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CONTRIBUTORS
PaulF.Agris
TheRNAInstitute,StateUniversityofNewYork,Albany,NY,UnitedStates
JuanD.Alfonzo
DepartmentofMicrobiology,OhioStateBiochemistryProgram,TheCenterforRNA
Biology,TheOhioStateUniversity,Columbus,OH,UnitedStates
PeterA.Beal
UniversityofCalifornia,Davis,CA,UnitedStates
GuillaumeF.Chanfreau
UniversityofCalifornia,LosAngeles,CA,UnitedStates
MeemanageD.DeZoysa
UniversityofRochesterMedicalCenter,CenterforRNABiology,Rochester,NY,
UnitedStates
EmilyEruysal
TheRNAInstitute,StateUniversityofNewYork,Albany,NY,UnitedStates
BrianD.Gregory
UniversityofPennsylvania;CellandMolecularBiologyGraduateProgram;Genomicsand
ComputationalBiologyGraduateProgram,UniversityofPennsylvania,Philadelphia,PA,
UnitedStates
AnthonyK.Henras
LaboratoiredeBiologieMol(cid:1)eculaireEucaryote,CentredeBiologieInt(cid:1)egrative,Universit(cid:1)e
deToulouse,CNRS,UPS,Toulouse,France
YvesHenry
LaboratoiredeBiologieMol(cid:1)eculaireEucaryote,CentredeBiologieInt(cid:1)egrative,Universit(cid:1)e
deToulouse,CNRS,UPS,Toulouse,France
Ya-MingHou
ThomasJeffersonUniversity,Philadelphia,PA,UnitedStates
OdileHumbert
LaboratoiredeBiologieMol(cid:1)eculaireEucaryote,CentredeBiologieInt(cid:1)egrative,Universit(cid:1)e
deToulouse,CNRS,UPS,Toulouse,France
OlgaKolaj-Robin
InstitutdeG(cid:1)en(cid:1)etiqueetdeBiologieMol(cid:1)eculaireetCellulaire(IGBMC);CentreNationalde
RechercheScientifique(CNRS)UMR7104;InstitutNationaldeSant(cid:1)eetdeRecherche
M(cid:1)edicale(INSERM)U964;Universit(cid:1)edeStrasbourg,Illkirch,France
IsaoMasuda
ThomasJeffersonUniversity,Philadelphia,PA,UnitedStates
ix
x Contributors
RyumaMatsubara
ThomasJeffersonUniversity,Philadelphia,PA,UnitedStates
KatherineM.McKenney
DepartmentofMicrobiology,OhioStateBiochemistryProgram,TheCenterforRNA
Biology,TheOhioStateUniversity,Columbus,OH,UnitedStates
AmithiNarendran
TheRNAInstitute,StateUniversityofNewYork,Albany,NY,UnitedStates
C(cid:1)eliaPlisson-Chastang
LaboratoiredeBiologieMol(cid:1)eculaireEucaryote,CentredeBiologieInt(cid:1)egrative,Universit(cid:1)e
deToulouse,CNRS,UPS,Toulouse,France
YvesRomeo
LaboratoiredeBiologieMol(cid:1)eculaireEucaryote,CentredeBiologieInt(cid:1)egrative,Universit(cid:1)e
deToulouse,CNRS,UPS,Toulouse,France
MaryAnneT.Rubio
DepartmentofMicrobiology,OhioStateBiochemistryProgram,TheCenterforRNA
Biology,TheOhioStateUniversity,Columbus,OH,UnitedStates
KathrynSarachan
TheRNAInstitute,StateUniversityofNewYork,Albany,NY,UnitedStates
BertrandS(cid:1)eraphin
InstitutdeG(cid:1)en(cid:1)etiqueetdeBiologieMol(cid:1)eculaireetCellulaire(IGBMC);CentreNationalde
RechercheScientifique(CNRS)UMR7104;InstitutNationaldeSant(cid:1)eetdeRecherche
M(cid:1)edicale(INSERM)U964;Universit(cid:1)edeStrasbourg,Illkirch,France
JoannaI.Sulkowska
CenterofNewTechnologies,UniversityofWarsaw,Warsaw,Poland
RyuichiTakase
ThomasJeffersonUniversity,Philadelphia,PA,UnitedStates
LeeE.Vandivier
UniversityofPennsylvania;CellandMolecularBiologyGraduateProgram,Universityof
Pennsylvania,Philadelphia,PA,UnitedStates
VilleY.P.V€are
TheRNAInstitute,StateUniversityofNewYork,Albany,NY,UnitedStates
YuruWang
UniversityofCalifornia,Davis,CA,UnitedStates
Yi-TaoYu
UniversityofRochesterMedicalCenter,CenterforRNABiology,Rochester,NY,
UnitedStates
YuxuanZheng
UniversityofCalifornia,Davis,CA,UnitedStates
PREFACE
The field of RNA modifications has been experiencing a renaissance in
recent years. Decades ago, some of the earliest studies focusing on RNA
modificationsrevealedtheirroleininfluencingthedecodingofgeneticmes-
sengerRNAsbytransferRNAs(tRNAs).WhileRNAmodificationswere
