Iron–sulfur proteins responsible for RNA modifications

BBAMCR-17441; No. of pages: 12; 4C: Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Review

Iron–sulfur proteins responsible for RNA modifications☆ Satoshi Kimura, Tsutomu Suzuki ⁎ Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bldg. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

a r t i c l e

i n f o

Article history: Received 16 September 2014 Received in revised form 8 December 2014 Accepted 9 December 2014 Available online xxxx Keywords: RNA modification Radical SAM enzyme Fe/S cluster

a b s t r a c t RNA molecules are decorated with various chemical modifications, which are introduced post-transcriptionally by RNA-modifying enzymes. These modifications are required for proper RNA function. Among more than 100 known species of RNA modifications, several modified bases in tRNAs and rRNAs are introduced by RNAmodifying enzymes containing iron–sulfur (Fe/S) clusters. Most Fe/S-containing RNA-modifying enzymes contain radical SAM domains that catalyze a variety of chemical reactions, including methylation, methylthiolation, carboxymethylation, tricyclic purine formation, and deazaguanine formation. Lack of these modifications can cause pathological consequences. Here, we review recent studies on the biogenesis and function of RNA modifications mediated by Fe/S proteins. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. © 2014 Elsevier B.V. All rights reserved.

1. Introduction RNA molecules contain various types of qualitative information that cannot be deduced from the genome sequence, including posttranscriptional modifications and editing, as well as terminal chemical structures that are required for proper RNA function. Over 100 different modifications have been identified in RNA molecules across all domains of life [1]. These modifications are chemically diverse, and include methylation, hydroxylation, acetylation, deamination, isomerization, selenylation, reduction, cyclization, and conjugation with amino acids and sugars. Several novel RNA modifications have been reported over the past few years, and as analytical technologies have developed, the chemical space of RNA modification is expanding. In contrast to the limited range of DNA modifications, the wide variety of RNA modifications appears to be a strategy by which RNA molecules acquire a greater variety of cellular functions. Modifications of RNA molecules stabilize tertiary structures, modulate affinity for RNA-binding proteins, regulate decoding of genetic information, and determine the subcellular localization and lifetime of RNAs. However, the exact function and biogenesis of many of these modifications remain to be determined. RNA modifications are introduced by members of various families of RNA-modifying enzymes, including Fe/S proteins (Fig. 1). Most Fe/S proteins involved in RNA modifications (Table 1) have radical S-adenosylmethionine (SAM) domains that produce 5′-deoxyadenosyl (5′dAdo) radicals, and there

☆ This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. ⁎ Corresponding author. Tel.: +81 3 5841 8752; fax: +81 3 5841 0550. E-mail address: [email protected] (T. Suzuki).

enzymes catalyze diverse chemical reactions, including C\C or C\S bond formation [2,3]. Here, we review recent studies on the biogenesis and function of RNA modifications mediated by Fe/S enzymes. 2. Methylation via radical SAM motif In Escherichia coli, 2-methyladenosine (m2A) is present at position 2503 in 23S rRNA (Fig. 2A) [4], as well as at position 37 of a subset of tRNAs (Fig. 2B) [5]. RlmN is a radical SAM-dependent methyltransferase responsible for m2A2503 formation in 23S rRNA [6,7]. The modified residue m2A2503 is located at the entrance of the ribosome exit tunnel, where it regulates translation elongation by interacting with nascent peptides. This modification is involved in resistance to several antibiotics that target the large ribosomal subunit [6], as well as in programmed ribosome stalling for regulated expression of some inducible antibiotic-resistance genes [8]. m2A2503 formation by recombinant RlmN in the presence of SAM has been successfully reconstituted in vitro (Fig. 2C) [7]. As its substrate, RlmN employs naked 23S rRNA, but not the fully assembled large ribosomal subunit, suggesting that m2A2503 formation takes place during ribosome assembly. To introduce m2A2503, RlmN primarily recognizes Helices 90–92 with the adjacent single-stranded RNA. In addition, RlmN is responsible for the synthesis of m2A at position 37 of some tRNAs [9]. RlmN introduces m2A37 on tRNA in vitro in the presence of SAM. Deletion of rlmN results in elevated misreading [9], suggesting that m2A37 plays a role in maintaining translational fidelity. The cfr gene, isolated in Staphylococcus sciuri, confers resistance to several antibiotics, including chloramphenicol, in Staphylococcus spp. and E. coli [10,11]. Like RlmN, Cfr is a radical SAM-dependent methyltransferase that targets A2503 in 23S rRNA, but Cfr catalyzes formation of 8-methyladenosine (m8A) (Fig. 2C) [12]. Although both RlmN and

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Please cite this article as: S. Kimura, T. Suzuki, Iron–sulfur proteins responsible for RNA modifications, Biochim. Biophys. Acta (2014), http:// dx.doi.org/10.1016/j.bbamcr.2014.12.010

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Fig. 1. Pathways of RNA modifications mediated by Fe/S proteins in eukaryotic cells.

