Post-transcriptional Modifications Modulate rRNA Structure and

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Post-transcriptional Modifications Modulate rRNA Structure and Ligand Interactions Jun Jiang, Hyosuk Seo, and Christine S. Chow* Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States

CONSPECTUS: Post-transcriptional modifications play important roles in modulating the functions of RNA species. The presence of modifications in RNA may directly alter its interactions with binding partners or cause structural changes that indirectly affect ligand recognition. Given the rapidly growing list of modifications identified in noncoding and mRNAs associated with human disease, as well as the dynamic control over modifications involved in various physiological processes, it is imperative to understand RNA structural modulation by these modifications. Among the RNA species, rRNAs provide numerous examples of modification types located in differing sequence and structural contexts. In addition, the modified rRNA motifs participate in a wide variety of ligand interactions, including those with RNA, protein, and small molecules. In fact, several classes of antibiotics exert their effects on protein synthesis by binding to functionally important and highly modified regions of the rRNAs. These RNA regions often display conservation in sequence, secondary structure, tertiary interactions, and modifications, trademarks of ideal drug-targeting sites. Furthermore, ligand interactions with such regions often favor certain modification-induced conformational states of the RNA. Our laboratory has employed a combination of biophysical methods such as nuclear magnetic resonance spectroscopy (NMR), circular dichroism, and UV melting to study rRNA modifications in functionally important motifs, including helix 31 (h31) and helix h44 (h44) of the small subunit rRNA and helix 69 (H69) of the large subunit rRNA. The modified RNA oligonucleotides used in these studies were generated by solid-phase synthesis with a variety of phosphoramidite chemistries. The natural modifications were shown to impact thermal stability, dynamic behavior, and tertiary structures of the RNAs, with additive or cooperative effects occurring with multiple, clustered modifications. Taking advantage of the structural diversity offered by specific modifications in the chosen rRNA motifs, phage display was used to select peptides that bind with moderate (low micromolar) affinity and selectivity to modified h31, h44, and H69. Interactions between peptide ligands and RNAs were monitored by biophysical methods, including electrospray ionization mass spectrometry (ESI-MS), NMR, and surface plasmon resonance (SPR). The peptides compare well with natural compounds such as aminoglycosides in their binding affinities to the modified rRNA constructs. Some candidates were shown to exhibit specificity toward different modification states of the rRNA motifs. The selected peptides may be further optimized for improved RNA targeting or used in screening assays for new drug candidates. In this Account, we hope to stimulate interest in bioorganic and biophysical approaches, which may be used to deepen our understanding of other functionally important, naturally modified RNAs beyond the rRNAs.

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INTRODUCTION Post-transcriptional modifications play important roles in regulating the functions of RNA, including mRNA1 and noncoding2 RNAs (ncRNA). Modifications such as methylation of rRNA in bacteria are closely associated with antibiotic resistance,3−5 and dynamic methylations (m6A) of mRNA are correlated with key biological roles such as human development.6 Methylation can occur on the nucleoside base (e.g., m6A, m3U, m5C, and m2G) or ribose (e.g., Cm) (Figure 1).2,7,8 © XXXX American Chemical Society

Post-transcriptional modification may also involve changes at the glycosidic bond, such as conversion of uridine to pseudouridine (Ψ) (Figure 1), or multiple modifications to a single nucleoside (e.g., m4Cm and m1acp3Ψ) (Figure 1).2 High-resolution (e.g., X-ray) structures of ribosomes reveal key structural features and chemical interactions involving Received: January 11, 2016

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DOI: 10.1021/acs.accounts.6b00014 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

locations is generally the first priority. Traditionally, modified nucleosides in RNA are identified by chromatography, electrophoresis, chemical derivatization, and mass spectrometry with enzyme-digested samples.11,12 Advances in mass spectrometry combined with other mapping tools have allowed both the types and locations of modified nucleosides to be identified in very large RNAs such as 16S and 23S rRNAs.13 With recent advances in high-throughput sequencing, populations of certain modifications (e.g., methylation or Ψ) in low-abundance ncRNA species such as tRNA fragments (tRFs)14 can also be rapidly identified. Because of these improved methods, identification of modifications in mRNA and ncRNA has outpaced structural and mechanistic studies to understand their biological roles. Synthesis, biophysical, and structure studies to elucidate the roles of modified nucleosides in rRNAs as well as other ncRNAs may help reveal redundant strategies such as enhanced chemical stability, specific intermolecular interactions, or structural motifs that can further our understanding of the biological functions.

