Charging the code — tRNA modification complexes Ro scisław Krutyho łowa 1,2,4 , Karol Zakrzewski 1,3,4 and
Sebastian Glatt 1
AlltypesofcellularRNAsarepost-transcriptionallymodified, constitutingthesocalled‘epitranscriptome’.Inparticular, tRNAsandtheiranticodonstemloopsrepresentmajor modificationhotspots.Theattachmentofsmallchemical groupsattheheartoftheribosomaldecodingmachinerycan directlyaffecttranslationalrates,readingframemaintenance, co-translationalfoldingdynamicsandoverallproteome stability.ThevarietyoftRNAmodificationpatternsisdrivenby theactivityofspecializedtRNAmodifiersandlarge
modificationcomplexes.Notably,theabsenceordysfunction ofthesecellularmachinesiscorrelatedwithseveralhuman pathophysiologies.Inthisreview,weaimtohighlightthemost recentscientificprogressandsummarizecurrentlyavailable structuralinformationofthemostprominenteukaryotictRNA modifiers.
Addresses
1MaxPlanckResearchGroupattheMalopolskaCentreof Biotechnology,JagiellonianUniversity,Krakow,Poland
2DepartmentofCellBiochemistry,FacultyofBiochemistry,Biophysics andBiotechnology,JagiellonianUniversity,Poland
3PostgraduateSchoolofMolecularMedicine,Warsaw,Poland
Correspondingauthor:Glatt,Sebastian(sebastian.glatt@uj.edu.pl)
4Theseauthorscontributedequally.
CurrentOpinioninStructuralBiology2019,55:138–146 ThisreviewcomesfromathemedissueonMacromolecular assemblies
EditedbyIlyaAVakserandAndrzejJoachimiak ForacompleteoverviewseetheIssueandtheEditorial Availableonline16thMay2019
https://doi.org/10.1016/j.sbi.2019.03.014
0959-440X/ã2019TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creative- commons.org/licenses/by-nc-nd/4.0/).
Introduction
The ‘Modomics’ database for RNA modification [1] cur- rently lists 200 unique chemical modifications of RNA, with around half of them being detected in tRNAs of all known species [2–4]. Despite their strong sequence vari- ation, all cellular tRNAs need to fold into almost identical three-dimensional structures to fit the relatively narrow tRNA binding sites of the ribosome during translation elongation. The possibility of incorporating chemical groups, which contribute additional biophysical proper- ties to the individual RNA bases, vastly expands the range of suitable sequences that can fold into the characteristic L-shaped tRNA structure. In addition, the modification
of RNA bases in and around the anticodon impacts on their intrinsic geometry and canonical Watson–Crick base pair interactions between codons and anticodons [5–7].
These alterations strongly influence the dynamics of tRNA selection at the ribosomal A-site [8] and subse- quently affect the local elongation speed, co-translational folding dynamics [9], proteome stability and cell survival [10]. tRNA modifications were initially thought to be routinely and uniformly added to their respective tRNA molecules. To date, it is becoming increasingly clear that most of them are dynamically regulated in response to environmental cues [11,12] and an intense cross talk between various modifications and their pathways emerges [13]. Here, we aim to provide a comprehensive summary of the respective modification enzymes that produce this plethora of posttranscriptional modifications patterns. We summarize available structural and func- tional knowledge concerning the most abundant families of tRNA modification enzymes. Our focus lies on the main modification cascades and known macromolecular assemblies that target the ASL in eukaryotes (Figure 1).
These partially highly complex molecular machines are not only important guardians of the proteome and regu- latory factors of translational elongation, but are also clinically very important. The pathophysiological conse- quences and clinical implications of disease-causing mutations in tRNA modifiers are very well covered by recent expert reviews [14–17].
(t)RNA methyltransferases
Methylations affect multiple properties of tRNA mole- cules, including folding dynamics, thermostability, mat- uration as well as protection from cleavage or priming for the synthesis of subsequent modifications [18,19].
Eukaryotic tRNA methyltransferases (TRMs) typically utilize S-adenosyl methionine (SAM) as a methyl group donor which results in formation of a S-adenosyl-
L- homocysteine and a methylated product [20]. In the following section, we aim to highlight structurally char- acterized TRMs and describe their selectivity for certain tRNA species and specific base positions within the respective ASLs (Figure 2).
