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tRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii Ningxi Yu, a Manasses Jora, a Beulah Solivio, a Priti Thakur, a Carlos G. Acevedo-Rocha, b Lennart Randau, b Valérie de Crécy-Lagard, c Balasubrahmanyam Addepalli, a Patrick A. Limbach a a Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio, USA b Max Planck Institute for Terrestrial Microbiology, Marburg, Germany c Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA ABSTRACT tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been de- termined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon- decoding strategies, we have used liquid chromatography and tandem mass spec- trometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii. A new posttran- scriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm 5 s 2 U), was dis- covered and localized to position 34. Moreover, data consistent with wyosine path- way modifications were obtained beyond the canonical tRNA Phe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our under- standing of archaeal tRNA modification profiles and decoding strategies. IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eu- karyotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, al- though position 37 appears to be modified to a greater extent than seen in H. vol- canii. KEYWORDS 5-cyanomethyl-2-thiouridine, archaea, LC-MS/MS, anticodon loop, modified nucleosides, posttranscriptional modification, tRNA A s key molecules involved in protein synthesis, tRNAs decode the mRNA, enabling the appropriate amino acid to be inserted into the growing polypeptide chain. While there are 61 sense codons in a standard genetic code, most organisms require far fewer tRNAs for decoding as described by the wobble hypothesis (1). The third base in a codon and position 34 in the tRNA anticodon loop are referred to as the wobble positions. It is now well established that chemical modifications to the nucleobase at position 34 in tRNAs are used by organisms to expand decoding approaches. For example, it was reported that the modified uridine, uridine 5-oxyacetic acid (cmo 5 U), at the wobble position of Escherichia coli tRNA UAC Val was able to efficiently decode all four Citation Yu N, Jora M, Solivio B, Thakur P, Acevedo-Rocha CG, Randau L, de Crécy-Lagard V, Addepalli B, Limbach PA. 2019. tRNA modification profiles and codon-decoding strategies in Methanocaldococcus jannaschii. J Bacteriol 201:e00690-18. https://doi.org/10 .1128/JB.00690-18. Editor Tina M. Henkin, Ohio State University Copyright © 2019 American Society for Microbiology. All Rights Reserved. Address correspondence to Patrick A. Limbach, [email protected]. Received 8 November 2018 Accepted 31 January 2019 Accepted manuscript posted online 11 February 2019 Published RESEARCH ARTICLE crossm May 2019 Volume 201 Issue 9 e00690-18 jb.asm.org 1 Journal of Bacteriology 9 April 2019 on September 12, 2020 by guest http://jb.asm.org/ Downloaded from

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Page 1: tRNA Modification Profiles and Codon-Decoding Strategies ... · ABSTRACT tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding

tRNA Modification Profiles and Codon-Decoding Strategies inMethanocaldococcus jannaschii

Ningxi Yu,a Manasses Jora,a Beulah Solivio,a Priti Thakur,a Carlos G. Acevedo-Rocha,b Lennart Randau,b

Valérie de Crécy-Lagard,c Balasubrahmanyam Addepalli,a Patrick A. Limbacha

aRieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio, USAbMax Planck Institute for Terrestrial Microbiology, Marburg, GermanycDepartment of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA

ABSTRACT tRNAs play a critical role in mRNA decoding, and posttranscriptionalmodifications within tRNAs drive decoding efficiency and accuracy. The types andpositions of tRNA modifications in model bacteria have been extensively studied,and tRNA modifications in a few eukaryotic organisms have also been characterizedand localized to particular tRNA sequences. However, far less is known regardingtRNA modifications in archaea. While the identities of modifications have been de-termined for multiple archaeal organisms, Haloferax volcanii is the only organism forwhich modifications have been extensively localized to specific tRNA sequences. Toimprove our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spec-trometry to characterize and then map posttranscriptional modifications on 34 ofthe 35 unique tRNA sequences of Methanocaldococcus jannaschii. A new posttran-scriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was dis-covered and localized to position 34. Moreover, data consistent with wyosine path-way modifications were obtained beyond the canonical tRNAPhe as is typical foreukaryotes. The high-quality mapping of tRNA anticodon loops enriches our under-standing of archaeal tRNA modification profiles and decoding strategies.

IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAsare also found in bacteria and eukaryotes, several that are unique to archaea wereidentified. By RNA modification mapping, the modification profiles of M. jannaschiitRNA anticodon loops were characterized, allowing a comparative analysis with H.volcanii modification profiles as well as a general comparison with bacterial and eu-karyotic decoding strategies. This general comparison reveals that M. jannaschii, likeH. volcanii, follows codon-decoding strategies similar to those used by bacteria, al-though position 37 appears to be modified to a greater extent than seen in H. vol-canii.

KEYWORDS 5-cyanomethyl-2-thiouridine, archaea, LC-MS/MS, anticodon loop,modified nucleosides, posttranscriptional modification, tRNA

As key molecules involved in protein synthesis, tRNAs decode the mRNA, enablingthe appropriate amino acid to be inserted into the growing polypeptide chain.

While there are 61 sense codons in a standard genetic code, most organisms require farfewer tRNAs for decoding as described by the wobble hypothesis (1). The third base ina codon and position 34 in the tRNA anticodon loop are referred to as the wobblepositions. It is now well established that chemical modifications to the nucleobase atposition 34 in tRNAs are used by organisms to expand decoding approaches. Forexample, it was reported that the modified uridine, uridine 5-oxyacetic acid (cmo5U), atthe wobble position of Escherichia coli tRNAUAC

Val was able to efficiently decode all four

Citation Yu N, Jora M, Solivio B, Thakur P,Acevedo-Rocha CG, Randau L, de Crécy-LagardV, Addepalli B, Limbach PA. 2019. tRNAmodification profiles and codon-decodingstrategies in Methanocaldococcus jannaschii.J Bacteriol 201:e00690-18. https://doi.org/10.1128/JB.00690-18.

Editor Tina M. Henkin, Ohio State University

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Patrick A. Limbach,[email protected].

Received 8 November 2018Accepted 31 January 2019

Accepted manuscript posted online 11February 2019Published

RESEARCH ARTICLE

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valine codons GUU/GUC/GUA/GUG, and cmo5U bound to U/C/A/G was confirmed bycrystal structures (2). The cmo5U·A and cmo5U·G pair adopted the normal Watson-Crickgeometry, and the cmo5U·U and cmo5U·C base pair has only one hydrogen bond toform a minor groove.

In addition to expanding decoding strategies, posttranscriptional modifications tonucleosides at positions 34 and 37 of tRNAs can enhance decoding accuracy andprevent frameshifting. For example, Yarian and coworkers found that the modifications2-thiouridine (s2U) and 5-methylaminomethyluridine (mnm5U) at position 34 alongwith the modification N6-threonylcarbamoyladenosine (t6A) at position 37 in tRNAUUU

Lys

were essential for recognizing the codons AAA and AAG and discriminating against thetwo asparagine codons, AAU and AAC (3). Later, Rozov and coworkers reported theX-ray structure of E. coli tRNAmnm5s2UUU

Lys bound to the codons, and t6A at position 37formed cross-strand stacking with the first nucleotide of the mRNA codon, whichenhanced the anticodon-codon stability (4).