Cfr share the same target residue in rRNA, their methylations occur independently. Thus, 2-, 8-dimethyladenosine can be produced at position 2503 by the serial action of the two enzymes. In contrast to general SAM methyltransferases, RlmN and Cfr both possess a radical SAM motif, and radical formation is required for their methylation reactions [7]. SAM is widely used as the methyl group donor for methylation of a substrate via the SN2 displacement reaction catalyzed by a SAM-dependent methyltransferase. In the case of RlmN and Cfr, however, only two of three hydrogens of the methyl group in SAM are transferred to the RNA substrate [13]. The remaining hydrogen of the methyl group is derived from the hydrogen originally bound to the C2 position of the adenine base of the substrate RNA [13]. In other words, RlmN and Cfr employ a unique catalytic mechanism in which the methyl group is generated on the substrate RNA. Several lines of evidence [14–18] suggest a plausible catalytic mechanism for the radical SAM-dependent methyltransfer reaction utilizing two SAM molecules (Fig. 2D). In this model, the conserved Cys residue (Cys355 in E. coli) on RlmN is methylated by SAM via a canonical SN2 displacement reaction. Consistent with this, methylated Cys355 was clearly visible in the crystal structure of apo form of RlmN with SAM [14]. In parallel, the Fe/S cluster on the radical SAM domain catalyzes a reductive cleavage of another SAM molecule to produce a 5′dAdo radical, releasing Met as a by-product. Next, the 5′dAdo radical abstracts the H-atom of the methylated Cys residue, generating a methylene radical. The methylene radical of RlmN adds to the C2 position of the adenine base, forming a covalent RlmN–RNA conjugate. After releasing an electron, deprotonation at the C2 position takes place. The other conserved Cys residue (Cys118 in E. coli) then forms a disulfide bond with Cys355 to release RlmN from the substrate RNA. When Cys118 is substituted with Ala, the RlmN–RNA conjugate can be detected [15,16]. Finally, the deprotonated H-atom is added to the ethylene group to complete the reaction.

3. Methylthiotransferases responsible for the tRNA modification Sulfur atoms are often incorporated in tRNAs as post-transcriptional modifications [19]. A subset of tRNAs that decode Ustarting codons (UNN) and A-starting (ANN) codons harbor 2methylthio-N6-isopentenyladenosine (ms2i6A) and 2-methylthioN6-threonylcarbamoyladenosine (ms2t6A), respectively, at position 37 (Fig. 3A). Because this position is 3′ adjacent to the anticodon, the methylthio group of ms2i6 A37 stacks on the codon–anticodon helix in the decoding center of ribosome [20], facilitating accurate decoding of tRNA. In addition, methylthio modification of ms2i6A37 is engaged in reading-frame maintenance [21] and is required for the attenuation activity of some operons involved in aminoacid biosynthesis [22]. Members of the methylthiotransferases (MTTase) protein family are involved in methylthiolation of RNAs and proteins. MTTases belong to a subset of radical SAM enzymes that contain two [4Fe-4S] clusters [3,23]. RNA MTTases are classified into two types, MiaB and MtaB, which are responsible for ms2i6A and ms2t6A formation, respectively (Fig. 3B) [24–27]. RimO is another MTTase that catalyzes methylthiolation of an Asp residue in ribosome protein S12 [28–30]. The biogenesis of ms2i6A begins with isopentenyl modification of the 6 N amino group of A37, catalyzed by MiaA, to form i6A [31]. Thereafter, a methylthio group is transferred to C2 position of i6A37 by MiaB (Fig. 3B)[24,25]. MiaB then recognizes the N6-isopentenyl group of i6A to introduce the 2-methylthio modification [32]. In the case of ms2t6A formation, t6A is first synthesized by four enzymes (TsaB, TsaC, TsaD, and TsaE) [33], and then methylthiolation of t6A is catalyzed by MtaB (Fig. 3B) [26]. As t6A is further modified to cyclic t6A (ct6A) catalyzed by TcdA in bacteria, fungi, plant and some protists [34], MtaB might be responsible for formation of 2-methylthio-cyclic t6A (ms2ct6A) in organisms in which both TcdA and MtaB are present.