In detail, Trm1, which conducts a double methylation of the exocyclic nitrogen of G26, which promotes a proper folding of multiple tRNA species by enforcing a water- mediated interaction of m
22
G
26with a nearby cytosine [21,22]. In yeast, Trm140 binds tRNA
Serand tRNA
Thrand catalyzes m
3C
32in a i
6A
37-dependent manner [23].
In human, Trm140 functionally corresponds to the
Methyltransferase-like (Mettl) 2, 6 and 8 proteins [24,25].
Although Mettl2/6/8 are currently not structurally charac- terized, structures of the Mettl3/Mettl14 complex that provides N
6-adenosine methylation [26–28] and the methyltransferase domain of Mettl16 [29] provide new insights into the METTL protein family. The known structure of human Ftsj2, a homolog of yeast Trm7, reveals a typical class I TRM fold [18]. In yeast, 2
0-O ribose methylation provided by Trm7 is guided by its interactions with Trm732 and Trm734, which drive the reactions at positions 32 and 34, respectively [30]. Trm4 catalyzes modifications at positions m
5C
48and m
5C
49; however, it is also capable of generating m
5C
34and m
5C
40[31]. Archaeal Trm4 was co-crystalized in the presence of a naturally occurring inhibitor sinefungin [32]. In human, Trm4 has two functional counterparts, Nsun2 and Nsun3 catalyzing m
5modifications in the nucleus and mitochondria, respectively. In addition to its tRNA modification activity, Nsun2 was reported to methylate miRNAs [33]. Methylation of the wobble cytosine provided by Nsun3 is required for initiation of 5-formylcytidine (f
5C
34) synthesis on tRNA
Met[34]. The first known structure of Nsun family member was solved
for Nsun6, which catalyzes the m
5C
72modification [35
].
Trm5 is another multifunctional enzyme, capable of con- ducting m
1modification at G
37or I
37. Interestingly, Trm5 also plays a role in some archaeal species during wybutosine (yW
37) synthesis. Trm5 was co-crystalized with tRNA
Pheand a SAM cleavage product [36
]. Available structure elucidates both the Trm5-tRNA interaction and the moon- lighting activity of Trm5 in archaea [36
]. Another member of the class Dnmt2, historically considered a DNA-specific methyltransferase [37], provides a m
5C tRNA modification at C
38and C
40[38]. m
5C
38was demonstrated to prevent the generation of tRNA-derived fragments [39], which appear due to the tRNA cleavage under stress conditions and may act as regulatory RNAs [40]. Although available structures do not provide an explicit explanation for tRNA recogni- tion, the enzymatic activity of a fungal Dnmt2 was recently found to be stimulated by the presence of queuosine [41
].
Pseudouridine synthases
Pseudouridylation is one of the most widely spread mod- ification in all types of RNAs, including tRNAs, snRNAs, rRNA, ncRNAs, and mRNAs, and occurs in each domain of life [42]. This altered form of a uridine base arises
Figure1
Methylation
Um
U/Cm Modifications at 34
5-carboxymethyluridine
Wybutosine
Adenine deamination Pseudouridilation
Thiolation
Ψ Ψ
Ψ Ψ
Ψ Ψ Ψ Ψ
Ψ
Ψ 44
28
27 40
26
39 38 37
36 35 34 33
32 31 30
m22G26 - Trm1m3C32 - Trm140 U/Cm32 - Trm7 (Ftsj2) Xm34 - Trm7 (Ftsj2) m5C34 - Trm4, Nsun2,3 mcm5U34 - Trm9 mchm5U34 - Abh8 m1I37 - Trm5 m1C37 - Trm5 m5C38 - Dnmt2
Um44 - Trm44 m5C40 - Dnmt2 m1
Ψ
39 - Nep1Q34 - Tgt ac4C34 - Nat10
f5C34 - Abh1 nm5U34 - Gtpbp3?
