultures.Moreover, considerable evidence points to a strongstress-dependence of cell-culture lifespan that again isat variance with the idea of a fixed mitotic clock.
Heterogeneity in replicative senescence
In a classic experiment, Smith and Whitney [8]demonstrated huge heterogeneity, and evenbimodality, in the doubling potential of individualcells from a clonally derived cell population. Otherstudies using BrdU labelling [9], Ki67 staining [10] ora p53-activity reporter assay [11] have shown that,with increasing population doubling level, fewer andfewer cells remain in the actively proliferating pool.This has been interpreted as indicating that a certainproportion of cells drop permanently out of the cellcycle at all population doubling levels, with theproportion of dropouts increasing with the age of theculture. Although this accords with the typical growthcurves of primary cells and readily explains theobserved heterogeneity in replicative senescence, itimplies that cells in a senescent culture display abroad distribution of replicative histories: some cellsbecome permanently arrested after very few celldivisions, whereas others might go through manymore divisions than indicated by the Hayflick limit ofthe population as a whole.
Can the current model of telomere shorteningexplain this heterogeneity? Unfortunately, telomerelength in cells that display a senescent phenotypeearly during the replicative lifespan has so far notbeen measured. Earlier mathematical modellingattempts assuming a fixed rate of telomereshortening per cell division were able to fit observed
Oxidative stress
shortens telomeres
Thomas von Zglinicki
Telomeres in most human cells shorten with each round of DNA replication,
because they lack the enzyme telomerase. This is not, however, the only
determinant of the rate of loss of telomeric DNA. Oxidative damage is repaired
less well in telomeric DNA than elsewhere in the chromosome, and oxidative
stress accelerates telomere loss, whereas antioxidants decelerate it. I suggest
here that oxidative stress is an important modulator of telomere loss and that
telomere-driven replicative senescence is primarily a stress response. This
might have evolved to block the growth of cells that have been exposed to a
high risk of mutation.
Thomas von Zglinicki
Dept Gerontology,University of Newcastle,Wolfson Research Centre,General Hospital,Westgate Road,Newcastle upon Tyne, UK NE4 6BE.e-mail: [email protected]
Opinion
growth curves reasonably well, especially if variationin cell-cycle times was included in the model [12,13].However, to model the heterogeneity in growthpotential successfully, it turned out to be necessary toassume an additional, stochastic component oftelomere shortening. Such a component might be dueto, for instance, random variation in the position ofthe most distal primer for lagging-strand synthesis[14], telomeric recombination [15] or stress-mediatedsingle-strand breakage [16]. Thus, all recentmodelling attempts agree that early-senescing cellshave telomeres as short as those in cells that reachsenescence only after many more divisions, and thatthe rate of telomere shortening must have beenhigher in early-senescing cells of a clone. I must stressagain that this idea still awaits experimentalverification. Premature senescence can be induced ina telomere-independent fashion by a range of stresses(see below). Thus, it is one view in the field that onlythose cells of a culture that divide until the very endexperience ‘real’ telomere-driven senescence; all othercells (i.e. most of them) would become senescent bytelomere-independent stress (‘culture shock’) [17].However, this is not in accordance with the data aboutpremature senescence and telomere shortening undermild stress (see below).
The second common view in the field is thatreplicative heterogeneity is driven purely by chancevariations in telomere shortening owing to, forexample, primer positioning variation or randomrecombination events. Thus, the average
telomere-shortening rate would still be anunequivocal indicator of replicative history, at least ina large cell sample (Fig. 1b). However, recombinationevents do depend on the prior existence of DNAdamage (i.e. single-strand breaks) [15]. Similarly, thepositioning of the most distal primer might depend onthe presence of damage sites and so cause atemporary stalling of the replication fork, which isanother possible contributor to ‘stochastic’ telomereshortening [18]. Thus, there is a good theoreticalchance that stress might influence the probability ofaccelerated telomere shortening and that telomere-driven and stress-induced senescence might be veryclosely interrelated indeed.