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Table 1 RNA modifications mediated by Fe/S enzymes. Enzyme

Modification

Substrate

Product

Reaction

RNA substrate

Position

Other substrates

Radical SAM

Number of FeS clusters

Human homolog

MiaB MtaB QueE Elp3 RlmN

ms2i6A ms2t6A Q mcm5U m2A

i6A t6 A CPH4 U A

ms2i6A ms2t6A CDG cm5U m2A

Methylthiolation Methylthiolation Ring contraction AcOH addition Methylation

Yes Yes Yes Yes Yes

2 2 1 1 1

CDK5RAP1 CDKAL1 – ELP3 –

m8A s2C yW m5U m5U

A C m1G U U

m8A s2C imG-14 m5U m5U

Methylation Thiolation Imidazoline ring formation Methylation Methylation

37 37 34 34 37 2503 2503 32 37 1939 747

Sulfur, SAM Sulfur, SAMa SAM SAM, acethyl-CoA SAM

Cfr TtcA TYW1 RlmD/RumA RlmC/RumB

tRNA tRNA tRNA tRNA tRNA rRNA rRNA tRNA tRNA rRNA rRNA

SAM Cysb, ATP SAM, pyruvate SAM SAM

Yes No Yes No No

1 1 2 1 1

– – TYW1/RSFAD – –

a The substrates are deduced from MiaB-catalyzed reaction. Although in vitro reconstitution of ms2t6A formation has not been succeeded, the requirements fo these co-factors are estimated by the reaction mechanism of homologous enzyme, MiaB and RimO. b A sulfur atom originates from persulfide of Cys mediated by cysteine desulferase IscS.

The methylthiolation of i6A on tRNA was first reconstituted in vitro for Thermotoga maritima MiaB with [4Fe-4S] clusters in the presence of SAM under anaerobic conditions [32]. MiaB uses two molecules of SAM for methylthiolation. During this reaction, one SAM is converted to SAH, whereas the other is converted to a 5′-dAdo radical. Although radical SAM-dependent methyltransferases contain a single Fe/S cluster, MiaB harbors two Fe/S clusters [23]; the first cluster is chelated by the radical SAM domain, and the second is bound by the N-terminal UFP0004 domain. Similar to the case of RlmN or Cfr, the first cluster in the radical SAM domain is responsible for the generation of a 5′-dAdo radical. The source of the sulfur atom of ms2i6A has not yet been determined. Structural studies of T. maritima MiaB and RimO identified a “pentasulfide bridge” consisting of exogenous sulfurs spanning between the two [4Fe-4S] clusters [35]. Addition of sulfide under strongly reducing conditions facilitates multiple turnover by MiaB without sacrificing Fe/S clusters as the source of sulfur, suggesting that exogenous sulfide is utilized as the sulfur donor for the methylthio group of ms2i6A [35]. Moreover, when MiaB is incubated with SAM without a reducing agent, methanethiol is released from the denatured MiaB, indicating that methyl group of SAM is transferred to an acid-labile sulfur on MiaB [36]. In fact, methylthiol can serve as a substrate of MiaB and be directly incorporated in ms2i6A [35]. Based on these observations, a catalytic mechanism of methylthiolation of ms2i6A has been proposed (Fig. 3C). First, exogenous sulfides are incorporated into the oligomeric sulfur bridge between two Fe/S clusters of MiaB, followed by methyltransfer from SAM to the terminal sulfur of the sulfur oligomer. The 5′dAdo radical produced by the first cluster abstracts the Hatom at the C2 position of i6A37 of the tRNA. Finally, the methylthiol of the oligomeric sulfur bridge is transferred to the activated C2 position to complete the reaction. In this catalytic mechanism, the second cluster plays a role in binding and activating the methylthio moiety [36]. In addition, the second cluster appears to be involved in turnover of the reaction [36]. Although source of such exogenous sulfides has not been determined, hydrogen sulfide generated from endogenous persulfides and polysulfides [37,38] might be the source of sulfur for ms2i6A. 4. Tricyclic base formation of wybutosine mediated by TYW1 Wybutosine (yW) and its derivatives (Fig. 4A) are fluorescent nucleosides found at position 37 of tRNAs from eukaryotes and archaea [39–42]. The base of yW is a tricyclic purine with a bulky side chain. yW and hydroxywybutosine (OHyW) are present in tRNAPhe from yeast and mammals, respectively. Wyosine (imG), isowyosine (imG2), and methylwyosine (mimG) are present in archaeal tRNAs. yW confers conformational rigidity on the anticodon loop of tRNA. On the ribosome, yW stabilizes the codon–anticodon interaction by stacking on the base pair formed by the first letter of the codon and the third letter of the