(m/n)cm5U34 - Elp123456
s2U34 - Mtu1, Ncs2/6 s2U37 - Cdkal1
yW37 - Tyw1,2,3,4
t6A37 - Sua5 i6A37 - Trit1
I34 - Adat2, Adat3 I37 - Adar2,(Adarb1) t6/i6 modifications
Ψ
27 - Pus1, Pus2Ψ
28 - Pus1Ψ
30 - Pus1Ψ
31 - Pus6Ψ
32 - Pus8, Pus9Ψ
34 - Pus1Ψ
35 - Pus1, Pus7Ψ
36 - Pus1Ψ
38 - Pus3Ψ
39 - Pus3τm5U34 - ?
f5C τm5U ac4C
nm5U s2U m5C Xm I
Q mchm5U m3C
m5C m5C
m2G; m22G
m1
Ψ
I
m1G; m1IyW
s2U t6A i6A3’
5’
Current Opinion in Structural Biology
tRNAmodificationsoccurringwithinthetRNAanticodonregion.
OverviewofthetRNAanticodonloopincartoonrepresentation.Individualmodificationsaregrouped,highlighted,andlabeled.Respectivelyfrom theleftmethylation(indigo),modificationofposition34(violet),5-carboxymethyluridin(lightgreen),pseudouridylation(rose),thiolation(yellow), wybutosine(yW;blue)N6-isopentenyladenosine(i6A)andN6-threonylcarbamoyladenosine(t6A)(red),adeninedeamination(teal).Modifications occurringwithinanticodonregionareplottedandhighlightedonamodeltRNAGlu(PDBID2CV2).
through the substitution of the canonical carbon-nitrogen glycosidic bond (C1–N1) with a carbon-carbon bond (C1–
C5) between the ribose and uracil. This simple isomeri- zation reaction is conducted by stand-alone enzymes called pseudouridine synthases (PUS). Interestingly, pro- karyotic and eukaryotic members of the five PUS fami- lies, namely TruA, TruB, TruD, RsuA, and RluA, display relatively low sequence identity, but show very similar folds of the catalytic domain [43,44
]. The conserved core of PUS enzymes is created by a central eight- stranded b-sheet and several surrounding helices, which properly position the catalytically active aspartate residue in proximity to the uridine base of the respective target
RNA. In the ASL of various eukaryotic tRNAs, several positions, namely U
27, U
28, U
30, U
31, U
32, U
34, U
35, U
36, U
38, and U
39, are specifically converted into pseudour- idine. PUS enzymes are found in the nucleus, the cyto- plasm, and mitochondria and for tRNA targets can be divided into two major groups, namely modifiers of cyto- plasmic or mitochondrial tRNAs.
In yeast, Pus1 is the main player for cytoplasmic tRNA ASL modifications, which displays broad specificity for target uridines (U
26, U
27, U
28, U
34,U
35,U
36) [45]. Pus7 shows specific activity at U
35and Pus8 appears as a highly specific enzyme for U
32bases. Additionally, Pus3 and
Figure2
Mettl3 • Mettl14 Trm1 (Trmt1) Dnmt2 (M.HsaIIP)
Trm5 (Trmt5) CTD
RRM RRM CTM
Mettl3 Mettl14 Ftsj2 (Trm7)
Trm4 (Ncl1, Nsun2) Trm9 • Trm112 Abh8
RRM
RRM RRM
Trm9 Trm112
AlkB-like Trm9-like domain
m22G26m5C40
m5C38
m5C37
m3C32
Nm32
m5C34 mcm5U34 mchm5U34
Mettl2,6,8
Current Opinion in Structural Biology
StructuraloverviewofmethyltransferasesactingontheASLoftRNA.
tRNAmethyltransferasesshareastructurallysimilarTRMdomain(darkblue)andanRNArecognitionmotif(RRM,orange).Ligandsandrelevant activesiteresiduesarehighlightedinyellow.Names,alternativenamesornamesofclosehomologsareshowninparentheses.Trm1from Pyrococcushorikoshiico-crystalizedwithaSAMmolecule(PDBID2EJT).HumanMettl3-Mettl14complexco-crystalizedwithSAM(PDBID5IL1).
HumanFtsj2,aTrm7homolog,co-crystalizedwithSAM(PDBID2NYU).Trm4fromMethanocaldococcusjannaschiicrystalizedwithaninhibitor (PDBID3A4T).Trm9–Trm112complexfromYarrowialipolytica.Trm9isshowninblue,Trm112(grey)activatesthecatalyticsubunit(PDBID 5CM2).N-terminalpartofahumanAbh8,whichconsistsanAlkB-likedomain(green)andaTrm9-likedomainmissingfromthestructure(PDBID 3THP).Trm5fromPyrococcusabyssico-crystalizedwithaSAMdegradationproductandatRNAPhe(PDBID5WT3).HumanDnmt2co-crystalized withSAH(PDBID1G55).