initiallyidentifiedinthesetRNAsandinribosomalRNAs(rRNAs)because
oftheirhighabundance,theadventofmodernsequencingtechnologieshas
enabled researchers to investigate the global extent of RNA modifications
on the transcriptome. These recent studies have revealed a large variety
of modifications and of modification sites in all major classes of cellular
RNAs. The enzymes that catalyze the addition of chemical modifications
toRNAareasdiverseasthechemicalgroupsthatareaddedtoRNAs.These
enzymes range from single protein chains to large ribonucleoprotein
complexes,inwhichthepresenceofanRNAguidetargetsthemodification
enzymetoitsRNAsubstrates.ThespecificityofRNA-modifyingenzymes
is essential, as misguided RNA modifications can negatively impact the
process of genetic decoding during translation. Moreover, the presence of
RNA modifications has a major influence on the metabolic fate of these
modified RNAs, as these modifications can promote their correct
folding, nucleocytoplasmic trafficking, translation, or turnover. Finally,
RNA modifications can provide recognition sites for proteins that interact
with modified RNAs. This volume of The Enzymes highlights some of
the most recent advances in the field of RNA modifications and attempts
toprovideacomprehensivereviewofthestructure,function,andspecificity
of RNA-modifying enzymes as well as a survey of the role of these modi-
fications on RNA metabolism. Throughout these chapters, it will become
apparent that these modifications providekey functional groups,as disrup-
tion of RNA modification reactions due to mutations in RNA-modifying
enzymes results in a variety of inherited diseases, highlighting the impor-
tance of these modifications in cellular metabolism.
I thank all the authors who generously contributed to this volume of
TheEnzymes.IwouldalsoliketoexpressmygratitudetoNaomiRobertson
at Elsevier for handling and copyediting this volume.
GUILLAUME F. CHANFREAU
xi
CHAPTER ONE
The Importance of Being Modified:
The Role of RNA Modifications in
Translational Fidelity
Paul F. Agris1, Amithi Narendran, Kathryn Sarachan, Ville Y.P. V€are,
Emily Eruysal
TheRNAInstitute,StateUniversityofNewYork,Albany,NY,UnitedStates
1Correspondingauthor:e-mailaddress:[email protected]
Contents
1. Introduction 2
1.1 UniversalGeneticCode 2
1.2 PosttranscriptionalRNAModification 4
1.3 RNAModificationandtheAccuracyandEfficiencyofTranslation 6
2. HumanMitochondrialtRNAMetDecodesthe1:3DegenerateCodonBox 9
2.1 HumanMitochondrialandCytoplasmictRNAMet 9
2.2 PosttranscriptionalModificationofMitochondrialtRNAMet 10
2.3 OtherHumanMitochondrialtRNAMetProperties 14
2.4 HumanMitochondrialtRNADisease 15
2.5 Summary 16
3. tRNADecodingoftheTwofold,2:2,DegenerateCodonBox 17
3.1 tRNALysTwofoldDegenerateCodonsAAAandAAG 17
3.2 WobblePositionUridine-34Modifications 18
3.3 ModificationsofAdenosine-37,30-AdjacenttotheAnticodon 22
3.4 Summary 24
4. tRNADecodingofCodonFourfoldDegeneracy 25
4.1 SingletRNAReadsAllFourCodonsoftheProkaryoteFourfoldDegenerate
CodonBox 25
4.2 EukaryotetRNAsThatWobbleinReadingFourfoldDegenerateCodons 27
4.3 RolesofBothWobblePositions-34and-37ModificationsinReading
FourfoldDegenerateCodons 28
4.4 Summary 30
5. ThreeAminoAcidsAreEncodedbySixSynonymousCodonsEach 30
5.1 SixfoldDegeneracyandReadingoftheArginineCodons 30
5.2 Modification-DependenttRNAReadingoftheArginineCodons 32
5.3 TheModification2-ThiocytidineModulatesInosineReadingofA,U,andC 33
5.4 tRNAArgSpeciesDecodingaTwofoldDegenerateCodonBox 35
5.5 Summary 37
TheEnzymes,Volume41 #2017ElsevierInc. 1
ISSN1874-6047 Allrightsreserved.