Stress dependency of replicative senescence
The irreversible loss of replicative capacity leads to asenescent phenotype, which is characterized by:irreversible growth inhibition, mostly in G1; changes in cell morphology such as flattening andenlargement; increased staining for senescence-associated β-galactosidase; a changed gene-expressionpattern; and upregulation of the activities of p53 orp21. Such arrest is produced not only afteraccumulated population doublings in culture, withconcomitant telomere shortening, but also as a resultof a wide variety of subcytotoxic stresses, includingmild chronic oxidative stress [19], acute treatmentwith a variety of DNA-damaging agents or ‘cultureshock’. Culture shock is mediated by p14ARF, at least inmouse fibroblasts [20], so transfection with oncogenes(e.g. activated RAS or p14ARF), overexpression of Rassignal mediators (e.g. mitogen-activated-proteinkinases) or treatment with histone deacetylaseinhibitors can all induce a senescent phenotypeindependently of telomere shortening [21]. Moreover,acute DNA-damaging treatments strong enough toimmediately induce a long-lasting growth arrest donot influence telomere length [22]. The question, then,is whether there is a significant overlap betweenreplicative senescence – the result of progressivetelomere shortening – and stress-induced senescenceor whether these processes are mechanisticallydifferent, as has recently been suggested [17]. In otherwords, is telomere shortening stress dependent?
Recent work has given an increasingly clearanswer to this question (Fig. 2). Accelerated
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Fig. 1. The telomere hypothesis of replicative senescence. (a) Maintenance of telomere length inhuman germ-line (and stem) cells (red) allows unlimited growth of these cells. Telomeres in humansomatic cells shorten by a constant amount with each division (green). At a crucial threshold length T0,which corresponds to the Hayflick limit (HL), senescence is triggered. There is only one trajectory tosenescence in this model and the HL is a constant. If the senescence checkpoint machinery iscompromised, cells overcome senescence and will shorten telomeres further until this leads to crisis.Those cells that can compensate for telomere erosion by activation of telomerase and/or alternativepathways of telomere maintenance (blue) can escape crisis and become immortal, even with shorttelomeres. (b) Chance events can increase the rate of telomere shortening. Accordingly, a proportion ofthe cells senesce prematurely but all cells senesce at the same telomere length. (c) The amount oftelomere shortening per cell division varies between subclones and from division to divisiondepending on the balance of oxidative stress and antioxidative defence (indicated by the width of theblue triangle). This allows a vast multitude of trajectories to senescence, which is still triggered at thesame threshold length T0. However, the Hayflick limit is no longer a constant but can vary depending onthe factors that influence the rate of telomere loss, such as oxidative damage and antioxidative defence.The minimum Hayflick limit (HLmin) is 1 population doubling (PD) because telomere shortening requiresat least one cell division. The maximum Hayflick limit (HLmax) is defined by the rate of telomereshortening in cells with perfect antioxidative defence (i.e. by the end-replication problem alone).
telomere shortening, together with decreasedreplicative lifespan, was found under a range of mild stresses. A mild stress is defined here as onethat allows at least some cell proliferation to occur, such as chronic hyperoxia [23–25] andtreatment with homocysteine [26], low doses oftert-butylhydroperoxide [27] or hydrogen peroxide(H2O2) [28,29]. A similar acceleration of telomereloss and reduction in proliferative lifespan is alsoseen in fibroblasts from subjects with Fanconianaemia (FA) [30], a condition that results inincreased oxidative stress. Out of 22 independentexperiments reported from seven differentlaboratories, an increased telomere shortening rateunder conditions of increased stress was confirmedin all but three experiments. In these three cases,telomere shortening rates were already very lowunder control conditions and, in fact, the intrinsicantioxidative capacity was high enough to buffer theeffect of hyperoxia [25,31], a nice example of ‘theexceptions proving the rule’.
The available data show that the acceleratedtelomere shortening seen under increased oxidativestress cannot be regarded simply as an observationartefact (Box 1). More importantly, antioxidants notonly reversed the accelerated telomere shorteninginduced by increased oxidative stress [26,32].Treatment of human endothelial cells and fibroblastswith either ascorbic acid 2-O-phosphate [33] or withthe free-radical scavenger α-phenyl-t-butylnitrone[28] even prolonged the replicative lifespan and
slowed down telomere shortening compared with cellsunder standard culture conditions.