Pus6 modify U
31and U
38/39in both cytoplasmic and mitochondrial tRNAs. The last two pseudouridine synthases involved in anticodon modifications are Pus2 and Pus9, which are responsible for editing uridine in mitochondrial tRNAs at U
26/27and U
32bases [42]. Strik- ingly, Pus enzymes modify the anticodon bases in cyto- plasmic tRNAs, whereas they don’t edit this important decoding region in mitochondrial tRNAs. In humans, TRUB2, RPUSD3, RPUSD4, and Pus1 have been pre- dicted to target mitochondrial tRNAs, but only Pus1 has been experimentally confirmed to modify mitochondrial tRNAs, at positions 27 and 28 [46]. The other three human mitochondrial pseudouridine synthases are less well characterized [47], but have been proposed to modify positions 31 and 32 [48] or 39 in mt-tRNA
Phe[49]. Stand- alone pseudouridine synthases have broad range of activ- ity, lack of which leads to reduced growth in yeast cells and major human diseases such as mitochondrial myopathy, sideroblastic anemia (MLASA), or intellectual disabilities [50,51].
The KEOPS complex
The bases in position 37 are highly modified in eukaryotic tRNAs. Besides previously described methylation, the formation of N
6-isopentenyladenosine (i
6A), N
6-
threonylcarbamoyladenosine (t
6A), wybutosine (yW) as well as thiolation can be distinguished. The tRNA isopen- tenyl transferases (IPTases) are responsible for i
6A, whereas yW is catalyzed by the concerted action of Trm5 [36
] and Tyw1-4, which are able to conduct SAM-dependent methyl-transfer reactions [52,53]. t
6A is universal and present in nearly all tRNAs, which decode
‘ANN’ codons. In all kingdoms of life, the first step of the biosynthesis of t
6A leads to the production of threonyl- carbamoyl adenylate (TC-AMP), which subsequently is used to transfer a threonylcarbamoyl group to adenosine.
The enzymes Sua5 and YRDC are responsible for the formation of TC-AMP in yeast and humans. These homo- logs belong to the Sua5/TsaC family, function as indepen- dent monomers and are localized both in the cytoplasm and mitochondria [54,55
,56
]. In eukaryotes, the product of the first reaction step is processed by the cytoplasmic KEOPS complex, containing five subunits called OSGEP/Kae1 (catalytic core), PRPK/Bud32, TPRKB/
Cgi121, LAGE3/Pcc1, and C14ORF142/Gon7 [54,57].
Despite some structural knowledge of the Sua5/TsaC family and the available structures of eukaryotic KEOPS complex components from yeast, the precise overall assem- bly of the complex, its mechanism of action as well as the role of particular subunits in the modification process
Figure3
Ψ
Ψ Ψ Ψ
Ψ
Ψ 30
28 27
37 36 35 34 Adar2 (Adarb1)
yW I
RRM DID DMATase
Sua5 Pus7
Adar2, dsRBD1/2
RRM
Cgi121
Bud32
I i6A t6A
Pus1 Tyw4 Mod5 (Trit1)
KELCH TRM
Kae1
KEOPS SUA5 TSAC
Current Opinion in Structural Biology
StructuraloverviewforothertRNAmodifiers.
VisualrepresentationofproteinstructuresincludingcolorillustrationfortRNAbindingdomains(orange),enzymaticcore(colorcorrespondingto respectivetypeofmodification).Names,alternativenamesornamesofclosehomologsareshowninparentheses.PseudouridinesynthasesPus1 (PDBID4IQM)andPus7(PDBID5KKP;bothrose),wybutosinebiosynthesisfactorTyw4(PDBID2ZW9;blue),yeastisopentenyltransferases Mod5(PDBID3EPJ;red),archaealSua5(PDBID6F89)andCgi121/Bud32/Kae1KEOPScomponents(PDBID3ENH;red),andthestructureof Adeninedeaminases(ADARs)(PDBID2L3J;teal).
remain poorly understood. The hetero-dimerization of certain KEOPS subunits and the presence of a metal cluster in the active site of catalytic core formed by OSGEP/Kae1 proteins were described [55
,56
]. The functional roles of other cascade components are still unknown, although dimerization of mitochondrial Qri7 seems to be important for its functionality [56
,58]. Despite the fact that detailed
aspects of t
6A biosynthesis remain elusive, recent studies showed that bicarbonate represents a rate-limiting factor for t
6A
37formation (Figure 3). Highlighting its importance, mutations in t
6A synthetase genes are correlated with neurodegenerative diseases, renal tubulopathy and Galloway-Mowat syndrome in humans (GAMOS) [59
,60,61].