http://dx.doi.org/10.1016/bs.enz.2017.03.005
2 PaulF.Agrisetal.
6. PosttranscriptionalModificationoftRNAIsRequiredforAccurateandEfficient
Decoding 38
Acknowledgment 39
References 40
Abstract
TheposttranscriptionalmodificationsoftRNA’santicodonstemandloop(ASL)domain
representathirdlevel,athirdcode,totheaccuracyandefficiencyoftranslatingmRNA
codonsintothecorrectaminoacidsequenceofproteins.ModificationsoftRNA’sASL
domain are enzymatically synthesized and site specifically located at the anticodon
wobble position-34 and 30-adjacent to the anticodon at position-37. Degeneracy of
the64UniversalGeneticCodesandthelimitationinthenumberoftRNAspeciesrequire
sometRNAstodecodemorethanonecodon.Thespecificmodificationchemistriesand
theirimpactonthetRNA’sASLstructureanddynamicsenableonetRNAtodecodecog-
nate and “wobble codons” or to expand recognition to synonymous codons, all the
whilemaintainingthetranslationalreadingframe.Somemodifiednucleosides’chem-
istriesprestructuretRNAtoreadthetwocodonsofaspecificaminoacidthatsharesa
twofolddegeneratecodonbox,andotherchemistriesallowadifferenttRNAtorespond
toallfourcodonsofafourfolddegeneratecodonbox.Thus,tRNAASLmodificationsare
critical and mutations in genes for the modification enzymes and tRNA, the conse-
quencesofwhichisalackofmodification,leadtomistranslationandhumandisease.
By optimizing tRNA anticodon chemistries, structure, and dynamics in all organisms,
modificationsensuretranslationalfidelityofmRNAtranscripts.
1. INTRODUCTION
1.1 Universal Genetic Code
The Universal Genetic Code of 64 triplet codons encoded in DNA and
transcribed into each messenger RNA (mRNA) is degenerate (Fig. 1). As
a general rule, 61 codons represent the 20 amino acids during translation
on the ribosome. Three codons are read by protein factors as translation
stops. TransferRNAs(tRNAs)decodethe mRNA codons whilebringing
thecognateaminoacidtotheribosomeforincorporationintothegrowing
peptidechain.Theribosomeisoneofthemostcomplexofenzymes.With
thebacterialandeukaryotedecodingoraminoacylsite(A-site)andthepep-
tidyl transferase center (PTC) consisting almost completely of RNA [1,2],
the ribosome is an RNA-based enzyme with large RNA substrates. Even
themitochondrialribosome,whichismorehighlyproportionedofproteins,
has an RNA-rich decoding and PTC [3]. In humans, the ribosome’s sub-
strates include some 25,000 mRNAs for proteins of less than 100 amino
ImportanceofRNABeingModified 3
Second
U C A G
xi6A37 U UUUUCU PPhhee Gm34 UUCCCU SSeerr 5xmU34 UUAAUC TTyyrr UUGGUC CCyyss CU
m1G37 UUA Leu U34 UCA Ser U; 34 UAA Stop UGA Stop A
UUG Leu 5m UCG Ser 5o UAG Stop UGG Trp G
x x
CUU Leu CCU Pro U34 CAU His CGU Arg U
mm12GA3377 C CCUUCA LLeeuu CCCCCA PPrroo 5U; xm34 CCAACA HGilsn 5xmU34CCGGCA AArrgg 2sC32 CA ble)
rst CUG Leu CCG Pro 5xo CAG Gln CGG Arg G wob
Fi mt66AA3377 A AAAAUUUUGUCA lllMMllleeeeett 5fC34 AAAACCCCGCUA TTTThhhhrrrr xo5U; xm5U3434 AAAAAAAAGUCA AALLyyssssnn 52xmsU34 AAAAGGGGGUCA SSAAeerrggrr 5xmU34 UCAG Third (
m6A37 GGUUCU VVaall 5mU34 GGCCUC AAllaa 5mU34GGAAUC AAsspp U34GGGGUC GGllyy UC
mm12GA3377 GGGUUGA VVaall 5oU; x34GGCCGA AAllaa 5oU; x34GGAAGA GGlluu 52xms GGGGGA GGllyy AG
x x
Fig.1 TheUniversalGeneticCode.Twofolddegeneratecodonsarehighlightedintan;
threefold(Ile)ingray,fourfoldinyellow,andsixfoldinblue.ThesinglecodonsofMetand
Trparehighlighted ingreen and orange,respectively, andthethreestop codons are
highlightedinred.Thefigureisannotatedwiththeabbreviationsforthosemodified
nucleosides found inthe anticodon domain of tRNAs responding to the codons and
discussedinthisreview.Thechemicalstructuresandfullnamesofthemodifications
arefoundinFig.3.