Figure 1. Structures of representative modified nucleosides. Base methylation in N6-methyladenosine (m6A), N3-methyluridine (m3U), C5-methylcytidine (m5C), and N2-methylguanosine (m2G), ribose methylation in 2′-O-methylcytidine (Cm), the C-glycoside in pseudouridine (Ψ), and multiple modifications in N4,2′-O-dimethylcytidine (m4Cm) and N1-methyl-N3-(3-amino-3-carboxypropyl)pseudouridine (m1acp3Ψ) are shown (modifications in bold).

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RIBOSOMAL RNA MODIFICATIONS Ribosomal RNAs provide the structural framework of ribosomes and play essential roles in translation.15−17 The 70S bacterial ribosome contains two highly modified RNAs, 16S rRNA in the small subunit and 23S rRNA in the large subunit. The sites and numbers of post-transcriptional modifications in rRNA vary across species, with 36 identified in Escherichia coli ribosomes (Figure 2).8 These modifications are located predominantly at the interface between the two subunits and tRNA binding sites, involving two functionally important regions known as the peptidyltransferase center (PTC) and decoding region.9,10 Since discovery of the first modified nucleoside (Ψ), numerous studies have focused on the rRNA modifying enzymes and phenotypes associated with their loss.8 The 13 modifications of domain V in E. coli 23S rRNA are located in the peptidyltransferase loop, P- and A-tRNA interacting loops,

modified nucleotides in rRNA,9,10 whereas solution-phase biophysical studies provide thermodynamic, kinetic, and structural information for RNA conformational changes or ligand interactions influenced by modifications. This Account focuses on the thermodynamic and structural effects, as well as ligand binding interactions of modified rRNA motifs. The established methodologies and knowledge gained in these studies with rRNAs can be applied to understand the roles of newly discovered modifications in an even wider variety of RNAs beyond the ribosome. Modifications appear to play important biological roles such as regulation of cellular activities, ligand binding, and antibiotic resistance, and their reversibility enables epitranscriptomic regulation of gene expression.1 Therefore, mapping their

Figure 2. Modifications in E. coli ribosomes. The A-, P-, and E-site tRNAs, in green, cyan, and purple, respectively, are located between the small (light green) and large (pink) ribosomal subunits. The anticodon loops of the A- and P-site tRNAs make direct contact with mRNA (gray). The rRNA modifications are indicated (various colors) in space-filling mode (image prepared using PDB 4V9D and PyMOL).8,18 Modification sites that are conserved between E. coli and human are shown in solid colors with residue numbering,19 and other modifications are indicated with transparent colors. B