anticodon, thereby maintaining the reading frame by preventing a frameshift [43,44]. Hypomodification of OHyW occurs in cells infected by human immune deficiency virus (HIV-1) [45]. Because programmed-1 ribosomal frameshifting at the gag-pol junction of HIV1 RNA is a highly conserved recoding event required for active viral expression [46], yW frequency in tRNA might be involved in the regulation of viral infection. The biogenesis of yW and its derivatives has been extensively studied. All the genes responsible for yW formation have been identified in yeast [47–49]. Five enzymes, TRM5 and TYW1–4, participate in the multi-step consecutive reactions involved in yW synthesis (Fig. 4B). yW biogenesis is initiated by 1-methylation of G37 to form m1G37, a reaction catalyzed by TRM5 [50]. TYW1 is the sole Fe/S protein involved in the biosynthetic pathway of yW [47]. Because 4-demethylwyosine (imG-14) accumulates in a TYW2 knockout strain [47,49], TYW1 has been proposed to be involved in the ring formation to synthesize imG14 from m1G. Although the enzymatic activities of yeast and mammalian TYW1 have not yet been directly demonstrated, formation of imG-14 from m1G, catalyzed by the archaeal homolog of TYW1, has been successfully reconstituted in vitro [51]. Archaeal TYW1 catalyzes ring formation of the tricyclic purine base on tRNA by condensation of m1G with two carbons of pyruvate to form imG-14. Next, TYW2 transfers the α-amino-α-carboxypropyl group from SAM to the tricyclic core of imG-14 to form yW-72 [47,52]. TYW3 methylates the N4 position of yW-72 to form yW-58 [47]. Finally, TYW4 catalyzes both methylation and methoxycarbonylation of yW-58 using two SAMs and one bicarbonate to synthesize yW [47,53]. In mammals and several species of fungi, including Aspergillus oryzae, the β-carbon of the side chain in yW is further hydroxylated to form OHyW [54]. An additional enzyme, TYW5, which harbors a Jumonji C domain, catalyzes hydroxylation of yW-72 to form OHyW-72 [54,55]. Subsequently, OHyW-72 is converted to OHyW in a reaction mediated by TYW4 [54]. NFS1 is a cysteine desulfurase required for Fe/S biogenesis [56]. Inhibition of NFS1 expression results in severe reduction of yW [47], indicating that Fe/S clusters play a critical role in yW synthesis. TYW1 contains a canonical radical SAM motif with three conserved Cys residues that potentially coordinate a Fe/S cluster [47]. The functional importance of these Cys residues for yW formation was confirmed by in vivo complementation studies in yeast [47,57]. Structural analysis revealed that archaeal TYW1 harbors two [4Fe-4S] clusters [57,58]. Young and Bandarian [51,59] succeeded in reconstituting formation of imG-14 from m 1 G, catalyzed by Methanococcus jannaschii TYW1, in vitro. Those authors screened several metabolites as the two-carbon source for imG-14 formation, and ultimately identified pyruvate as the substrate for the TYW1-catalyzed reaction. Stable-isotope labeling experiments using [13C] pyruvate revealed that the C2 and C3 carbons of pyruvate are incorporated to imG-14. Based on biochemical and spectroscopic approaches, a catalytic mechanism involving two Fe/S clusters

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Fig. 2. Methylation by RlmN and Cfr. The secondary structures of a part of domain V of E. coli 23S rRNA (A) and tRNAGlu(B) with post-transcriptional modifications; N2-methylguanosine (m2G), pseudouridine (Ψ), 2′-O-methylcytidine (Cm), 5-hydroxycytidine (ho5C), 2-methyladenosine (m2A), 2′-O-methyluridine (Um), 5-methylaminomethyl-2-thiouridine (mnm5s2U), and 5-methyluridine (m5U). C, Biosynthesis scheme of m2A and m2,8A. The reactions mediated by RlmN and Cfr are independent. D, Proposed catalytic mechanism to form m2A catalyzed by RlmN (see text). H-atoms of methyl group from SAM and H-atom of C2 of adenine base are colored in red and blue, respectively.

has been proposed for imG-14 formation (Fig. 4C) [51,60]. The first Fe/S cluster is chelated by three Cys residues in the radical SAM domain, which catalyzes reductive cleavage of SAM to generate 5′-dAdo radical (·Ado). Based on the EPR and Mössbauer data, the second Fe/S cluster is thought to coordinate pyruvate directly [60]. Alternatively, it is speculated

that the pyruvate is fixed through a Schiff base with a conserved essential Lys residue [51,59]. The 5′-dAdo radical produced by the first cluster might directly (or indirectly, through some residues of TYW1) abstract the hydrogen from the methyl group of m1G. This methyl radical then adds to C2 of pyruvate, followed by homolytic scission of the C1–C2

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Fig. 3. Methylthiolaion by MiaB and MtaB. A, Secondary structures of E. coli tRNATrp (left panel) and human tRNALys (right panel) with post-transcriptional modifications; 4-thiouridine (s4U), dihydrouridine (D), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2-methylthio-N6-isopentenyladenosine (ms2i6A), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), 5-methylcytidine (m5C), 5,2′-O-dimethyluridine (m5Um), and 1-methyladenosine (m1A). B, Biosynthesis scheme of ms2i6A and ms2t6A. Modifying enzymes are colored in green. C, Proposed catalytic mechanism to form ms2i6A from i6A catalyzed by MiaB (see text).

bond, elimination of CO2, and release of one electron. Next, the amino group of guanine attacks C3 carbon of pyruvate via nucleophilic addition to form the ring of imG-14, with release of a water molecule.