acidsinlengthtomorethan20,000aminoacids[4];GTP;anumberofpro-
tein factors; and some 40 transfer RNAs (tRNA) each aminoacylated with
one specific amino acid. The ribosome’s enzymatic activity in translating
mRNAcodonsintotheaminoacidsequencesisa“processive”mechanism
ofaction.Theribosomesequentiallyandcovalentlylinksthegrowingpep-
tidechaintotheaminoterminusoftheincomingaminoacidthatisboundto
itsspecifictRNAthroughanesterbond.tRNAsareaminoacylatedattheir
0
universal 3-terminal adenosine by amino acid-specific, aminoacyl-tRNA
synthetases.TheaminoacylatedtRNAisbroughttotheribosomebyapro-
tein,elongationfactor,thatrecognizesthatthetRNAisaminoacylated,but
is not engaged in codon recognition and specificity.
CodonrecognitionbyaminoacylatedtRNA,acceptanceandaccommo-
dationofthetRNAbytheribosome,peptidebondformation,translocation
4 PaulF.Agrisetal.
of the peptidyl-tRNA from the decoding or aminoacyl site (A-site) to the
peptidyl-site (P-site), and the movement of unacylated tRNA from the
P-sitetotheexit(E-site)areprocessive.WithoutleavingthemRNAtem-
plate, the ribosome progresses from codon to codon from the mRNA’s
translational start sequence and first codon AUG for methionine to the
0
3- termination codons UAG, UAA, and UGA, and release sequences.
The three nucleosides of each aminoacylated tRNA’s anticodon recognize
athree-nucleotidemRNAcodonandbindtothecodoninasequence-and
frame-specific manner to deliver the 20 common amino acids for protein
synthesis. The aminoacylated tRNA is accepted at the A-site in response
tothecomplementarymRNAcodon,andaproofreadingmechanismcon-
sisting of some nine hydrogen bonds formed between the ribosomalRNA
(rRNA), tRNA, and codon ensures a high fidelity in translation. Accurate
interactionoftheanticodonwithcodonisverymuchdependentontheini-
tial Watson–Crick(A●U;G●C)complementarityof thehydrogenbond-
ingofthefirsttwobasepairs.Otherenzymeshavingprocessivemechanisms
ofactionincludetheDNA-andRNApolymerases.Whileproteinsynthesis
is accurate to 1 in 10,000 or 20,000 amino acids at a rate of 10–20 peptide
bondsformedpersecond[5,6],DNA-andRNApolymeraseshaveerrorsof
lessthan1in109at50–100sofbasepairspersecondincludingproofreading
steps[7,8]and1in105atarateof8–85basespersecond[9,10],respectively,
in vivo.
tRNAhastobeconsideredoneofthemostuniqueofenzymesubstrates.
The ribosome structure and its processive catalytic mechanism [2] require
thetRNAsubstratestohaveauniformityinchemistryandstructure.Nev-
ertheless, some 40 tRNA molecules, 22 within the human mitochondria,
possess enough unique chemical diversity and structural malleability to be
recognized by 20 aminoacyl-tRNA synthetases and to decode 61 mRNA
codonsontheribosome.HowdotRNAsachieveuniformityofchemistry
and structure and yet a necessary distinctiveness for protein synthesis?
Although seemingly contradictory, tRNAs use posttranscriptional modifi-
cation chemistriesto achieve both astablestructure mandatedby the ribo-
someandtheoriginalityrequiredforproteinrecognitiondeterminantsand
codon reading.
1.2 Posttranscriptional RNA Modification
AllRNAsareinvolvedinactivitiesthatcontributedirectlyor indirectlyto
theregulation,accuracy,andefficiencyofgeneexpressionthatisfulfilledin
translatingthemRNAcodonsintotheaminoacidsequencesofproteins.As
ImportanceofRNABeingModified 5
in translating one language into another, the accuracy of that translation is
dependentnotonlyonaliteralword-to-wordequivalencybutalsooncon-
text,frame,andpunctuation.Theenzymaticallysynthesized,sitespecifically
positioned,posttranscriptionalmodificationsofRNA(Fig.2)couldbecon-
sidered the punctuation marks in the translation of the genetic code. It is
difficult to find among RNA species (mRNA; tRNA; ribosomal, rRNA;
smallnuclear,snRNA;micro,miRNA;longnoncoding,lncRNA)onethat
is not posttranscriptionally modified [11].