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The use of small constructs allows investigation of the thermodynamic contributions and roles in structural modulation by site-specific modifications. Such thermodynamic information is obtained in solution and complements highresolution structure studies. The modifications in h31, h44, and H69 are highly representative of those in other species, including conserved methylated residues in h31 and h44 and Ψ residues in H69. To obtain modified RNAs, the phosphoramidite method was employed for solid-phase synthesis (summarized in Figure 4).28−30 Chemical synthesis allows for highly controlled, site-specific incorporation of varying numbers and types of modifications as well as non-natural analogues into RNA. Therefore, individual or cooperative effects of modifications on RNA structure or stability can be determined. The major techniques employed in our work include UV melting, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy,28−31 and the results were used to support data obtained in the context of complete ribosomes. The functionally important h31 (Figure 5) resides in the 3′ major domain of the small subunit rRNA. Base methylations at G966 and C967 are conserved in bacteria, and U966 (E. coli numbering) in yeast and human is hypermodified to m1acp3Ψ (Figure 1 and 5A).2 By using our combined synthetic and biophysical approach, we showed that methylations at G966 and C967 in E. coli h31 thermodynamically destabilize the RNA hairpin when both are present.28 In crystal structures of the small subunits, three residues in the h31 loop region, m2G966m5C967-A968 in E. coli (Figure 5B) and m1acp3Ψ1191-C1192A1193 in yeast,9,32 form a continuous base-stacking motif. A similar motif was also observed in an NMR structure of the modified h31 construct, but not in the corresponding unmodified h31.23 In the modified h31, G971 is extended outward from the helix.9 This structural feature is missing in the unmodified h31, suggesting that stacking is enhanced by base modification and modulates the conformation of G971. Extensive interactions between G971 and h30/h43 may also contribute to the h31 loop conformation and further modulate its interactions with the P-tRNA.9 Another key modified helix in the small subunit rRNA is h44 (Figure 6). The A/P site of h44 hosts key nucleotides responsible for translational fidelity.35 In E. coli, there are three conserved modifications, m4Cm1402, m5C1407, and m3U1498 (Figure 6A).27 In Thermus thermophilus, C1400 and C1404 are also methylated (Figure 6A).27 Thermal melting experiments on our synthetic constructs revealed that ° of −1.3 dimethylation at m4Cm1402 stabilizes h44 (ΔΔG37 kcal/mol when E. coli h44 duplexes with either C1402 or m4Cm1402 are compared).30 With Cm1402, the ΔΔG°37 value decreased to −0.6 kcal/mol, suggesting that the methylations of m4Cm1402 have additive effects in modulating RNA folding. The 2′-O-methylation favors a C3′-endo sugar pucker, which helps orient the Watson−Crick edge of m4Cm1402 toward A1500, while N4-methylation contributes to enhanced base stacking.10,36 In the context of bacterial ribosomes, N4-methylation may assist in interactions between the base moiety of m4Cm1402 and the mRNA codon interacting with the P-tRNA.10 The neighboring m3U1498 together with C1403 and A1499 forms a triple-base platform.10 Methylation at m3U1498 alone exhibits no stabilizing effects on h44 but modulates the impact of m4Cm1402, indicating a cooperative effect of the two modified residues.30 Methylation of U1498 blocks hydrogen-bonding interactions involving N3, which may play a role in organizing

and peripheral regions of the PTC. Residues Cm2498 and m2A2503, which reside between helices 89 and 90 (H89 and H90; note that helices in the small and large subunit rRNAs are denoted with a lowercase h or uppercase H, respectively) in the peptidyltransferase loop within 15 Å of the A- and P-tRNA 3′ termini,9,10 are of interest because they provide a growth advantage in E. coli in competition with methylation-deficient strains.8 Outside of the PTC, several modified nucleosides are of interest because they interact either directly or indirectly with translation factors. For example, h31, which contains m2G966 and m5C967, interacts with initiation factor 3 (IF3) in bacteria.20 Similarly, h31 is positioned within 10 Å of the Nterminus of eukaryotic initiation factor 1α (eIF1A) in the initiation complex of Lachancea kluyveri.21 A continuous basestacking motif consisting of residues 966-967-968 (E. coli numbering) is observed in L. kluyveri and E. coli ribosomes.18 Both m2G966 and m5C967 play roles in proper translation initiation,22 possibly by modulating the h31 loop structure.23 Out of 11 conserved modification sites between E. coli and human rRNAs, seven are clustered in the intersubunit bridge regions. Bridges B2a and B2b are established by extensive interactions between H69 in the large subunit and h44 in the small subunit.24 In E. coli H69, Ψ occurs at residues 1911, 1915, and 1917,25 and Ψ1915 is N3-methylated.26 As with PTC modifications, the highly conserved Ψ’s also provide growth advantages to bacteria.25 In h44, methylation occurs at N3 of U1498, C5 of C1407, and N4 and 2′-O of C1402.27 Several other modified nucleotides are located nearby, namely, m62A1518, m62A1519, and m7G527.10 In this case, a more defined biological role has been identified in which N4methylation of C1402 is important for controlling non-AUG initiation and read-through of the UGA stop codon.27 Given the key locations (Figure 3) and primary importance of h31,

Figure 3. Locations of h31, h44, and H69 in the ribosomal subunit interface. Direct contacts occur between h31 and P-tRNA with AtRNA in the vicinity. Helix 44 plays a role in decoding by monitoring A-tRNA anticodon and mRNA base pairing. Helix 69 interacts simultaneously with the A- and P-tRNAs.10

h44, and H69 in ribosomal function, our laboratory has focused on in vitro biophysical studies with these helices. More specifically, studies were done with synthetic RNA models to elucidate the structural and stabilizing roles of the individual as well as combined modifications.