Figure4
structure unknown structure known structure unknown structure known
PDB: 107 PDB: 23
apo (6) tRNA (6) ligand (11)
structure unknown structure known structure unknown structure known
apo (33) ligand (63) tRNA (11)
45 20 32 23
20 22 29 8
87 189 39 184
Elp3
Elp3 Elp1
Elp1
Elp2
Elp2
Elp1 Elp3
Elp2
Elp1
Elp3
Elp456
Sit4 Sap185 Sap190 Kti12
Cbr1 Mcr1
structure unknown structure known structure known
structure unknown
apo (18)ligand (27)tRNA (9) apo (13) ligand (30) tRNA (4)
PDB: 54 PDB: 47
PDB: 166 apo (55)
ligand (7)
RNA (104)
Elp456
Hrr25 90°
PDB: 489
apo (94) ligand (364) RNA (31) Ribosome
GO: 0005840
Total hits: 50397 eukarya archeae bacteria
6998 2816 40583 eukarya
archeae bacteria
461 883 11566 Total hits: 12910 Total hits: 9336 eukarya archeae bacteria
1034 385 7917
tRNA modifiers
GO: 6400
tRNA ligases
GO: 4812
Saccharomyces cerevisiae Homo sapiens
(a)(b)
Current Opinion in Structural Biology
Elp2
Kti11
•Kti13
Trm9
•Trm112
TheElongatorcomplexandstructuralknowledgeoftRNAmodifiersingeneral.
(a)AnintegrativemodeloftherecentlycharacterizedElongatorcomplexanditssubunitsisshownfromthefrontandthetop.Thecomplex harborssixpairsofproteins,namelyElp1(orange),Elp2(yellow),Elp3(catalyticsubunit,pink)andElp456(green,blueandbrown).Known accessoryproteinsordownstreamfactorsareshownbelow.Trm9–Trm112methyltransferase(PDBID5CM2)inblueandgrey,Kti11–Kti13(PDB ID4XHL)ingreenandbrownandHrr25/Kti14(PDBID5CYZ)inchocolate.Additionalaccessoryproteinsofunknownstructurearelisted(right).
(b)AcomparisonofavailabledatabaserecordsfortRNAmodifiers,tRNAligasesandribosomes.ASWISSPROTdatabasewasqueriedusingGO terms(tRNAmodifiers–GO:6400excludingGO:4812,tRNAligases–GO:4812,ribosome–GO:0005840).Distributionofrecordsacrossthree domainsoflifeisshownontheleftpanel.Species-specificsearchesforSaccharomycescerevisiaeandHomosapiensallowedtoevaluatethe numberofannotatedproteinsandthenumberofproteinswithavailablestructuralinformation(rightpanel,upperbars).Theiraccessionnumbers wereusedforasubsequentsearchinthePDBdatabasetouncoverthetotalnumberofknownstructures(rightpanel,lowerbars).Everystructure wascategorizedintoligand-freestructures(lightestshade),ligand-boundstructures(mediumshade)and(t)RNAboundstructures(darkestshade).