Further investigations will be required to elucidate the detailed mechanism of the catalytic reaction. In addition, the enzymatic activity of eukaryotic TYW1 has not yet been directly demonstrated. Eukaryotic

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Fig. 4. Tricyclic base formation of wybutosine. A, Secondary structure of S. cerevisiae tRNAPhe (left panel) with post-transcriptional modifications; N2-methylguanosine (m2G), dihydrouridine (D), N2,N2-dimethylguanosine (m22G), 2′-O-methylcytidine (Cm), 2′-O-methylguanosine (Gm), wybutosine (yW), pseudouridine (Ψ),5-methylcytidine (m5C), 7-methyladenosine (m7A), 5-methyluridine (m5U), and 1-methyladenosine (m1A). Chemical structures (right panels) of wybutosine (yW) and hydroxywybutosine (OHyW). B, Biosynthesis scheme of yW. Functional groups added at each step are colored in blue. Modifying enzymes are colored in green. C, Proposed catalytic mechanism to form imG-14 from m1G catalyzed by TYW1 (see text). C2 and C3 of pyruvate are shown in red.

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TYW1 contains a N-terminal flavotoxin domain in addition to the Cterminal radical SAM domain [47]. An additional factor, such as FMN, might be required for the reaction catalyzed by eukaryotic TYW1. 5. The Elongator complex is essential for tRNA wobble modifications Several eukaryotic tRNAs responsible for NNA codons bear 5-methoxycarbonylmethyluridine (mcm 5 U) and its derivatives, including 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5 Um), 5carbamoylmethyluridine (ncm5 U), 5-carbamoylmethyl-2'-Omethyluridine (ncm 5 Um), and 5-methoxycarbonylhydroxymethyluridine (mchm5U) at the wobble position (Fig. 5A)[61–63]. The Elongator complex (Elp) associated with actively transcribing RNA polymerase II [64] is responsible for this wobble modification [65]. Elongator is composed of six components, ELP1–6 [66,67]; ELP3 is a Fe/S protein that has been reported to be a histone acetyltransferase [68]. Deletion of each component of the complex elicits pleiotropic phenotypes, including slow growth, temperature sensitivity [66,69, 70], reduced histone acetylation [67], defects in exocytosis [71], elevated sensitivity to DNA damage [72], defects in telomeric gene silencing [72], deficient demethylation of the paternal genome [73], impairment of migration and differentiation by cortical neurons [74], and reduced resistance to the Kluyveromyces lactis killer toxin [69]. Intriguingly, the defects in transcriptional elongation, exocytosis, telomeric gene silencing, and DNA damage response observed in the Elongator mutants are suppressed by overexpression of tRNAs that contain mcm5s2U at the wobble position in the wild-type background [75,76]. Moreover, deletion of tuc2 (ncs2), which is responsible for 2-thiouridine formation of mcm5s2U, results in phenotypes similar to those observed in the Elongator mutants [75,76]. These observations suggest that the telomeric gene silencing and DNA-damage response mediated by the Elongator complex can be explained by the function of tRNA wobble modifications, which might be required for accurate and efficient expression of gene products involved in those processes. The lack of 5-methoxycarbonylmethyl modification of mcm5s2U in tRNAs could influence the decoding ability of their respective cognate codons. In Elongator mutants of S. cerevisiae, in vivo ribosome distribution analysis revealed a clear accumulation of ribosomes at AAA, GAA, and CAA codons, indicating that these codons were translated less efficiently by hypomodified tRNAs [77]. Telomeric gene silencing of S. cerevisiae is mediated by Sir4 function. Elongator mutants express lower levels of Sir4, which contains abundant AAA codons, due to the accumulation of hypomodified tRNALys lacking the mcm5 modification [75,76]. Lys is specified by both AAA and AAG codons; however, AAG codons are redundantly deciphered by a tRNALys with the mcm5s2UUU anticodon and another with the CUU anticodon. By contrast, AAA codons are deciphered only by tRNALys with the mcm5s2UUU anticodon. Thus, lack of the mcm5 modification could cause a severe reduction in AAA decoding. In fission yeast, Cdr2, a protein kinase required for cell cycle progression, is translated less efficiently in the elp3 mutant, due to the abundant usage of Lys codons in the Cdr2 gene [78]. Intriguingly, synonymous alteration of Lys codons from AAA to AAG rescues expression of the Cdr2 protein. A similar mechanism may be involved in the expression of Atf1 and Pcr, which are transcription factors induced by oxidative stress [79]. These findings provide an attractive mechanism by which tRNA wobble modifications mediated by the Elongator complex might regulate translational efficiency of target mRNAs as a function of their codon usage, thereby making a fundamental contribution to cellular functions. Among the components of the Elongator complex, only Elp3 harbors enzymatic domains, including a radical SAM domain and histone acetyltransferase (HAT) domain. Genetic mutations of these domains fail to rescue the phenotypes observed in the Elp3 deletion strain [76], suggesting that both enzymatic activities are necessary for Elp3 function. In all Elongator components, only Elp3 is widely distributed in