The data suggest that differences between cellstrains in telomere-shortening rates under constantexternal stress should be due to different capacitiesfor antioxidative defence. This was confirmed by acomparison of fibroblast strains from different humandonors using several indicators of oxidative stress anddefence capability, such as dihydrochlorofluoresceinfluorescence [28,31], steady-state protein carbonyllevels [34] or rates of lipofuscin accumulation [35].The single most important factor for maintaining lowintracellular peroxide concentrations and a low rateof telomere shortening in human fibroblasts appearedto be the production and activity levels of superoxidedismutases [36] (T. von Zglinicki, unpublished).Preliminary evidence that telomere shorteningin vivo is linked with disease states in which oxidativestress plays a causative role comes from recentfindings that short telomere length in lymphocytes iscorrelated with the incidence of vascular dementia[31], atherosclerosis [37] or aplastic anaemia [38].These data are often seen as an indication of fastercell turnover in the stem-cell compartment inaccordance with the idea of constant telomereshortening rates. However, oxidative stress issupposed to play a strong causal role in all of thesediseases, and there is an intriguing possibility thatsystemic oxidative stress, whether caused by eitheran unusually low antioxidative capacity or anunusually high exposure to oxidative stressors, canexplain this association. If this idea is correct, itsuggests that telomere length might serve as abiomarker of cumulative exposure to stress and aprognostic indicator for risk of late-life diseases.
How does oxidative stress cause telomere shortening?
The lowest telomere shortening rates in humanfibroblasts are around 10–20 bp per populationdoubling (Fig. 2). This shows that polymerase α canposition the most distal primer very close to thephysical end of the chromosome. Faster telomereshortening rates are observed in other cell strains,which also have higher peroxide levels, indicating lessefficient antioxidant defence [25,31]. The clearimplication is that the contribution to telomere losscaused by oxidative damage is, in many cases, muchgreater than the contribution from the end-replicationproblem alone. Telomeres, as triple-G-containingstructures, are highly sensitive to damage byoxidative stress [39], alkylation [40] or ultraviolet(UV) irradiation [41]. Accordingly, high-intensitystresses can cause telomere shortening without DNAreplication by inducing telomeric double-strandbreaks at high frequency [32,41]. Whether this hasany physiological relevance is unclear because suchtelomere shortening will always be accompanied bysignificant damage elsewhere in the genome.
By contrast, telomere shortening under mildstresses, as under control conditions, requires DNA
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Fig. 2. Stress dependency of telomere shortening: data on telomere-shortening rates under conditionsof decreased oxidative stress (red), standard cell culture (green) and increased oxidative stress (blue).The cells and conditions used were: 1, human embryonic fibroblasts (HEF) WI-38, normoxia/40%hyperoxia [23]; 2, HEF MRC-5, PBN/normoxia/hyperoxia [28]; 3, HEF WI-38, normoxia/hyperoxia [24];4, human foreskin fibroblast (HFF) BJ, normoxia/hyperoxia [24]; 5, HEF BJ, normoxia/hyperoxia [25];6–14, human skin fibroblast (HSF) from different donors, normoxia/hyperoxia [31]; 15, HFFnormal/glucose-6-phosphate-dehydrogenase-deficient [49]; 16, HSF normal/Fanconi anaemia [30];17, HEF WI-38 control/bolus tert-butylhydroperoxide stress [29]; 18, HEF WI-38 control/repeatedtert-butylhydroperoxide stress [29]; 19, HEF WI-38 control/H2O2 [29]; 20, HEF MRC-5 control/H2O2 [28];21, human endothelial cells (HUVEC) Ascorbate–2-O-phosphate/control/H2O2 [33]; 22, HUVECcontrol/homocysteine [26]. Abbreviation: PD, population doubling.
replication [23,42–44]. That indicates that theinduction of double-strand breaks is not the maincause of damage-induced telomere shortening but thatthe presence of unrepaired nucleotide or base damageinterferes with the replication fork at telomeres in away that enlarges the proportion of unreplicated ends.Whereas nucleotide excision repair of UV-inducedpyrimidine dimers in fibroblast telomeres was similar
to that in other non-transcribed loci [45], telomereswere significantly less proficient in repair ofsingle-strand breaks formed by oxidative or alkylativeDNA damage than non-transcribed minisatellites orthe bulk of the genome [40]. Restriction with limitedamounts of S1 nuclease [23,43,46–48] and alkaline gelelectrophoresis [28,40] indicated a much higherdensity of single-stranded sites in telomeres thanelsewhere in the genome. Two-dimensionalelectrophoresis confirmed these sites as single-strandbreaks, which are rather homogeneously distributedalong the telomere [28].
Cells that are held for a time in a non-proliferativestate (e.g. at confluence) accumulate telomeric single-strand breaks. If confluent cells are replated andallowed to proliferate, their telomeres initially shortenmore quickly until the frequency of single-strandbreaks returns to normal [28,43]. Although fastertelomere shortening was not observed in anotherstudy following prolonged periods of confluence usinga slightly modified setting [44], the positive correlationbetween telomere single-strand-break frequency andshortening rate has recently been confirmed [47,48]. Itis not known at present why single-stand break repairis less proficient in telomeres than in interstitialnoncoding regions of the genome. Also, the mechanismby which the presence of single-strand breaksaccelerates telomere shortening is not clear. Any of thepossibilities named above might be important.