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MODIFICATIONS IN h31, h44, AND H69 Our laboratory has determined the biophysical effects of RNA modifications by using synthetic models of h31, h44, and H69. C


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Figure 4. General scheme for solid-phase RNA synthesis. B and Bx represent unmodified and modified bases, respectively, PG1−PG4 are various protective groups (ACE = bis(2-acetoxyethoxy)methyl; TOM = triisopropylsilyloxymethyl; DMT = dimethoxytrityl; BzH = benzhydryloxybis(trimethylsilyloxy)silyl; CNEt = cyanoethyl; Me = methyl; DPC = diphenylcarbamoyl; Ac = acetyl), and S is the solid support (CPG = controlled pore glass; PS = polystyrene).

Figure 6. Secondary structures, modifications, and drug interactions of h44. (A) The secondary structures of h44 from E. coli and T. thermophilus are shown. Modified nucleotide residues are highlighted (red). (B) In the ribosome crystal structure,33 modified bases of h44 are in red. (C) Helix 44 is shown to participate in interactions with streptomycin (PDB ID 4DR3)37 and amicoumacin A (PDB ID 4W2F).38 Modified nucleotide residues important for antibiotic binding are highlighted (red).

Figure 5. Secondary structures, modifications, and drug interactions of h31. (A) The secondary structures of h31 from E. coli and yeast are shown. Modified nucleotide residues are highlighted (red). (B) In the ribosome crystal structure,33 a base-stacking motif with m2G966m5C967-A968 is observed in h31 (modified residues in red). (C) In the translation initiation complex from yeast (PDB ID 4UER),21 eIF1A resides close to h31. In the bacterial ribosome,33 h31 makes direct contacts with the P-tRNA. The tetracycline binding site overlaps with the A-tRNA (PDB ID 4V9A).34

for modulation of tertiary structure and dynamic behavior.31 Helix 69 participates in almost every step of translation and establishes direct contacts with multiple translation factors.39 This helix was also identified as an antibiotic binding site.40,41 With synthetic 19-mer RNA constructs of H69, the contribution of each residue to global thermodynamic stability was determined,29 allowing for a deeper understanding of the effects of mutations or loss of modifications in H69.31,42 The differential contributions of each Ψ to the global thermodynamic stability of E. coli H69 were quantified using three separate synthetic RNA constructs (Figure 7A).29 Residue Ψ1911 is stabilizing, whereas Ψ1915 and Ψ1917 are destabilizing.29 In human H69, Ψ residues 3731 and 3733 (1915 and 1917 in E. coli) are less destabilizing than their

the triple-base platform. Similarly, methylation of m5C1407 in combination with m4Cm1402 destabilizes the helix, suggesting long-range crosstalk between m4Cm1402 and m5C1407.30 In ribosome crystal structures, m5C1407 interacts with A1919 of H69 in the large subunit and is located next to A1492 and A1493 of h44 (Figure 6B), suggesting a role in translational fidelity.33 The relatively small changes in global stability of the modified versus unmodified h44 duplexes from E. coli and T. thermophilus suggest a role for methylation in fine-tuning the RNA structure, including long-range effects in the ribosome.30 Across the subunit interface lies H69 (Figure 7), whose highly conserved sequence and Ψ modifications are important D


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neighboring residue, A1913, is subjected to rather dramatic conformational changes upon formation of the intersubunit bridge B2a (Figure 7C),9,43 making direct contacts with A1493 of h44. Given the essential roles of A1492 and A1493 in decoding,35 it is not surprising that mutations or changes in the H69 modification status affect translational fidelity.48 The constructs for h31, h44, and H69 are good representations of modified RNAs because they contain a range as well as the most abundant types of modifications (methylations and Ψ). Furthermore, these modifications are located within different secondary structure environments (e.g., hairpin loop, stem duplex, base mismatches, and internal loops) and favor the formation of base-stacking arrangements, which in turn enhance RNA folding. Conversely, base-stacking motifs are observed to be energetically unfavorable within certain single-stranded RNA structures due to penalties in loop closures (h31 and H69). Such destabilizing effects may play roles in modulating local RNA conformation and dynamic behavior, thus enabling the flexibility needed for carrying out biological functions. We hope to stimulate interest in using this combined synthesis and biophysical approach to obtain thermodynamic and solution structure information that can complement high-resolution structure studies and biological assays.