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archaeal species. Recently, Selvadurai and colleagues [80] succeeded in reconstituting formation of 5-carboxymethyluridine (cm5U) (Fig. 5B) in tRNA by Methanocaldococcus infernus Elp3 protein (MinElp3) in the presence of Na2S2O4, SAM, and acetyl-CoA under reducing conditions. In this reaction, Elp3 transfers the carboxymethyl group of acetyl-CoA to the C5 position of a uracil base (U34) at the wobble position of the tRNA. This experiment clearly demonstrated that Elp3 is the sole component of the Elongator complex that generates a tRNA wobble modification. Elp3 contains the CxxxxCxxC motif in its radical SAM domain, and [4Fe-4S] cluster formation is necessary for catalysis in vitro. The reconstituted reaction was accompanied by the release of 5′-dAdo and CoA as by-products, indicating that Elp3 generates the 5′-dAdo radical and hydrolyzes the thioester of acetyl-CoA. On the basis of biochemical analyses, a mechanism for the Elp3-catalyzed reaction has been proposed (Fig. 5B). In this model, after the association of Elp3 with tRNA, the radical SAM domain catalyzes the reductive cleavage of SAM to generate the 5′-dAdo radical. The 5′-dAdo radical then abstracts the H-atom of the methyl group of acetyl-CoA bound to the HAT domain, thereby generating acetyl radical, which in turn adds to the C5 carbon of U34 to form a C\C bond. Loss of an electron and the abstraction of the proton at C5 results in the formation of tRNA-acetyl-CoA conjugate, and hydrolysis of this conjugate produces cm5U 34 on tRNA. In S. cerevisiae, cm5U is not a final modification in tRNA [81], and might represent an intermediate in the synthesis of mcm5U and ncm5U. cm5U is further methylated to form mcm5U by the SAM-dependent methyltransferase Trm9p [82]. To form ncm5U, ammonium might attack the carbonyl carbon of the thioester of the tRNA-acetyl-CoA conjugate; however, this has not yet been experimentally verified. The proposed reaction scheme suggests that both radical SAM and HAT domains work coordinately to synthesize the wobble modification. Structural studies will reveal how Elp3 recognizes tRNA, and how the two domains catalyze the composed chemical reaction. In eukaryotes, other components of Elongator complex are essential for tRNA modifications [65]. The Elp4-5-6 subcomplex, which adopts a hexameric RecA-like ATPase structure, is involved in tRNA binding [83], indicating that eukaryotic wobble uridine formation occurs via a mechanism more complex than that of the archaeal system, even though the main catalytic function of Elp3 is likely to be conserved. In addition, many regulatory factors are present in eukaryotic Elongator complex [65,84]. Further analyses are necessary for a deeper understanding of the function and regulation of Elongator complex in eukaryotes. 6. Deazapurine formation by QueE Queuosine (Q) is a guanosine derivative with a 7-deazapurine core structure found at the wobble position of tRNAs responsible for NAY codons: tRNAs for Tyr, His, Asp and Asn (Fig. 6A) [85,86]. Q plays a role in maintaining translational accuracy [45]. As Q is also required for the virulence of Shigella flexneri [87,88], biogenesis of Q is a pharmaceutical target for antibacterial inhibitors [89]. Although Q and its derivatives are present in tRNAs of most eukaryotes and bacteria [86], Q is only synthesized in bacteria, and eukaryotes lack the Q biosynthetic pathway [90]. Eukaryotes acquire queuine, which is a free base of Q, as a dietary nutrient and/or from microflora. In bacteria, preQ1, a precursor of Q, is synthesized from GTP though multi-step reactions mediated by a series of enzymes, including GCHI, QueD, QueE, QueC, and QueF (Fig. 6B) [2]. Thus synthesized preQ1 is incorporated to tRNA by replacing the guanine base at position 34 via transglycosylation catalyzed by tRNAguanine transglycosylase (TGT) [91]. In eukaryotes, eukaryotic TGT (eTGT) incorporates the free Q-base taken from diet or microflora to tRNA via transglycosylation reaction. In bacterial Q biogenesis, QueE is a sole radical SAM enzyme which catalyzes a ring contraction reaction to generate the 7-deazapurine core structure of Q [2,92]. QueE catalyzes conversion of 6-carnoxy5,6,7,8-tetrahydropterin (CPH4) to 7-carboxy-7-deazaguanine (CDG) in the presence of SAM and Mg2 +. Based on structural studies of

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Fig. 5. Carboxymethylation by ELP3. A, 5-Methoxycarbonylmethyluridine (mcm5U) and its derivatives are found at the wobble position (position 34) of eukaryotic tRNAs (left panel). Chemical structures of mcm5U, mcm5s2U, mcm5Um, mchm5U, ncm5U and ncm5Um are shown in the right panels. B. Proposed catalytic mechanism to form 5-carboxymethyluridine (cm5U) catalyzed by archaeral Elp3.