Posttranscriptional modifications in tRNAs are ubiquitous, and compilations ofthese are readily accessible (5–7). Within archaea, the first in-depth studies of tRNAmodifications were performed by McCloskey and coworkers (8, 9). These studiesrevealed that archaea have some modified nucleosides in common with bacteriaand eukaryotes, such as 1-methylguanosine (m1G), dihydrouridine (D), and N6-threonylcarbamoyladenosine (t6A). However, some modifications have been iden-tified only (thus far) within archaea (10), including archaeosine (G�) (position 15)(11) and agmatidine (C�) (position 34) (12, 13) (see Fig. S1 in the supplementalmaterial).

Our understanding of tRNA modification profiles (i.e., the specific modifications andlocations in each tRNA) in archaea is limited (14). To date, the only archaeal tRNAsequences whose modification profiles are almost completely characterized are fromHaloferax volcanii (15, 16). Whether this organism is a good representative of howarchaea have adapted their tRNA-based decoding machinery can be determined onlyby compiling information from additional archaeal tRNAs.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is one of the mostuseful analytical approaches available for characterizing posttranscriptional modifica-tions in RNA (17). In addition to LC-MS/MS determination of the types and abundanceof modified nucleosides, this approach can also be used to localize specific modifica-tions onto unique tRNA sequences. This RNA modification mapping approach is verypowerful for tRNAs, as they are characterized by a wide diversity and high density ofposttranscriptionally modified nucleosides that limit the applicability of other ap-proaches, especially those based on reverse transcriptase (18). Here we have usedLC-MS/MS and modification mapping to conduct an in-depth examination of theposttranscriptionally modified nucleosides in the anticodon loop of tRNAs from Metha-nocaldococcus jannaschii (formerly Methanococcus jannaschii). These anticodon modi-fication profiles are compared against those found in H. volcanii, and insights intoarchaeal codon-decoding strategies are identified in comparison to those used bybacteria and single cell eukarya.

RESULTSModified nucleosides in M. jannaschii total tRNAs. A total of 30 posttranscrip-

tionally modified nucleosides in M. jannaschii tRNAs were identified by LC-MS/MSanalysis of total tRNA nucleoside digests (Table 1). The extracted ion chromatogramsand MS and MS/MS spectral data of all 30 modified nucleosides are provided in Fig. S2to S31 in the supplemental material. Of these, 13 would be expected to be localized inthe anticodon arm (5). Four modified nucleosides (mnm5U, mnm5s2U, cnm5U, andagmatidine) are typically found at the wobble position (position 34). Six modifiednucleosides (m1G, imG-14, imG, mimG, t6A, and ms2t6A) are known to be localized toposition 37 of the anticodon loop (19–21). Three modified nucleosides, Um, s2C, andCm, are often found at position 32.

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The majority of these modified nucleosides have been characterized before, eitherin archaea or in other organisms (5). However, several modified nucleosides were eitherunique or required additional confirmation. For example, cnm5U has recently beenreported in four archaea (H. volcanii, Haloarcula marismortui, Halobacterium salinarum,Methanococcus maripaludis) (22), and we report here the presence of both this position34 modification and its 2-thiolated analog, cnm5s2U, which has not been previouslycharacterized. The identification of cnm5s2U was easily enabled by higher-energycollisional dissociation (HCD) LC-MS/MS of nucleosides (23). Figure 1A shows the HCDMS/MS spectrum obtained when m/z 284, corresponding to cnm5U, was dissociated.The peak at m/z 152.0455 corresponds to the modified nucleobase ion of cnm5U. Thepeak at m/z 125.0346 is a diagnostic base fragment ion that was observed previously(22). A similar HCD fragmentation pattern was seen when m/z 300, corresponding tocnm5s2U, was dissociated (Fig. 1B). Peaks at m/z 168.0232 and m/z 141.0123 weredetected, which match the addition of sulfur in place of oxygen in the elementalformula, compared against m/z 152.0455 and m/z 125.0346 for cnm5U. The high-pressure liquid chromatography (HPLC) elution profile for cnm5U was similar to thatpreviously reported (22), and as is common for other 2-thiolated nucleosides (e.g.,mnm5U/mnm5s2U [Fig. S12 and S13]), the 2-thio analog elutes later than the 2-oxoversion. The HCD MS/MS spectral data of mnm5U and mnm5s2U were also obtained(see Fig. S32 in the supplemental material) to confirm the fragment ion relationshipsbetween 2-thio and 2-oxo modified uridines.

N6-Hydroxynorvalylcarbamoyladenosine (hn6A) and 2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine (ms2hn6A) were identified in M. jannaschii. A particularchallenge in identifying hn6A is that the modified adenosine, m6t6A, has the same massas hn6A. Here, hn6A was confirmed by a combination of HPLC elution time and HCDfragmentation pattern (see Fig. S33 in the supplemental material) (23). The HCD spectra

TABLE 1 Detected posttranscriptional modifications in M. jannaschii

Nucleosidea Retention time (min) M � H CIDb fragment(s)

m5C 4.76 258.1082 126.0661Cm 6.62 258.1082 112.0506s2C 3.66 260.0698 128.0278C� 28.73 356.2037 224.1617� 1.51 245.0766 209.0556, 179.0450. 155.0450m1� 3.50 259.0923 223.0712, 193.0607, 169.0608s4U 10.07 261.0537 129.0117Um 12.34 259.0923 113.0346cnm5U 6.81 284.0876 152.0454cnm5s2U 17.97 300.0646 168.0226mnm5U 1.90 288.1188 156.0767mnm5s2U 5.07 304.0959 172.0538Am 29.38 282.1194 136.0617m1A 4.05 282.1194 150.0773m6A 31.17 282.1194 150.0773m1I 17.73 283.1034 151.0613t6A 30.94 413.1413 281.0990hn6A 32.44 427.1568 295.1147ms2t6A 32.69 459.1291 327.0865ms2hn6A 33.71 473.1445 341.1023G� 29.76 325.1251 193.0831Gm 17.34 298.1143 152.0566m2G 20.26 298.1144 166.0724m2

2G 29.20 312.1299 180.0878m2

2Gm 32.57 326.1456 180.0878m2Gm 31.27 312.1298 166.0722m1G 18.30 298.1143 166.0723imG-14 31.75 322.1143 190.0722imG 32.39 336.1299 204.0878mimG 34.35 350.1456 218.1035aThe abbreviations of modifications are from the MODOMICS database (5).bCID, collision-induced dissociation.

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of hn6A and m6t6A differ significantly. Adenosine, t6A, and hn6A share similar ions atm/z 136.0619 and m/z 119.0354, which arise from the adenine nucleobase. In contrast,the HCD spectrum for m6t6A is more similar to that for m6A, as both contain ions at m/z150.0777 and m/z 123.0667, arising from the N6-methyladenine nucleobase and itsfragment ion. The fragmentations of adenine and N6,N6-dimethyladenine have beenstudied before (24, 25), and these studies indicated that the methyl group at position6 affected the dissociation of the adenine nucleobase. In our study, the same fragmen-tation effects were observed: the labile groups (N6-hydroxynorvalylcarbamoyl groupfrom hn6A and the N6-threonylcarbamoyl group from t6A) at position 6 of the adeninenucleobase are dissociated to produce protonated adenine.