The Elongator complex
One of the chemically most complex modifications is catalyzed by a similarly complicated relay system. In eukaryotes, the highly conserved Elongator complex [62] carboxymethylates (cm
5) 11 out of 13 yeast tRNAs carrying a U
34in the wobble position [63]. The core Elongator complex harbors two copies of each of its six subunits, Elp1-6 and displays an overall molecular weight of more than 850 kDa [64]. Furthermore, the asymmetri- cally shaped complex [62] is regulated by various tempo- rarily associated regulatory factors, including Kti11, Kti12, Kti13, Kti14/Hrr4, Sit4, Sap185, Sap190, Cbr1, and Mcr1 [65]. In recent years, the crystal structures of the dimeric Elp1 C-terminus [66], Elp2 [67
,68], the enzymatically active Elp3 subunit [69], the hetero-hexameric Elp456 subcomplex [64], and the temporarily associated regula- tory factors Kti11, Kti13, and Kti14/Hrr25 [70,71] became available (Figure 4a). In addition, the overall architecture of the fully assembled Elongator complex has been recently determined, using negative stain electron microscopy in combination with an integrative modeling approach based on the known high resolution crystal structures and additional spatial restraints from cross linking mass spectrometry [67
,72].
The pivotal addition of cm
5to U
34can subsequently leads to the synthesis of 5-methoxycarbonylmethyluridine (mcm
5U), 5-carbamoylmethyluridine (ncm
5U), 5-meth- oxy-carbonyl-methyl-2-thiouridine (mcm
5s
2U), 5-methox- ycarbonylmethyl-2
0-O-methyluridine (mcm5Um), or 5-(carboxyhydroxymethyl)uridine methyl ester (mchm
5U) by other enzymatic cascades. These small additional groups on the ‘Elongator’ modification at the fifth carbon (C5) are attached by Trm9 [73] and Abh8 [74], both of which require an obligatory activator Trm112 (Figure 1) [74,75
, 76]. The identity of the protein that synthesizes ncm
5, either directly or by conversion of mcm
5, remains a mystery.
Last but not least, the additional thiolation on the second carbon (C2) is catalyzed by the Urm1/Uba4 pathway [77], which in eukaryotes includes Nfs1, Tum1, Urm1, Uba4, Ncs2, and Ncs6 [78]. In summary, around 25 individual proteins are involved in wobble uridine modifications, which are highly conserved among eukaryotes and also have related counterparts in bacteria and archaea. It remains to be shown if some of these factors form dynamic inter- mediates with the Elongator complex and how they influ- ence each other.
Conclusion
In summary, tRNA modifiers represent a highly dynamic network of a large number of macromolecular complexes and individual proteins. Because of space restrictions, we are not able to cover structural details regarding the enzymes that conduct adenine deamination [79], queuo- sine biosynthesis [80], wybutosine [53], thiolation [78], and other more unique modifications, like 5-taurino- methyluridine [81] or N4-acetylcytidine [82].
Nevertheless, we aimed to numerically evaluate the overall state of the field and compared the number of known tRNA modifiers in publicly available databases [83] to other well-established tRNA-dependent mecha- nisms, like ribosomes and tRNA aminoacyl transferases, also known as tRNA ligases (Figure 4b). We performed species-specific queries to more precisely identify the number of known structures of tRNA modifiers, tRNA ligases and ribosomes. While the structures of most ribosomal proteins and more than a half of all human tRNA ligases have been determined, tRNA modifiers seem to still offer many possibilities for exploratory research projects. In particular, RNA-bound or tRNA- bound structures amount to a relatively small fraction of available records in the structural databases [84,85].
Undoubtedly, structural characterization of modifiers in their tRNA-bound state most significantly improves our understanding of their specificity and catalytic mecha- nisms. We assume, that the ascent of cryo-EM will have a similar impact on the tRNA modification field as it had for the other fields [86].
Interestingly, expansion of tRNA gene copy numbers and iso-acceptor tRNAs correlate well with an increase in different tRNA modification enzymes [87]. This obser- vation suggests co-evolutionary mechanisms, which expand the decoding potential of the available tRNA pool and constantly optimize translational efficiency and accuracy via novel tRNA modification pathways [87]. Last but not least, the assembly of large complexes seems advantageous for some modifications, whereas other pathways seem to rely on stand-alone enzymes.
It remains to be shown, if additional molecular assemblies that spatially link different modification pathways will be discovered and characterized.
Conflict of interest statement Nothing declared.
Acknowledgements
WethankMonikaGaik,TingYu-Lin,andMikołajSokołowskiforcritical commentsonthemanuscript.ThisworkwassupportedbytheOPUS10 grant(UMO-2015/19/B/NZ1/00343;RK,KZandSG)fromtheNational ScienceCentreandtheFirstTeamgrant(FirstTEAM/2016-1/2;SG)from theFoundationforPolishScience.
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