Conclusion
Cellular replicative senescence is heterogeneous evenwithin a clonally derived cell population growingunder standard (i.e. low-stress) conditions, and therates of cellular ageing and telomere shortening inbulk culture depend on the balance between oxidative
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It has been suggested that the accelerated telomere shortening seenunder increased oxidative stress might be an artefact resulting fromthe increased replicative demand on a subpopulation when asignificant proportion of the cells are forced by stress to drop out ofthe cell cycle [29]. This might be true if the cells leaving the cyclingcompartment died quickly (e.g. by apoptosis). However, in the humanfibroblast system, the proportion of cells driven into apoptosis by mildoxidative stress is negligible. If all cells divide equally, the finalnumber of cells after n population doublings (NF) would be given by Eqn 1,
NF = 2n N0 [1]
where N0 is the starting cell number and (as a first-order approximation)n ≈ tobs ÷ tcyc (where tobs is the observation time and tcyc the duration ofone cell cycle). In this case, the observed telomere shortening ∆TF perpopulation doubling would equal the (constant) telomere shortening percell division ∆T0.
Let us now assume that a only proportion f = N0,growth/ N0 of cellsgrows after stress, with all other cells (N0,blocked) being arrested without telomere shortening. Evidently, the growing cells have todivide ns times (ns > n) for the culture to reach the same final cellnumber NF (Eqn 2).
[2]
The observable telomere shortening rate per population doublingcan now be calculated as an average of early dropouts (no telomereshortening) and compensatory fast-cyclers (telomere shortening by nsunits) (Eqn 3).
[3]
Dumont et al. [2,9] treated fibroblasts with low doses oftert-butylhydroperoxide and measured values of f = 0.16, n = 1,∆T0 = 105±12 bp, ∆TF = 381±139 bp. The above model results inns = 2.85 and ∆TF = 173±20bp, which is less than half then their measured∆TF. Measuring fibroblast growth rates and telomere shortening ratesbefore and after extended periods of confluence, Sitte et al. [43] found∆T0 = 124±14 bp, ∆TF = 349±93 bp. However, the growth rate f was still at~72% at confluence, indicating that the above model would account fornot more than a 10% increase in ∆T. Similarly, the growth rate f in fiveFanconi anaemia fibroblast lines was half that in controls, whereas thetelomere shortening rate was twice as large [30]. The above modelresults in not more than a 25% increase of ∆T.
In conclusion, observed increases in telomere shortening rates undermild stress conditions are too large to be explained solely bycompensatory cycling without the assumption of an additional increasein the amount of telomere sequence lost per cell division. In other words,mild stress accelerates telomere shortening.
Box 1. Is accelerated telomere shortening under increased oxidative stress an observation artefact?
0n
growth0n
blocked0F Nf2f1N2NN ss ])[(,,+−=+=
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2fnTnN
TnN20T
ss ∆=
∆+=∆ ,
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Str
ess
leve
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Fig. 3. Replicative senescence as a tumour-suppression mechanism. Mutational risk increases notonly with increasing number of cell divisions but also with stress-induced damage between divisions.If senescence only counted cell divisions (light vertical bar) then it would allow many cells to growwith a high mutational risk (white tracks) and would be ineffective at tumour suppression, especiallyin a stressful environment. If telomeres, being less well repaired, monitor the probability of genomicdamage, senescence (dark diagonal bar) inhibits the growth of all cells before their mutational riskbecomes too high (yellow tracks).
stress and antioxidative defence. This stronglysuggests that the main mechanism of senescenceunder low and mild stress conditions is telomereshortening, and that telomere shortening itself isstress dependent. The rate of telomere shortening percell division is not an innate constant. Rather, itchanges from cell to cell, possibly from one divisioncycle to the next, as a function of (external) oxidativestress and (internal) antioxidant defence. Dependingon this balance, many trajectories to senescence arepossible (Fig. 1c), with widely different averagetelomere shortening rates.
This leads to two important conclusions. First,telomeres are not merely ‘cell-division counters’.A proportion of the oxidative damage inflicted upontelomeres remains unrepaired and determines theamount of shortening in the next round of replication.This proportion is related to the total amount ofdamage in the bulk of the genome. Although most ofthat damage has been repaired, it is the residual,unrepaired fraction that determines the probability ofmutation. Thus, telomere shortening counts not onlycell divisions but also the cumulative probability ofmutations occurring, and short telomeres triggersenescence in response to oxidative stress and
mutational probability. I suggest that telomeres actas cellular ‘sentinels’ for genomic damage and remove‘dangerous’ cells from further proliferation.