Wyosine (imG), which is a modified nucleoside found in archaea and eukaryotes, wasidentified in M. jannaschii. It has the same mass as its isomer isowyosine (imG2);however, these isomers can be differentiated through nucleobase fragmentation pat-terns. Nucleobase fragmentation of imG shows the loss of CH4, CO, and HCN, while onlyCO and HCN fragments are derived from the nucleobase of imG2 (20). HCD MS/MSidentified the loss of CH3, CO, and HCN from the wyosine nucleobase (see Fig. S34 inthe supplemental material), which is consistent with the identification of imG in M.jannaschii.

In several instances, modified nucleosides arising from a single modification path-way were detected, and the relative levels of these modifications can provide someinsight into tRNA (hypo)modification status. For example, t6A and ms2t6A were bothdetected. Comparing the peak areas for these two nucleosides as a means to estimatetheir relative abundances revealed that the 2-methylthio modification was rare (t6A/ms2t6A ratio of 21:1). In contrast, hn6A and ms2hn6A were found to be nearly equivalent(hn6A/ms2hn6A ratio of 1.5:1), suggesting that M. jannaschii tRNAs utilize the MtaBhomolog (26) in a tRNA- or nucleoside-dependent manner. Another position 37 mod-ification pathway, that of methylwyosine, yielded three components (excluding m1G,which is the first step in the pathway [20]): imG-14, imG, and mimG. The peak areas ofimG-14, imG, and mimG were calculated from the nucleoside analysis data to estimatetheir relative abundances. This semiquantitative analysis yielded imG-14/imG/mimGpeak area ratios of 20:23:1, suggesting that imG-14 can accumulate and that mimG isa rare modified nucleoside in this sample.

FIG 1 HCD spectral data of cnm5U (A) and cnm5s2U (B). A1 and B1 are the base ions, and A2 and B2 are the fragment ionsof A1 and B1, respectively.

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Modification mapping of M. jannaschii tRNA anticodon stem-loops. Once thecensus of modified nucleosides was obtained and the identities of all modificationsconfirmed, the next step was to conduct RNA modification mapping by LC-MS/MS. Theprotocol for such analyses is well established even when analyzing the pool of totaltRNAs (17, 27). However, in this study, our particular interest was to focus on obtaininghigh-quality mapping data for the anticodon loop, to enable an enhanced understand-ing of the decoding strategy for this archaeon. One feature that simplified assigningand annotating the LC-MS/MS data for anticodon loop regions was that the majority ofthe RNase digestion products were found to be signature digestion products (SDPs), anantecedent of which was used by McCloskey and coworkers to characterize tRNAanticodon loop modification profiles in yeast and Sulfolobus solfataricus (28).

SDPs are RNase digestion products detected by mass spectrometry that are uniqueto a single RNA (usually tRNA) sequence (29–32). SDP specificity arises through acombination of oligonucleotide base composition, sequence, and modification status.The combination of unique mass (from base composition and modification[s]) andsequence enables these digestion products to be used as specific identifiers of partic-ular tRNAs. Here, once the MS/MS data from the RNase digestion product wereinterpreted and the product found to be an SDP, those MS/MS data could then beannotated to only a single M. jannaschii tRNA sequence, uniquely defining the modi-fication profile of tRNA anticodon loops.

M. jannaschii contains 35 unique tRNA sequences. Sufficient LC-MS/MS data wereobtained to characterize the anticodon loops of each unique tRNA sequence except forselenocysteine (tRNA-Sec). The various RNase digestion products that generated oligo-nucleotides containing position 34 and 37 for these 34 tRNA sequences are listed inTable 2. Those RNase digestion products that were found to be SDPs for a particulartRNA are denoted by italics. Additional RNase digestion products that confirm the dataobtained from SDPs are also included in Table 2. Detailed MS/MS spectral data andpeak annotations are listed in Fig. S35 to S92 in the supplemental material.

Several digestion products were detected that do not fit the SDP criteria but rathercould map onto at least two different tRNAs. However, by understanding and applyingcommon rules for localizing modified nucleosides within a tRNA sequence, only a singletRNA sequence was the logical solution for mapping. For example, CCUAGp is an insilico-predicted RNase T1 digestion product that could arise from four different tRNAs:tRNAGCG

Arg , tRNAUCUArg , tRNAUAG

Leu , and tRNAGGASer . When the LC-MS/MS data were acquired,

analyzed, and interpreted, this digestion product was found to correspond toCC[cnm5s2U]AGp. The 2-thio-5-cyanomethyluridine modification should occur at posi-tion 34, as noted above. The only tRNA among the four where CCUAGp arises fromposition 32 through position 36 is tRNAUAG

Leu , thus specifically mapping this potentiallyambiguous RNase T1 digestion product back onto a single tRNA sequence.

A single RNase digestion product sometimes will not cover the entire anticodonloop. In those cases, multiple digestion products were required to ensure that bothposition 34 and position 37 were accurately mapped. For example, GACU[cnm5U]GUpand U[hn6A]AUCAGp were identified to cover the anticodon loop in tRNAUGU

Thr . The useof multiple RNases allowed all anticodon loops to be mapped with SDPs or withdigestion products that cannot logically be mapped elsewhere in the sequence.

When possible, position 32 within each tRNA sequence was also mapped. Position32 modifications were found to be rare in M. jannaschii tRNAs. Of the three putativemodified nucleosides for this position identified above, Cm was identified at position32 of tRNAUUU

Lys , tRNACAUMet , tRNAGCC

Gly , and tRNAGACVal . Um was identified at position 32 of

tRNAGGCAla , and s2C was identified at position 32 of tRNACCA

Trp . Other sites of modificationthat could be unambiguously identified in the data were also assigned, and a consen-sus modification sequence is shown in Fig. 2.

The use of SDPs for localizing modifications to specific tRNA sequences is predicatedon purifying only the total pool of tRNAs, with no additional RNAs (e.g., rRNA or mRNAfragments) that could confound the mapping process being present in significant

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abundance. While the likelihood of significant contamination is low (17), Table S1 in thesupplemental material was generated by examining all possible M. jannaschii RNAsequences rather than just limiting SDPs to tRNA sequences. Even with this muchbroader criterion, all tRNA anticodon loops could be mapped by SDPs or digestionproducts that logically cannot map elsewhere, except for tRNAGGC

Ala and tRNAGACVal ,

providing additional confidence in the accuracy of these anticodon loop modificationassignments.

Many modifications outside the anticodon arm could also be localized in this work.One challenge with mapping regions outside the anticodon arm is that tRNA sequencesimilarities reduce the utility of the SDP approach. For example, the T-arm is highlyconserved in many M. jannaschii tRNAs, leading to RNase digestion products that couldarise from more than one tRNA. In those cases, modifications were localized to specificregions, although it remains to be determined whether every possible tRNA possessesthat modification. The selected MS/MS spectral data and peak annotations for assign-ments outside the anticodon arm are provided in Fig. S93 to S130 in the supplementalmaterial. Combining the high-quality anticodon mapping data with these other local-ization data results in the first compilation of modified tRNA sequences from M.jannaschii (Fig. 3).