TheUniversalGeneticCodeforthe20aminoacids,thefirstcode,and
the operational RNA code for the recognition of tRNA by its cognate
aminoacyl-tRNA synthetase, the second code [12], did not take into
accounttheposttranscriptionalmodificationsofRNA,forlittlewasknown
about them at the time, except that they existed. The ubiquitously occur-
ring,conservedmodifiednucleosidesconstituteayettobefullyappreciated
third code in regulating translation, its accuracy, and efficiency [13]. Even
Aminoacyl
stem
D stem
and loop T stem
and loop
Extra loop
Anticodon
stem and loop 29 41
ψ
30 40
ψ
31 39
s2C
ψ
32 38
33 37
t6A
34 m6A
36 i6A
35 ms2i6A
f5C m2A
τm5s2U m1G
mnm5U
mnm5s2U
mcm5s2U
ncm5U
mcm5U
cmo5U
Fig. 2 tRNA and its anticodon stem and loop (ASL) domain. Left: General cloverleaf
structure of a 76-nucleotide tRNA with the aminoacyl-accepting stem in green,
dihydrouridine (D) stem and loop in black, ASL in red, extra loop in blue, and
ribothymidine(T)stemandloopinplum.Right:AnenlargementoftheASLdomain,with
theabbreviationsoftheimportantmodificationsdiscussedinthisreviewlisted.Adarker
colorhueinplumrepresentsmoreimportantmodificationsites.
6 PaulF.Agrisetal.
withmorethanhalfacenturyofresearch,morethan100differentposttran-
scriptionalmodificationsofRNAsremainmostlyanundeterminedregulat-
ing factor to translation [14,15]. The posttranscriptional modification of
RNA, designatedastheepitranscriptome[16],nowusuallyrefersexclusively
to mRNAs. mRNAs have a half-dozen different types of modifications
identifiedtodate[17].Theserepresentsomeofthemostcommonofallmod-
0
ifications,the2-Omethylationsofthefourmajornucleosidesadenosine(Am),
guanosine(Gm),cytosine(Cm),anduridine(Um);theisomerofuridinewith
acarbon–carbonglycosidicbondpseudouridine(Ψ);andthebasemethylations
N6-methyladenosine (m6A), 1-methyladenosine (m1A), 7-methylguanosine
(m7G), 5-methylcytosine (m5C), and 5-hydroxymethylcytosine (hm5C)
[18]. Though these are simple chemical alterations, their influence in
regulatingtranslationappearstobetrulysignificant [19–21].Weareonlyjust
beginning to understand the relevance of mRNA modifications to
decoding [22].
Inrecentyears,thecodingregionsofmRNAhavebeenfoundpunctu-
ated with posttranscriptional modifications [17]. Early in the study of
mRNAbiochemistryitwasrecognizedthatmRNAhadmodifiednucleo-
0
sides in noncoding regions, such as the 5-CAP [23]. Some of the mRNA
modificationsaretransientinthatmethyltransferases“write”amethylation
and demethylases “erase” the methyl group while other proteins, as
“readers,” recognize the “mark” and bind the RNA, affecting its structure
and function [24]. Some have been proven to affect translation and gene
expression [25–28]. The biochemical mechanism by which translation is
affected is at present speculative. However, nucleobase modifications of
nucleosides within the mRNA coding region will alter recognition of
codonsbytRNA.AnymodificationoftheWatson–Crickhydrogenbond-
ing face of a nucleoside will change tRNA’s anticodon base pairing to the
codon. Thus, the methyl of an m6A in an mRNA coding region negates
canonicalWatson–CricktRNAbasepairing,possiblychangingtheaccuracy
andrateofdecodingoraffectingprematuretermination,andeventheeven-
tualacceptanceofthemRNAfortranslation.Theerasemechanismcanthen
functionalize that codon for tRNA reading.
1.3 RNA Modification and the Accuracy and Efficiency
of Translation
Accuracy of decoding mRNA at the ribosome’s aminoacyl- or A-site
dependsontRNArecognitionofacomplementarymRNAcodon.tRNA
asthedecoderofmRNAisalsoposttranscriptionallymodifiedfortheaccu-
racy and efficiency of reading mRNA codons [11,29–33]. More than