Figure 7. Secondary structures, modifications, and drug interactions of H69. (A) The secondary structures of H69 from E. coli and T. thermophilus are shown. Modified nucleotide residues are highlighted (red). (B) In the NMR solution structure of H69 (PDB ID 2MER),31 loop residues (Ψ1915 to A1919) that participate in a continuous basestacking motif are shown (ball-and-stick). Ψ1911 and A1919 form a canonical Watson−Crick base pair. The base of C1920, which is modified to Cm in T. thermophilus, is highlighted (black). (C) The conformational switching of A1913 (top panel, PDB IDs 1NKW and 2WRO)33,43 in H69 is important for subunit association. The Ψ1911− A1919 pair is stabilized by a water-mediated hydrogen bond (bottom panel) (PDB ID 2MER, the water molecule is modeled into the NMR structure).31 (D) H69 (red) binds to capreomycin (PDB ID 4V7M),44 which simultaneously interacts with h44 (yellow).

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MODIFICATIONS MODULATE LIGAND INTERACTIONS WITH rRNAs The distribution of post-transcriptional modifications in ribosomes overlaps with functionally important regions. Furthermore, modifications in structural motifs that reside at or near the subunit interface appear to have particularly important roles in controlling RNA thermodynamics and conformational states. In addition, modifications display cooperative effects when used in certain combinations or sequence contexts. Therefore, it is not surprising that modifications may also impact drug targeting by stabilizing unique conformational states at the binding sites or inhibiting biologically important interactions, thus conferring drug sensitivity or resistance. Aminoglycosides are major players in antibiotic use and development.3,4 They interact with the decoding region (h44) and subunit interface (h44 and H69) of the ribosome where many modified nucleosides are distributed (Figure 2).40 Overuse of aminoglycosides has led to resistance due to drug modification (e.g., N-methylation, N-acetylation, and Ophosphorylation) and altered transport49 or target modification such as methylation or mutations.3,4 Such modifications could inhibit binding interactions either directly or indirectly through RNA conformational changes. Deepening our understanding of this relationship will aid in future drug development. In addition to aminoglycosides, tetracycline (Figure 8) also interacts with rRNA at the subunit interface40 contacting both h31 and h44 (Figure 5C).20 As mentioned previously, the loop of h31 has two modified residues, m2G966 and m5C967; however, methylation at 967 only exists in bacteria, suggesting possible species-specific interactions with ligands.2 Nearby, a number of other antibiotics contact h44. Streptomycin (Figure 8) binds near the codon recognition site and causes misreading,50,51 as well as interacting with a central pseudoknot in 16S rRNA, protein S12, and a methylated nucleotide in h18 (m7G527) (Figure 6C).50 Loss of methylation at G527 causes low-level streptomycin resistance in E. coli and T. thermophilus50,51 but high-level resistance in B. subtilis.52 Future studies

bacterial counterparts, with the difference in thermodynamic contributions of the modifications being attributed to the loop sequence (A1918 in bacteria compared with G1918 in eukaryotes).45 Later studies in our laboratory revealed stem− loop crosstalk in H69, indicating that differences in the stem sequences of bacterial and human H69s can be propagated to the loop regions.42 Results from NMR studies on a synthetic H69 construct show that Ψ modulates RNA folding by enhanced base stacking or additional hydrogen-bonding interactions involving the N1H proton.31 The Ψ residues within the loop or loop-closing position contribute differently to the overall structure of modified H69. More specifically, Ψ1911 forms a Watson−Crick pair with A1919 (Figure 7B), which is absent in the corresponding unmodified RNA. In addition, a platform consisting of Ψ1911-A1919, on which the loop structure is built, is strengthened by a hydrogen bond between Ψ1911 N1H and a backbone nonbridging oxygen atom (Figure 7C), likely mediated by a solvent water molecule as observed in other RNAs.46 Protection of the Watson−Crick edges from solvent for both Ψ1911 and A1919 was also observed in chemicalprobing experiments.47 The other two modifications, Ψ1915 and Ψ1917, reside in the 3′ half of the H69 loop region. These residues mediate continuous base stacking from Ψ1915 to the 3′ terminus of modified H69 (C1924) (Figure 7B), whereas two breaks at U1915−A1916 and U1917−A1918 were observed in the corresponding unmodified H69.31 We hypothesize that increased structural rigidity resulting from base stacking in the 3′ half of the modified H69 loop would make the turn and loop closure more energetically demanding, which is reflected by an overall destabilizing effect of Ψ’s at positions 1915 and 1917 (note that this effect was also observed in h31). Thus, the three Ψ’s appear to work synergistically to modulate the folding and dynamic behavior of H69, especially in the 5′ half of the H69 loop region where changes in dynamic behavior of C1914 are observed.31 The E