TABLE 2 Detected oligonucleotides from anticodon stem-loops

tRNA-anticodon Detected oligonucleotide(s) (enzyme)a

Ala-GGC CA[Um]UGp (T1), UGGCUGp (MC1)Ala-UGC CU[cnm5U]GCp (Cus), CAAGp (T1)Arg-GCG GGCCUGCp (A), GCGGAGCCp (A)Arg-UCG [cnm5U]CG[imG-14]AUp (A)Arg-UCU CCU[cnm5U]CU[t6A]AGp (T1), CCU[cnm5s2U]CU[t6A]AGp (T1)Asn-GUU GUU[t6A]AUp (A)Asp-GUC GGGACUGUp (A), UCACUCCCGp (T1)Cys-GCA UGCA[m1G]AUCCGCCp (MC1), UGCA[imG-14]AUCCGCCp (MC1),

CA[imG-14]AUCCGp (T1)Gln-UUG U[mnm5U]UG[m1G]ACCCCp (Cus), U[mnm5s2U]UG[m1G]ACCCCp (Cus)Glu-UUC CCU[mnm5s2U]UC[m1G]AGp (T1)Gly-GCC GGGC[Cm]UGCp (A), CCACGp (T1)Gly-UCC CCU[mnm5U]CCAAGp (T1)His-GUG GUG[m1G]AUCp (A)Ile-GAU GAU[t6A]ACp (A), GAU[hn6A]ACp (A)Ile-CAU CU[C�]AU[hn6A]ACCGp (T1)Leu-GAG GAGGGUCUp (A)Leu-UAA [cnm5U]AA[m1G]AUCp (A), [cnm5U]AA[imG]AUCp (A),

[cnm5s2U]AA[m1G]AUCp (A)Leu-UAG CC[cnm5s2U]AGpb (T1), [m1G]ACCCAGp (T1)Lys-UUU [Cm]U[cnm5s2U]UU[t6A]ACCAGp (T1),

[Cm]U[cnm5s2U]UU[hn6A]ACCAGp (T1)Met-CAU1 CU[Cm]AU[hn6A]ACCGp (T1), CU[Cm]AU[ms2hn6A]ACCGp (T1)Met-CAU2 CUCAUAACCCGp (T1)Phe-GAA AA[m1G]AUCCAGp (T1), GAA[m1G]AUp (A), GAA[imG]AUp (A)Pro-GGG GGG[m1G]GGCp (A)Pro-UGG AUU[cnm5U]Gp (T1), [m1G]AUCCUGp (T1)Ser-GCU GGGACUGCp (A), CU[hn6A]AUCCCAUUGp (T1)Ser-GGA GGA[m1G]AUCpb (A), GGA[imG]AUCpb (A)Ser-UGA U[cnm5s2U]GA[m1G]AUCp (A)Thr-GGU GGU[hn6A]AGCp (A)Thr-UGU GACU[cnm5U]GUp (A), U[hn6A]AUCAGp (T1)Trp-CCA A[s2C]UCCA[m1G]AUCCCUGp (T1)Tyr-GUA GUA[imG-14]AUp (A)Val-CAC CCCUCACAAGp (T1)Val-GAC CC[Cm]UGpb (T1), ACACGp (T1)Val-UAC CCU[cnm5U]ACp (A), CCCU[cnm5U]ACAAGp (T1)aThe enzyme used to generate the digestion product is indicated. T1, RNase T1; A, RNase A; MC1, RNaseMC1; Cus, cusativin; U2, RNase U2. Anticodon positions 34, 35, 36, and 37 are identified by boldface. The M.jannaschii tRNAs are italicized.

bThe digestion products can logically be mapped to only a single tRNA sequence location based on priorlocalization of particular anticodon loop modifications.

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DISCUSSIONModified nucleosides in M. jannaschii. M. jannaschii is a hyperthermophilic ar-

chaeon in the Euryarchaeota phylum. It is adapted for a high-pressure (200 atm) andhigh-temperature (48°C to 94°C) environment. A methanogen, M. jannaschii survives oncarbon dioxide and hydrogen, producing methane as the end product (33). M. jann-aschii is the first archaeal organism whose genome was completely sequenced (34), andthe genes involved in amino acid biosynthesis, protein synthesis, cellular processes,metabolism, nucleosides, and nucleotides synthesis have been annotated (35).

McCloskey and colleagues previously characterized 25 posttranscriptionally modi-fied nucleosides in M. jannaschii tRNAs (8). All of those were also found here except forinosine and what they identified as an unknown nucleoside with a molecular mass of422 Da, which is likely yW-86 (20). McCloskey and colleagues noted that the presenceof inosine might result from artifactual deamination of adenosine. As new modifiednucleosides from archaea have been discovered since the work of McCloskey et al. dueto advances in LC-MS technology, it is not surprising that an additional seven modifiednucleosides were identified here: s2C, agmatidine, cnm5U, cnm5s2U, imG-14, mimG, andms2hn6A.

The census of modified nucleosides in M. jannaschii tRNAs reveals a common suiteof structural and functional archaeal modifications. With the exception of cnm5s2U, allmodified nucleosides have been identified before (5). Several modifications (t6A, m1G,and Cm) are found throughout all kingdoms. Others, such as mnm5U, mnm5s2U, hn6A,and s2C, are also found in bacterial tRNAs. Four modifications, cnm5U, cnm5s2U,agmatidine, and mimG, have only been discovered in archaea to date. Our analyses

FIG 2 Consensus modification sequence for M. jannaschii tRNAs, listing both experimentally determined sequencelocations and the expected enzyme(s) required for modification.

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revealed the presence of imG and imG-14, which are believed to be within thewybutosine pathway and have previously been found in eukaryotes (20, 36).

There are 19 species of archaea (including M. jannaschii) whose tRNA modificationshave been studied before (8, 9, 15, 16, 37). Table S2 in the supplemental materialsummarizes the distribution of 38 different posttranscriptionally modified nucleosidesamong these 19 species. The modified nucleosides that are found in Euryarchaeota andCrenarchaeota are compared in Fig. S1 in the supplemental material. When focusing onthe anticodon loop, there are five position 34-modified uridines (mnm5U, mnm5s2U,mcm5s2U, cnm5U, and cnm5s2U) that are found in Euryarchaeota but not in Crenar-chaeota. All except mcm5s2U were found here. Four position 37 modifications, t6A,ms2t6A, hn6A, and ms2hn6A, have been found in both Euryarchaeota, including M.jannaschii, and Crenarchaeota. As discussed further below, the wyosine pathway variesbetween Euryarchaeota and Crenarchaeota (20).

FIG 3 Compilation of modified total tRNA sequences from M. jannaschii. Anticodons with identified modifications indicated in red. Modifications that wereidentified by signature digestion products are indicated by bold black. Modifications that were identified by nonsignature digestion products are indicated bygray.

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RNA modification mapping of archaeal total tRNA pools. Despite the availabilityof new technologies and analytical methods, very little data are available on the tRNAmodification profiles for archaeal organisms. A review of the Modomics database findsonly 61 archaeal tRNA sequences whose modification profiles are known or partiallyknown. As noted before, the vast majority (41 of 61) arise from Haloferax volcanii (15,16). The remainder are distributed among six other archaea: Halobacterium salinarum(formerly Halobacterium cutirubrum), 12 sequences (38, 39); Thermoplasma acidophilum,three sequences (14, 40–42); Methanobacterium thermoautotrophicum, two sequences(43); and Halococcus morrhuae (40), Methanosarcina barkeri (44), and Sulfolobus acido-caldarius (14, 40), one sequence each.