Burkholderia multivorans QueE in complex with SAM and several substrates of pre- and post-reaction states [93], a detailed mechanism has been proposed for ring compaction catalyzed by QueE (Fig. 6C). 5′dAdo radical, produced by reductive cleavage of SAM, may abstract the H-atom of C6 of CPD4; Mg2+ coordinates with CPD4 and may facilitate this reaction. Generation of the five-membered pyrrole ring could occur via several distinct routes. Next, the amino-centered radical abstracts the H-atom of dAdo to regenerate the 5′-dAdo radical, which can be used for the next round reaction. Finally, CDG is formed by the elimination of the amino group. 7. 2-Thiocytidine formation of tRNA by TtcA In tRNAs from E. coli and other bacteria, there are four species of sulfur-containing modifications; 4-thiouridine (s4U) at position 8, 2thiouridine (s2C) at position 32, 5-methylaminomethyl-2-thiouridine (mnm5s2U) at position 34, and ms2i6A at position 37 [19]. In addition to ms2i6A, described above, s2C32 is also synthesized via a Fe/Sdependent pathway, whereas s4U8 and mnm5s2U34 are synthesized independently of Fe/S formation [94]. TtcA (two-thio-cytidine), which is responsible for s2C32 formation [95], contains a PP-loop motif in its Nterminal region and two CXXC motifs. Recently, TtcA was found to contain [4Fe-4S] cluster chelated by three essential Cys residues [96]. Moreover, formation of s2C32 on tRNA by TtcA and IscS has been successfully reconstituted in the presence of ATP and L-Cys [96]. Because the PP-loop is a motif of the N-type ATP pyrophosphatase involved in the formation of adenylate intermediate of the substrate [97], TtcA probably adenylates the C2 carbonyl oxygen using ATP. Subsequently, the

persulfide sulfur from Cys is transferred to the C2 carbon, concomitant with the release of AMP. However, no Cys residue in TtcA that forms persulfide with a sulfur donor has been identified. While, unliganded iron in Fe/S cluster is supposed to serve as the acceptor of the sulfur atom, in the similar manner in which the second cluster of RimO binds to the free sulfur atom. 8. Fe/S cluster involved in RNA recognition and stabilization of protein folding As described above, Fe/S cluster plays a critical role in various enzymatic reactions involving electron transfer. However, in some cases, Fe/ S clusters are involved in other processes, including substrate recognition and stabilization of protein folding. Three methyltransferases responsible for 5-methyluridine (m5U) are present in E. coli; TrmA/RumT [98] for m5U54 in tRNAs, RlmC/RumB for m5U747 in 23S rRNA, and RlmD/RumA for m5U1939 in 23S rRNA [99, 100]. Although all of these enzymes share the similar amino acid sequence, only RlmC/RumB and RlmD/RumA harbor Fe/S cluster binding motif [101]. Structural analysis of RlmD/RumA complexed with rRNA fragment and SAM revealed that the Fe/S cluster is not responsible for the catalytic reaction, but involved in the substrate rRNA binding [102,103]. One sulfur atom of the Fe/S cluster is exposed to the solvent, and interacts with the uracil base of U1940 mediated by two water molecules. This observation verified that the Fe/S cluster of RlmD/RumA is involved in the RNA binding. Tryptophanyl-tRNA synthetase (TrpRS) containing Fe/S cluster is another example for the Fe/S cluster to be involved in RNA recognition [104,105]. In Bacillus subtillis, RlmC/RumB

Please cite this article as: S. Kimura, T. Suzuki, Iron–sulfur proteins responsible for RNA modifications, Biochim. Biophys. Acta (2014), http:// dx.doi.org/10.1016/j.bbamcr.2014.12.010

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Fig. 6. Ring compaction to form 7-deazapurine. A, Queuosine (Q) is present at the wobble position (position 34) of tRNAs with QUN anticodons (left panel) and its chemical structure (right panel). B, Biosynthesis scheme of Q. Intermediates are 7,8-dihydroneopterin triphosphate (H2NTP), 6-carboxy-5,6,7,8-tetrahydropterin (CPH4), 7-carboxydeazaguanine (CDG), 7-cyano7-deazaguanine (preQ0), 7-aminomethyl-7-deazaguanine (preQ1), and epoxyqueuosine (oQ). Modifying enzymes are colored in green. C, Proposed catalytic mechanism to form CDG from CPH4 catalyzed by QueE. H-atom at C6 of CPH4 is colored in red.