Second, telomere-driven senescence depends onthe culture conditions. Indeed, replicative senescenceis a stress response in the vast majority of cases.There is still an upper limit to the proliferation ofsomatic human cells, set by the end-replicationproblem. However, in most cases, telomere-drivensenescence occurs much sooner owing to imperfectprotection from damage, which results in acceleratedtelomere loss. From the evolutionary point of view,telomere-driven senescence becomes much moreplausible as a tumour-suppression mechanism if itlimits proliferation not just after some magic numberof cell divisions but also in response to possibledamage to the genome (Fig. 3). This newinterpretation of telomere-driven cell senescence doesnot detract from previous experimental work on thissystem but it offers a richer set of possibilities toexplain the apparent contrast between senescencecaused by mitotic clocks and that caused by cultureshocks. It also opens exciting new possibilities forinterventions in cellular ageing and for usingtelomere length as a biomarker.
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Acknowledgements
My work leading to thisarticle was supported byVerum FoundationMunich, the DeutscheForschungsgemeinschaft,Germany and the MedicalResearch Council (UK).I thank T. Kirkwood forcritically reading thearticle.
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TRENDS in Biochemical Sciences Vol.27 No.7 July 2002
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344 OpinionOpinion
The biosynthesis of rRNA includes the formation ofmodified nucleotides in pre-rRNA. The nucleotidemodifications are of three main types: (1) conversionof uridine to pseudouridine (Ψ); (2) methylation of2′ hydroxyls (Nm); and (3) alterations to bases, mostof which undergo methylation at different positions(mN) (Fig. 1). The extent and complexity ofmodifications varies between and within the threephylogenetic kingdoms. Examples for Bacteria andEukarya include Escherichia coli [ten Ψs (includingone modified Ψ), four Nms (including onebase-modified Nm) and 19 mNs], Saccharomycescerevisiae [44 Ψs (including one Ψ with additionalmodifications), 54 Nms, and ten mNs] andHomo sapiens (~91 Ψs, 105 Nms and ten mNs) [1–3].Current values for the Archaea range substantially,with some having considerably more Ψ and Nmnucleotides than Bacteria [1,4,5].
There is an interesting dichotomy in the modifyingmachinery. Modifications in E. coli are mediated byprotein-only enzymes that are site- or region-specific.
By contrast, the Ψ and Nm modifications ineukaryotes are created by two families of site-specific,small nucleolar RNA–protein complexes (snoRNPs).One such family is responsible for most, and possiblyall, pseudouridylations and another for2′-O-methylations. In both processes, sites areselected by the RNA component through base pairing(guide function), and catalysis is mediated by a type-specific protein component [6]. The Archaea use anevolutionarily related RNP for ribose methylation [4]and proteins related to those in the Ψ-formingsnoRNPs have been identified [7].
The effect of nucleotide modification on rRNA isone of the oldest questions in RNA science andinformation is still limited [8]. The chemical propertiesof the modified nucleotides do not a priori point tospecific functional roles, but it is clear that a range ofstructural changes can occur. Effects include alteredsteric properties in all cases, different hydrogen-bondingpotential, enhancement of local base stacking (Ψ) and
rRNA modifications
and ribosome
function
Wayne A. Decatur and Maurille J. Fournier
The development of three-dimensional maps of the modified nucleotides in the
ribosomes of Escherichia coli and yeast has revealed that most (~95% in E. coli
and 60% in yeast) occur in functionally important regions. These include the
peptidyl transferase centre, the A, P and E sites of tRNA- and mRNA binding,
the polypeptide exit tunnel, and sites of subunit–subunit interaction.
The correlations suggest that many ribosome functions benefit from
nucleotide modification.
O OH
OO
NHHN
O
O
N
N
R2
O
R1
N
N N
N
R1
R3
R4
Ti BS
N
O O
OO
CH3Nm Ψ
(a)
(c)
(b)
Fig. 1. Major types of rRNA modification. (a) 2′-O-methylation (Nm).(b) Isomerization of uridine to pseudouridine (Ψ), ‘the fifth nucleoside’.(c) Most other modifications involve base methylation at variouspositions (arrows). This includes Ψ – a few are acetylated or otherwisemodified at such positions [47].