Although multiple techniques exist to localize modifications onto RNA sequences,mass spectrometry-based approaches have been the most successful for analyzingtRNAs, which can be challenging due to both the structural diversity of modifiednucleosides and the relatively high density of modifications per tRNA sequence (5).Mass spectrometry has the advantage of unbiased detection: every modification thatresults in a mass change to the canonical nucleoside can be detected directly. More-over, the mass change alone is often sufficient to identify the modification. When thesecharacteristics are combined with the standard tandem mass spectrometry approachfor sequencing oligonucleotides (17, 45, 46), the specific sequence location and identityof modified nucleosides can be localized onto tRNA sequences.

The strategy used here was adapted from our previous approach to total tRNAmodification mapping (17, 27, 45). The key to obtaining high-quality data covering theanticodon loop of M. jannaschii tRNAs was the use of multiple RNases. In this work,RNase T1 was used to establish the baseline mapping coverage, as this enzymereproducibly cleaves tRNAs at unmodified guanosines and m2G. The RNase T1 digestswere complemented with four other RNases: RNase A, RNase U2, MC1, and cusativin. Atotal of 10 experimental LC-MS/MS analyses of RNase digests were performed. For eachanalysis, the resulting MS/MS data were first interpreted using RNAModMapper asdescribed in Materials and Methods. High-quality interpretations that yielded SDPswere annotated directly onto the appropriate tRNA sequence. In those cases, otherenzyme digests were used to validate the SDP annotation. In some instances, differentenzymes were required to generate SDPs that could cover the entire anticodon loop.Below we separately examine those modifications found outside the anticodon armand those localized within the anticodon. The responsible enzyme, when known, andspecific tRNAs found to contain each modification are discussed.

M. jannaschii tRNA modifications outside the anticodon. (i) m2G(6). Trm14,previously identified in M. jannaschii (MJ0438), is believed to responsible for m2G atposition 6 (47). Our mapping analyses detected the digestion product GG[m2G]G[s4U]p.This digestion product could arise from tRNAGCA

Cys , tRNAGUGHis , or tRNACAU1

Met . Previously m2Gwas localized to position 6 of tRNAGCA

Cys in M. jannaschii (47), which suggests that thistRNA is more likely than the others to contain this modification.

(ii) s4U(8). The sulfur transferase ThiI (MJ0931) is most likely responsible for s4U atposition 8 (48). This modification could be localized at this position in many possibletRNAs, including tRNAUCU

Arg , tRNAGUUAsn , tRNAGCC

Gly , tRNAUGASer , and tRNAUAC

Val .(iii) m2G(10)/m2

2G(10). The methylated guanosine modification N2-methylguanosine(m2G) was localized to position 10 in tRNAUCU

Arg , tRNAUUULys , and tRNAGAU

Val . The dimethylatedguanosine modification (m2

2G) could be localized to position 10 of tRNAGUGHis . These

modifications are proposed to arise from TrmG10/PAB1283 in Pyrococcus abyssi (49).While the protein family for PAB1283 is conserved in archaea, its homolog (MJ0710) hasnot been experimentally verified in M. jannaschii.

(iv) G�(15). Archaeosine (G�) is widely found at position 15 of archaeal tRNAs. Thepathway for this modification is complex, with multiple steps (11, 50), and the wholepathway can be identified in M. jannaschii: the four enzymes for synthesis of the preQ0

precursor (MptA/MJ0775, QueD/MJ1272, QueE/MJ1645, and QueC/MJ1347); TGT(MJ0436), which inserts preQ0 in tRNA (51); and ArcS (MJ1022), which converts preQ0-

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tRNA to G� (52). Archaeosine could be identified at this position in a large number ofM. jannaschii tRNAs.

(v) m22G(26)/m2

2Gm(26). The position 26 dimethylguanosine nucleobase modifi-cation was reported to arise from Trm1 in Pyrococcus horikoshii (53), and a Trm1ortholog (MJ0946) is present in M. jannaschii. Either m2

2G and m22Gm was found at

position 26 in nearly all of the tRNA sequences possessing G26. In most cases, both thedimethyl- and trimethylguanosines were found in the same digestion products, sug-gesting that 2=-OH methylation occurs after nucleobase methylation. While C/D boxsmall RNAs (sRNAs) are often responsible for ribose methylations (54, 55), even intRNAs, M. jannaschii tRNA introns do not contain guide RNAs. As M. jannaschii does nothave a homolog of TrmH, unlike several other archaea (14), the enzyme and mechanismresponsible for ribose methylation at this site are unknown.

(vi) m5C(48/49). m5C has been found at multiple positions, including positions 48and 49, in various archaea (56). The Trm4 methyltransferase (MJ0026) is responsible form5C formation in M. jannaschii (57). In this study, m5C was localized to position 48 intRNAGCA

Cys and to position 49 of multiple tRNAs; no other tRNA positions were found tocontain m5C.

(vii) �(54)/m1�(54)/�(55). Pseudouridine was detected in the nucleoside analy-sis. The archaeal Pus10 protein (MJ0041) generates �54 and �55 in archaeal tRNAs(58), although pseudouridine at position 55 can also be generated through an H/ACAguide RNA mechanism (59–62). As no pseudouridine-specific mapping was performed(63, 64), the localization of this modification to positions 54 and 55 of specific tRNAs isexpected but not verified here. The DUF358 SPOUT-methyltransferase MJ1640 (re-named TrmY) acts in concert with Pus10 to catalyze the biosynthesis of m1� at position54 (65–67). Data consistent with m1�(54) were obtained from tRNAGAC

Val .(viii) Cm(56). The Trm56 2=-O-methyltransferase MJ1385 (68) is responsible for Cm

formation at position 56 in M. jannaschii, and Cm was localized to position 56 intRNAUUG

Gln and tRNAGACVal in this study.

(ix) m1I(57). Mapping localized m1I to position 57 in M. jannaschii tRNAGUGHis and

tRNAGGUThr . It has been shown that the pathway to m1I57 goes through m1A57. Methyl-

ation has been shown to be catalyzed by the TrmI methylase (69–71), but the geneencoding the deaminase is yet to be identified in any archaea.

(x) m1A(58). TrmI, responsible for m1A at position 58, has been validated inPyrococcus abyssi (69). The TrmI ortholog is present in M. jannaschii (MJ0134). m1A waslocalized to tRNAGUU

Asn , tRNAUUGGln , tRNAGAU

Ile , tRNACAUMet , tRNACAC

Val , and tRNAGACVal .

An abundant RNase digestion product, [Cm][m1I][m1A]AUCCp (see Fig. S94 in thesupplemental material), was found during analysis. Due to the strong sequence simi-larity in the T-arm, this digestion product could map onto multiple M. jannaschii tRNAslocalizing Cm56, m1I57, and m1A58.

(xi) m2G(67). Our data localized m2G to position 67 in tRNAUCUArg , tRNAGUU

Asn , tRNAGCCGly ,

tRNACAUIle , and tRNAUAC

Val . Confidence in this position assignment was noted because anRNase T1 digestion product consistent with [m1A]AUCUCCCC[m2G]p (see Fig. S126 inthe supplemental material) was detected. This modification is typically found at theopposing location, G6, in the acceptor stem (see above), but prior studies on tRNAUUU

Lys

from the squid species Heterololigo bleekeri (72) have identified this modification at G67.It may be likely that M. jannaschii tRNAs utilize m2G6 or m2G67 for folding, stability, orcharging. It is not yet known whether Trm14 (MJ0438) would also be responsible formodifying G67 or if a different methyltransferase is required.