and RlmD/RumA are fused to form a single methyltransferase, called RlmCD, which catalyzes the formation of both m5U747 and m5U1939 in 23S rRNA [106]. As RlmCD harbors the Fe/S cluster binding motif, RlmCD is likely to use the Fe/S cluster for rRNA recognition. The single mutation of one of Cys residues which chelate the Fe/S cluster resulted in decreased solubility of RlmD/RumA [102]. In addition, decomposition of the Fe/S cluster by oxidation precipitated RlmD/RumA [101]. These findings clearly indicate that the Fe/S cluster of these methyltransferases is required for protein stability or folding. 9. Alteration of RNA modification under environmental conditions Fe/S clusters easily react with reactive oxygen species (ROS), resulting in the loss of ferric ions, which fuel the Fenton reaction to produce more ROS [107]. Hence, RNA modifications mediated by Fe/S

cluster enzymes are sensitive to oxidative stresses. In addition, the cellular concentration of Fe2+ is another factor that modulates the activity of Fe/S cluster enzymes. In fact, 2-methylthio modification of ms2i6A in tRNAs from E. coli, Staphylococcus typhimurium, and Pseudomonas aeruginosa is decreased upon iron depletion [108]. Because animals harbor various iron-chelating proteins, including transferrin and lactoferrin, the levels of ms2i6A or other Fe/S-dependent modifications can be altered when pathogenic bacteria infect host cells. Indeed, lack of 2-methylthio modification of ms2i6A has been observed in E. coli injected into host animals [109], suggesting that pathogenic bacteria can regulate the levels of RNA modifications by sensing the iron concentration in host cells. Considering that translational efficiency and accuracy can be controlled by tRNA modifications, altered frequency of tRNA modification would affect decoding properties of tRNA, leading to a global alteration of the proteome. Consistent with this idea, loss

Please cite this article as: S. Kimura, T. Suzuki, Iron–sulfur proteins responsible for RNA modifications, Biochim. Biophys. Acta (2014), http:// dx.doi.org/10.1016/j.bbamcr.2014.12.010

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of 2-methylthio modification of ms2i6A relieves attenuation of operons involved in aminoacid biogenesis [22], suggesting that iron depletion can impinge on aminoacid metabolism.

Sports, and Culture of Japan (to T. S.). We apologize to those of our colleagues whose many contributions related to Fe/S cluster in RNA modifications could not be cited in this review due to space limitations.

10. Human diseases associated with Fe/S-dependent RNA modifications

References

Lack of RNA modification can cause functional defects of RNA, leading to the molecular pathogenesis of various diseases. Our group previously reported that the lack of tRNA modification is a primary cause of mitochondrial encephalomyopathic diseases [110, 111]. These findings provided the first confirmation of a human disease caused by an RNA modification disorder. In addition, large-scale disease-associated exome analyses have identified numerous genes encoding tRNA/rRNA-modifying enzymes [112]. More than 200 million people worldwide suffer from Type 2 diabetes (T2D), which is characterized by deficient insulin secretion from pancreatic β-cells and insulin resistance. Whole-genome association studies reveal that mutations in the CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1) (Table 1) are one of the most reproducible risk factors for T2D [113,114]. CDKAL1 is the human homolog of bacterial MtaB, which is responsible for 2-methylthio modification of ms2t6A at position 37 of tRNALys [26]. Knockout of CDKAL1 results in complete loss of 2-methylthio modification of ms2t6A [115]. Furthremore, β-cell-specific CDKAL1 knockout mice exhibit pancreatic islet hypertrophy and impaired blood glucose control. These mice are also hypersensitive to the high-fat diet–induced endoplasmic reticulum (ER) stress response. These observations suggest that mistranslation induced by hypomodified tRNALys could produce aberrant proteins, including proinsulin, in β-cells. Mutations in components of the Elongator complex are associated with some neurological disorders. Allelic variants of ELP3 detected in three human populations have been associated with amyotrophic lateral sclerosis (ALS) [116]. In flies, a mutagenesis screen for genes required for neuronal function and survival identified two different loss-offunction mutations in ELP3. In addition, knockdown of Elp3 in zebrafish resulted in motor axonal abnormalities [116]. Other components of the Elongator complex are also associated with neurological disorders. Pathogenic mutations in human ELP1 (IKAP/IKBAP) are associated with familial dysautonomia (FD) [117–119], and ELP1 has also been associated with bronchial asthma in children [120]. On the other hand, ELP4 gene has been associated with Rolandic epilepsy [121]. 11. Conclusion and perspective Recent studies provide mechanistic insights into biogenesis of several RNA modifications mediated by radical SAM enzymes. 5′dAdo radical generated by Fe/S cluster initiates complex chemistries including methylation, methylthiolation, carboxymethylation, tricyclic purine formation, and deazaguanine formation. Some radical SAM enzymes have second Fe/S cluster required for binding of substrate and intermediate. In methylation and methylthiolation, radical SAM enzymes employ two molecules of SAM to catalyze two different reactions; methylation and 5′ dAdo radical formation. Radical SAM enzymes would have evolved to catalyze various types of reactions to expand chemical diversities of RNA modifications. Since Fe/S cluster is sensitive to ROS, and RNA modifications can be regulated by iron concentration, it is plausible to speculate that radical SAM enzymes play a regulatory role in protein synthesis by sensing cellular metabolic status. Acknowledgment We are grateful to the members of the Suzuki laboratory for many insightful discussions. This work was supported by a JSPS Fellowship for Japanese Junior Scientists (to S.K.), and by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education Science,

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