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Figure 8. Representative ribosome-targeting antibiotics. The chemical structures of (A) tetracycline, (B) streptomycin, (C) amicoumacin A, and (D) capreomycin IB are shown.

might include testing the dual modifications in h44 and h18 for possible synergistic effects. Amicoumacin A (Figure 8) is an antibiotic that stalls initiation by stabilizing interactions between the mRNA and A site of h44.38 The highly conserved bacterial h44 contains m4Cm1402, m5C1407, and m3U1498, which reside close to the binding site.9,27 Loss of dimethylation at A1518 and A1519 (h45) allosterically alters the amicoumacin A binding site, including disruption of an extensive hydrogen-bonding network between residues in h44 and h45 (e.g., U1495−U1406−G1517, C1496−G1517, and G1497−A1518−A1519).38,51 These observations support the idea that modifications may impact antibiotic sensitivity indirectly. In contrast, resistance to 4,6aminoglycosides such as gentamicin and tobramycin can be induced by modifications such as methylation at N7 of G1405 and N1 of A1408.53 Such modifications alter the conformation of h44 directly, which further impacts ribosome assembly.54 Several peptide antibiotics target the ribosome. Capreomycin (Figure 8) and related viomycin interact with h44 and H69 in the intersubunit bridge B2a.44 In M. tuberculosis, capreomycin and viomycin interact with C1409 in h44 and C1920 in H69 (Figure 7D).55 Drug binding decreases upon loss of 2′-Omethylation at both C1409 in h44 and C1920 in H69, possibly due to a loss of hydrophobic interactions in the complex.55 These peptide antibiotics are also associated with resistance mechanisms such as peptide or target modification.56 One drug-development approach involves the incorporation of alternative building blocks (e.g., non-natural amino acids) into the compounds;57 however, other strategies are still needed in order to remain ahead of the resistance problem. A number of antimicrobial peptides have been developed,57 but only a few target the ribosome. In our laboratory, phage display was used to screen for small peptides (7- or 12-mers) that target various rRNA motifs (Figure 9), including the A site of h44, h31, and H69. The synthetic rRNA constructs (E. coli sequences) were biotinylated and used as targets in phage display, while the corresponding human analogues, unmodified RNAs, and partial-motif fragments were used in counterselection steps.58−60 Among the selected peptides, amidated HPVHHYQ was shown to bind to the A site by using ELISA, enzymatic footprinting, electrospray ionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC).60 HPVHHYQ-NH2 exhibited moderate affinity (low micromolar) and (5-fold) selectivity for the E. coli sequence over the human analogue or unrelated TAR RNA.60 Peptides TRLPWPA, TLWDLIP, and CVRPFAL from another selection

Figure 9. Peptide selection and RNA binding studies. To a phage library with randomized heptapeptides, biotinylated target RNA and a competitor RNA are added, followed by incubation. The bound phage is washed and eluted, followed by amplification in E. coli. This process is repeated 3−4 times, followed by sequencing and solid-phase peptide synthesis. Binding studies include chemical and enzymatic probing, ESI-MS, SPR, ITC, and NMR to determine Kd values, stoichiometry, binding site, and structure.