M. jannaschii tRNA anticodon modifications. (i) �. Pseudouridine was detectedin the nucleoside analysis; however, as no pseudouridine mapping was conducted, itremains to be determined what locations and which tRNAs contain pseudouridine inthe anticodon arm.

(ii) Cm/Um(32). The sugar methylated residue Cm was mapped to position 32 intRNAGCC

Gly , tRNAUUULys , tRNACAU1

Met , and tRNAGACVal . Similarly, the sugar methylated residue Um

was mapped to position 32 in tRNAGGCAla . As M. jannaschii tRNA introns do not contain

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guide RNAs, the enzyme(s) responsible for Cm/Um formation remains unidentified in M.jannaschii. TrmJ is responsible for Cm32 but not Um32 in Sulfolobus acidocaldarius (73);however, the candidate TrmJ gene in M. jannaschii is still unidentified. Cm was alsoreported to be found at position 34 in tRNACCA

Trp , which was guided by the intron-encoded and trans-acting box C/D ribonucleoprotein of pre-tRNACCA

Trp (74), but Cm34was not found in tRNACCA

Trp here.(iii) s2C(32). 2-Thiocytidine was localized to position 32 in tRNACCA

Trp . A tRNA2-thiolation protein candidate, MJ1157, is likely responsible for s2C formation.

(iv) Agmatidine(34). Agmatidine, introduced by TiaS (75), was found at position 34in tRNACAU

Ile , as expected. The TiaS ortholog is present in M. jannaschii (MJ1095).(v) xm5(s2)U(34). U34 of tRNAUCU

Arg , tRNAUAALeu , and tRNAUCU

Arg was modified either ascnm5U or as the 2-thio analog, cnm5s2U. Similarly, we found that U34 of tRNAUUG

Gln wasmodified to either mnm5U or its 2-thio analog, mnm5s2U. While the data obtained allowonly an estimation of the relative levels of xm5U versus xm5s2U modifications, thesehypomodification states suggest that the xm5U (cnm5U and mnm5U) modificationpathways might be sufficient alone to guarantee accurate decoding, independent ofthe s2U modification pathway. Archaeal Elp3 can catalyze the tRNA modification at C5of U34 (76), and the Elp3 homolog, MJ1136, is present in M. jannaschii. Two methyl-transferases, Trm9 and Trm112, were necessary for the formation of mcm5s2U in H.volcanii (77). Only Trm112 (MJ_RS09395) is present in M. jannaschii, and thus it remainsto determine all of the proteins involved in the biosynthesis of xm5(s2)U modifiednucleosides.

(vi) (ms2)x6A(37). t6A is found throughout all domains, and its biosynthesis path-way in archaea has been described (78). t6A could be mapped to position 37 intRNAUCU

Arg , tRNAGUUAsn , tRNAGAU

Ile , and tRNAUUULys . The enzymes responsible for t6A biosynthesis

have been identified in archaea (79). The first step is catalyzed by threonylcarbamoyl-AMPsynthase (TsaC/MJ0062), which forms the carboxythreonyladenylate that is transferredto target tRNAs by the KEOPS/t6A synthase complex components (MJ1130, MJ0594a,and MJ0187) (80). The modified nucleoside t6A can be further modified to ms2t6A byMtaB in bacterial cells. The MtaB homolog (MJ0867) is found in M. jannaschii (26), and,while ms2t6A was detected during nucleosides analysis, our modification mapping didnot identify the tRNA(s) possessing this modification, most likely due to its low relativeabundance in this sample.

(vii) (ms2)hn6A(37). hn6A was initially reported in various thermophilic bacteria andarchaea (8) and was mapped here to position 37 of tRNAGAU

Ile , tRNACAUIle , tRNAUUU

Lys ,tRNACAU1

Met , tRNAGCUSer , tRNAGGU

Thr , and tRNAUGUThr . ms2hn6A was also localized to position 37

of tRNACAU1Met . Previously, Perrochia et al. suggested that the archaeal Sua5 and KEOPS

could be responsible for the synthesis of hn6A (78); however, the detailed biosynthesispathway of hn6A is still unknown. Interestingly, A37 of tRNAGAU

Ile and tRNAUUULys was

detected in two different modification states: t6A and hn6A. What is unique is that thedata suggest that t6A and hn6A share either a common pathway or a common tRNAsubstrate target.

(viii) Wyosine(37) and derivatives. Archaeal wyosine (imG) and methylwyosine(mimG) formation follows the pathway illustrated in Fig. 4. As noted above, nucleosideanalysis identified all four components of this pathway (m1G, imG-14, imG, and mimG).The methyltransferase Trm5 (MJ0883) was reported to yield m1G at position 37 (81).Taw1 (MJ0257), which further modifies m1G to imG-14, and Taw3 (MJ1510), which isresponsible for imG and mimG, have been identified in M. jannaschii (20, 82).

M. jannaschii tRNAGAAPhe was found to contain both m1G and imG but no imG-14 at

position 37 (see Fig. S72 and S73 in the supplemental material for m1G and Fig. S74 for

FIG 4 Wyosine (imG) modification pathway and predicted or identified modifying enzymes.

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imG). Previous bioinformatics analysis suggests that this organism should be capable ofgenerating 7-aminocarboxypropylwyosine (yW-72) at G37 (20), and the immediateprecursor to this modified nucleoside (yW-86) (83) was likely present in the priornucleoside analysis of M. jannaschii tRNAs conducted by McCloskey et al. (8). Theabsence of these wyosine derivatives in the tRNAs analyzed here could be related toculturing conditions or due to limited Tyw2 (MJ1557) activity.

We were surprised to identify five additional tRNAs that contained wyosine-relatedmodifications at G37: tRNAUCG

Arg (imG-14) (see Fig. S41 in the supplemental material),tRNAGCA

Cys (imG-14) (see Fig. S47 and S48 in the supplemental material), tRNAUAALeu (imG)

(see Fig. S63 in the supplemental material), tRNAGGASer (imG) (see Fig. S81 in the

supplemental material), and tRNAGUATyr (imG-14) (see Fig. S87 in the supplemental

material). tRNAUCGArg was found to contain solely imG-14 at position 37. In contrast, both

m1G and imG, but no imG-14, were found at position 37 of tRNAUAALeu and tRNAGGA

Ser . Withthe exception of tRNAUCG

Arg , which contains guanosine at position 36, these tRNAs sharea sequence motif of 36-AGAUCC-41 that might explain the additional tRNAs found tocontain wyosine derivatives. The other two tRNAs that have this anticodon sequencemotif, tRNAUGA

Ser and tRNACCATrp , were found to have m1G at position 37. As noted above,

semiquantitative analysis at the nucleoside level yielded peak imG-14/imG/mimG arearatios of 20:23:1, which is consistent with multiple tRNAs containing one or more ofthese wyosine-related modifications.

Methylwyosine is an archaeon-specific guanosine 37 derivative, but it was notlocalized to any tRNA of M. jannaschii, including its canonical substrate, tRNAGAA

Phe . Themost likely rationale for not mapping mimG is that its abundance is below the limits ofdetection for the RNA modification mapping method used here. Overall, these resultssuggest that wyosine modification to G37 might be more prevalent in archaea thanpreviously thought.

Archaeal codon-decoding strategies. RNA modification mapping of the total tRNApool from M. jannaschii allowed us to generate anticodon modification profiles for 34tRNAs (Table 3). Because H. volcanii is the only other archaeal organism whose totaltRNA pool has been extensively characterized, below we compare the anticodonmodification profiles between these two archaea before describing the general char-

TABLE 3 Codon-anticodon decoding for M. jannaschii tRNAs

Amino acid Codon(s) Positions 34, 35, 36, 37a

Phe UUU, UUC GAAm1G, GAAimGLeu UUA, UUG cnm5UAAm1G, cnm5UAAimG, cnm5s2UAAm1GLeu CUU, CUC, CUA, CUG GAGG, cnm5s2UAGm1GIle AUU, AUC, AUA GAUt6A, GAUhn6A, C�AUhn6AMet AUG CAUA, CmAUhn6A, CmAUms2hn6AVal GUU, GUC, GUA, GUG GACA, CACA, cnm5UACASer UCU, UCC, UCA, UCG GGAm1G, GGAimG, cnm5s2UGAm1GPro CCU, CCC, CCA, CCG GGGm1G, cnm5UGGm1GThr ACU, ACC, ACA, ACG GGUhn6A, cnm5UGUhn6AAla GCU, GCC, GCA, GCG GGCU, cnm5UGCATyr UAU, UAC GUAimG-14

UAA, UAG StopHis CAU, CAC GUGm1GGln CAA, CAG mnm5UUGm1G, mnm5s2UUGm1GAsn AAU, AAC GUUt6ALys AAA, AAG cnm5s2UUUt6A, cnm5s2UUUhn6AAsp GAU, GAC GUCAGlu GAA, GAG mnm5s2UUCm1GCys UGU, UGC GCAm1G, GCAimG-14

UGA StopTrp UGG CCAm1GArg CGU, CGC, CGA, CGG GCGG, cnm5UCGimG-14Ser AGU, AGC GCUhn6AArg AGA, AGG cnm5UCUt6A, cnm5s2UCUt6AGly GGU, GGC, GGA, GGG GCCA, mnm5UCCAaPosition 34 of the anticodon is in bold; position 37 of the anticodon is underlined.

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acteristics of codon-decoding strategies across the three domains, using E. coli as thecomparative organism for bacteria, S. cerevisiae as the comparative organism forsingle-cell eukaryotes, and our data for M. jannaschii (Table 4).

There are a total of 46 identified tRNAs in H. volcanii (84). H. volcanii and M.jannaschii have several decoding strategies in common. Both organisms use an A34sparing strategy to decode all pyrimidine-ending codons. Both M. jannaschii and H.volcanii use tRNACAU

Ile with modified C34 to decode the isoleucine AUA codon but notthe methionine AUG codon. In our study, we have identified agmatidine at position 34of tRNACAU

Ile ; while agmatidine has not been experimentally verified in H. volcanii, thegenes for this modification are present (85). Based on the modifications that could belocalized in M. jannaschii and H. volcanii tRNAs, as well as the tRNA modifications foundin other archaea, cnm5U, cnm5s2U, and agmatidine appear to be archaeon-specificposition 34 modifications.

H. volcanii always uses three isoacceptor tRNAs to decode the 4-codon boxes, but M.jannaschii only uses two isoacceptor tRNAs to decode 4-codon boxes except valine

TABLE 4 Comparison of decoding strategies of M. jannaschii, H. volcanii, E. coli, and S.cerevisiaea

aBlack, M. jannaschii; orange, H. volcanii (xU indicates the experimental unidentified uridine derivative); red, E.coli; blue, S. cerevisiae. The H. volcanii anticodons that are in parentheses indicate that these codons are foundin tRNA genes but were not yet identified in the experimental data.

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codons. In addition, U34 is modified in M. jannaschii tRNAs, which likely accounts for themore efficient decoding of the 4-codon boxes. Both archaea have modifications atposition 32. H. volcanii has only Cm at position 32 of tRNAUUU

Lys , tRNACUULys , tRNACCA

Trp , andtRNAGUA

Tyr . The modifications at position 32 in M. jannaschii tRNAs are more diversified.We identified Cm in tRNAUUU

Lys and tRNAGACVal , Um in tRNAGCC

Ala , and s2C in tRNACCATrp . We also

found that except for tRNACAUIle , no other modified C34 was identified in M. jannaschii.

In contrast, H. volcanii uses ac4C34 for decoding serine, proline, glutamine, and lysinecodons.

(i) One-codon box. There are two 1-codon boxes for methionine and tryptophan.M. jannaschii is unique in that C34 in tRNACCA

Trp is unmodified. In contrast, H. volcanii, E.coli, and S. cerevisiae use Cm34 to decode G3 of the tryptophan codon (UGG). Cm34 isbelieved to stabilize codon-anticodon interactions. As C34 is unmodified in M. jann-aschii, additional stabilization might occur via the s2C32 modification. In contrast, Cm34was detected in M. jannaschii tRNACAU

Met , likely to differentiate between the methioninecodon (AUG) and the isoleucine codon (AUC), similar to the strategy used by E. coli,which contains ac4C34.

(ii) Two-codon box. There are 12 2-codon boxes, which can be divided into twogroups. The first group has pyrimidine-ending codons: Phe (UUU/UUC), Tyr (UAU/UAC),His (CAU/CAC), Asn (AAU/AAC), Asp (GAU/GAC), Cys (UGU/UGC), and Ser (AGU/AGC).The second group of amino acids has purine-ending codons: Leu (UUA/UUG), Gln(CAA/CAG), Lys (AAA/AAG), Glu (GAA/GAG), and Arg (AGA/AGG). A G34-containingtRNA is used to decode pyrimidine-ending codons across all domains, with the mostcommon feature being that position 37 is modified in various manners to enhanceanticodon-codon interactions. For purine-ending codons, tRNA requirements appear tobe determined by U34 modification status. Similar to the general trends seen in E. coliand S. cerevisiae, M. jannaschii only uses a single U34-containing tRNA, which is thenposttranscriptionally modified to more efficiently decode each codon box in this group.

(iii) Three-codon box. M. jannaschii follows the proposed rule for archaeal decod-ing of the isoleucine (AUA) codon, which is based on the bacterial model of modifyingC34 to differentiate from methionine (AUG) decoding.

(iv) Four-codon box. There are eight 4-codon boxes. M. jannaschii only uses twotRNAs (UNN and GNN) to decode all 4-codon boxes except valine. The G34-containingtRNA is able to decode C3 and U3 codons, while U34 is modified to cnm5(s2)U ormnm5U for decoding A3 and G3 codons. Besides U34 modifications, E. coli and S.cerevisiae have other decoding strategies to decode particular 4-codon boxes. Serine,proline, threonine, and alanine codons can be decoded by modifying A34 to inosine inS. cerevisiae. To decode the arginine codons (CGU/CGC/CGA/CGG), both E. coli and S.cerevisiae use I34. Because there is no A34 anticodon in M. jannaschii and H. volcanii, theinosine-based decoding strategy is not required.