were shown by surface plasmon resonance (SPR) to have dissociation constants in the high nanomolar to low micromolar range for modified h31 and greater than 10-fold selectivity over unmodified h31.59 For modified H69 (containing Ψ1911, m3Ψ1915, and Ψ1917), NQVANHQ was selected and showed a 2-fold selectivity for the modified over unmodified H69 (U1911, U1915, and U1917) and a 4-fold preference over human H69.58 Phage display performed under conditions that favor an alternative H69 conformation led to a peptide with low micromolar affinity and a 2-fold selectivity for modified over unmodified H69.61 It is noteworthy that this peptide was shown by NMR to affect the loop region of H69 upon binding,61 which may be important due to the key role of this loop in ribosome function.39 Since the aminoglycoside neomycin binds to the stem region of H69,62 future directions include conjugation of peptides to aminoglycosides to improve their RNA affinities as well as transport across cell membranes, a strategy that has been successful in other cases.63 In recent work by Arya and co-workers, aminoglycoside−peptide conjugates have been shown to exhibit enhanced bacterial growth inhibition.64 Synthetic peptide chemistry has also allowed for dimerization effects on RNA binding to be determined by the Santos laboratory.65 Overall, phage display allows screening of large peptide libraries against a variety of RNA structural motifs that vary with modification status. The selected parental sequences can be further optimized, taking advantage of the broad range of chemical diversity available for the building blocks. Such approaches may be useful with rRNAtargeting peptides in order to optimize their binding affinities, chemical stabilities, specificities, and cell penetration abilities.

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CONCLUSIONS AND OUTLOOK The thermodynamic and structural contributions of modified nucleosides are important for the biological activities of rRNA. F


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Accounts of Chemical Research Some of the key functional roles include ligand interactions, which could impact antibiotic resistance. Overlap between the modification sites and functional regions in the ribosome suggest that the modifications are important for protein synthesis. Although many of the modifications have been suggested to merely have fine-tuning roles in regulation of ribosomal activity,8 the interplay between modifications, local structure, and extended structural contexts are clearly important, and the effects of multiple modifications can be additive. Furthermore, many ribosomal modifications impact the competitiveness and survival rates of organisms within complex environments.8 A growing body of literature indicates that localized structural effects around these modifications, as well as long-range crosstalk between modified regions and other regions of the ribosome, also lead to antibiotic sensitivity or resistance. During RNA processing, post-transcriptional modifications may play roles in modulating RNA local structure and global folding as with the modified rRNAs. Future work will involve developing a deeper understanding of the spatial and temporal characteristics of modifications in rRNAs and other RNA species (e.g., mRNAs or ncRNAs). The dynamics of methylation (m6A) in mRNA has been recently established,66 whereas a similar link between inducible modification (methylation)67 and demethylation68 in rRNA is lacking. The recently discovered fat mass and obesity associated protein (FTO), which is an m6A demethylase, plays roles in reversible and dynamical methylation in long ncRNAs (lncRNAs), which might also include rRNA.69 Inducibility has also been observed in pseudouridylation of RNA,70 so the next challenge will be identification of “eraser” mechanisms for the C-glycosides. Structural alterations from site-specific modifications could further affect RNA fate and activity through interactions with endogenous molecules. For example, small molecules may discriminate between the modification states of RNAs due to differences in their local or tertiary structures. In conclusion, research on RNA modifications is important not only to understand their direct effects on RNA stability, structure, and function but also to aid in the development of compounds that specifically target these regions under specific modification states. The structural and biological consequences of many of the natural modifications, as well as their induction of dynamics in rRNAs and other RNAs, remain elusive. With accumulation of data on rRNA modifications and identification of recurring themes, this knowledge may be applied to other functionally important RNAs and employed in future drug development.

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laboratory at Wayne State University. Her interests include ligand interactions with helix 69 of bacterial ribosomes. Christine S. Chow received her A.B. (1987) from Bowdoin College and Ph.D. (1992) from Caltech (advisor J. K. Barton) and conducted postdoctoral research at MIT (advisor S. J. Lippard). She joined Wayne State University in 1994 and is currently a professor in the Department of Chemistry.

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ACKNOWLEDGMENTS C.S.C. acknowledges the many contributions of her co-workers and collaborators to the studies described herein and financial support from the National Institutes of Health (Grants AI061192 and GM087596) and Wayne State University.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Jun Jiang received his Ph.D. in Chemistry (2012) from Wayne State University (advisor J. SantaLucia, Jr.), then joined Chow′s group at Wayne State University as a postdoctoral researcher. His research focuses on the biophysical properties of rRNA pseudouridylation. Hyosuk Seo received her B.S. (2008) and M.S. (2010) degrees in Chemical and Biological Engineering from Seoul National University (advisor Y.-S. Lee). She is currently a graduate student in the Chow G


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