Conclusion. The posttranscriptionally modified nucleosides in M. jannaschii tRNAshave been characterized and then mapped to specific tRNAs through modern LC-MS/MS approaches. Significant diversity in anticodon position 34 and 37 modificationswas found, including a number of (hypo)modified states such as xm5(s2)U34,(ms2)x6A37, and wyosine pathway derivatives at G37. In addition to identifying a newmodified nucleoside, cnm5s2U, wyosine derivatives were found on multiple tRNAsbeyond the canonical tRNAGAA

Phe . By establishing the tRNA modification profile for M.jannaschii, these results reveal that this archaeal organism, like H. volcanii, followscodon-decoding strategies that are similar to those of bacteria, although M. jannaschiipossesses a greater diversity of anticodon modification states than H. volcanii. Theseresults add to our growing understanding of codon-decoding strategies and modifi-cation profiles in the archaeal kingdom.

MATERIALS AND METHODSCulturing and tRNA isolation. M. jannaschii (DSM 2661) cells were a kind gift of Karl O. Stetter and

Michael Thomm. The cells were grown anaerobically in a 300-liter bioreactor on H2 and CO2 (80:20) at85°C under a pressurized headspace of 200 kPa at the Archaeenzentrum of the Universität Regensburg,

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Germany. The cells were harvested at the midpoint of their exponential growth phase and subsequentlystored at �80°C. Total RNA was isolated from 3.2 g of cells with TRIzol (Invitrogen) reagent according tothe manufacturer’s instructions.

Qiagen-Tip 2500 was used to purify tRNAs (approximately 4 mg) from total RNA (86). A QiagenQ-2500 column was equilibrated with 50 ml fresh equilibration buffer (0.05 M morpholinepropanesulfo-nic acid [MOPS], pH 7.0) two times. The total RNA was resuspended in 0.1 M MOPS and loaded onto theequilibrated Q-2500 column. The column was washed with 200 ml of wash buffer (0.05 M MOPS, 0.2 MNaCl, pH 7.0) and 10 ml elution buffer (0.05 M MOPS, 0.75 M NaCl, 15% ethanol, pH 7.0). The tRNA waseluted using an additional 20 ml of elution buffer. The flowthrough was collected, loaded back onto thecolumn two more times, and treated as described. The final purified tRNA was precipitated with onevolume of isopropanol before storage at �20°C overnight. The sample was centrifuged for 30 min, andthe pellet was washed with 70% ethanol. The pellet was lyophilized and stored at �20°C.

Total nucleoside preparation and LC-MS/MS analysis. Total tRNA of M. jannaschii was denaturedby heating at 100°C for 3 min and then rapidly placed in an ice water slush. The denatured total tRNAwas added to 1/10 volume of 0.1 M ammonium acetate and mixed. Next, 0.1 U of nuclease P1 was addedfor every 1 �g of total tRNA, and the mixture was incubated at 45°C for 2 h. Then, 0.0001 U snake venomphosphodiesterase and 0.003 U alkaline phosphatase were added per �g of tRNA, and this mixture wasincubated at 37°C for 2 h. The resulting sample of hydrolyzed nucleosides was dried in a vacuumconcentrator. The nucleoside digest was reconstituted in mobile phase A (MPA) (5 mM ammoniumacetate, pH 4.5) and separated on a Waters HSS T3 column (100 Å, 1.8 �m, 2.1 by 50 mm). Mass spectraldata were obtained on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer, followingthe chromatographic and mass spectrometry conditions previously reported (23).

RNase digestion and LC-MS/MS analysis. Total tRNA of M. jannaschii was denatured as describedabove. RNase T1, RNase A, RNase U2, RNase MC1, and cusativin were used to digest the total tRNAmixture into smaller oligonucleotides amenable to LC-MS/MS RNA modification mapping. For eachRNase digest, 4 �g of total tRNA was prepared in 1/3 volume of 200 mM ammonium acetate. Fifty unitsof RNase T1 was added for every 1 �g of total tRNA, and the mixture was incubated at 37°C for 2 h. Forevery 1 �g of total tRNA 0.001 U of RNase A was added, and the mixture was incubated at 37°C for30 min. Two micrograms of RNase U2 was added for every 1 �g of total tRNA, and the mixture wasincubated at 65°C for 30 min (87). For every 1 �g of total RNA 1.5 �g of RNase MC1 was added, and themixture was incubated 37°C for 2 h (88). One microgram of cusativin was added for every 1 �g of totaltRNA, and the mixture was incubated at 60°C for 1 h (89).

The RNase digestion products were lyophilized and rehydrated in MPA (200 mM hexafluoroisopro-panol [HFIP], 8.15mM triethylamine [TEA], pH 7.0), and then separated on a Waters XBridge C18 column(3.5 �m, 1 by 150 mm) at a flow rate of 60 �l/min and a temperature of 50°C with a gradient of 5% MPB(50% MPA, 50% [vol/vol] methanol, pH 7.0) to 20% MPB in 5 min, 20% MPB to 95% MPB in 43 min, a holdat 95% MPB for 5 min, and reequilibration of the column for 15 min at 5% B. Mass spectrometry analysiswas conducted using a Thermo LTQ-XL mass spectrometer in negative polarity with a capillary temper-ature of 275°C, spray voltage of 4 kV, capillary voltage of �100 V, and sheath gas, auxiliary gas, andsweep gas at 40, 10, and 10 arbitrary units, respectively. The sample was analyzed over an m/z range from500 to 2,000 for the full scan, followed by four data-dependent acquisition scans.

Data analysis. The tRNA sequences of M. jannaschii were obtained from GtRNAdb (http://gtrnadb.ucsc.edu/) (90). The only isoleucine tRNA listed in GtRNAdb was tRNAGAU

Ile . As for other archaea (12), thetRNACAU

Met sequence in GtRNAdb is most likely the tRNACAUIle sequence and was used for all data interpre-

tation and annotation steps. The complete list of genomic tRNA sequences used for modificationmapping is in Table S3 in the supplemental material.

LC-MS/MS data from RNA modification mapping experiments were analyzed using variable sequenceposition modification of RNAModMapper (91). All the .RAW data files were converted to .MGF files byMSConvert from ProteoWizard, and the mass tolerance of both precursor ion and fragment ion was setto 1 Da. For RNase T1, the 3= end of digestion products was set to phosphate, and the number of missedcleavages was set to 0. For RNase A, RNase U2, RNase MC1, and cusativin, the 3= end of digestionproducts was set to cyclic phosphate, and the number of missed cleavages was set up to 4. The P scoreand dot product score threshold were set to 70 and 0.8, respectively. Only oligonucleotides (RNasedigestion products) having at least 80% of c- and y-type ions identified were used to annotate the tRNAgene sequences. For oligonucleotides (RNase digestion products) found to contain a modified nucleo-side, tRNA sequences were annotated with these data only when the corresponding c- or y-type ionsnecessary to localize the position of this modification in the digestion product were identified. Whenpossible, signature digestion products were used to establish the final localization of modificationswithin the anticodon stem-loop of tRNAs following the previously reported procedure (29–32).

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/JB

.00690-18.SUPPLEMENTAL FILE 1, PDF file, 3.5 MB.

ACKNOWLEDGMENTSFinancial support of this research was provided by the National Institutes of Health

(GM70641 to V.D.C.-L), the LOEWE Center for Synthetic Microbiology (SYNMIKRO) and

M. jannaschii tRNA Modification Profiles Journal of Bacteriology

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the Max Planck Society to L.R., and the National Science Foundation (NSF CHE 1507357to P.A.L.). The generous support of the Rieveschl Eminent Scholar Endowment and theUniversity of Cincinnati for these studies is also appreciated.

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