The Importance of Being Modified: The Role of RNA Modifications in Translational Fidelity

Abstract

The posttranscriptional modifications of tRNA's anticodon stem and loop (ASL) domain represent a third level, a third code, to the accuracy and efficiency of translating mRNA codons into the correct amino acid sequence of proteins. Modifications of tRNA's ASL domain are enzymatically synthesized and site specifically located at the anticodon wobble position-34 and 3'-adjacent to the anticodon at position-37. Degeneracy of the 64 Universal Genetic Codes and the limitation in the number of tRNA species require some tRNAs to decode more than one codon. The specific modification chemistries and their impact on the tRNA's ASL structure and dynamics enable one tRNA to decode cognate and "wobble codons" or to expand recognition to synonymous codons, all the while maintaining the translational reading frame. Some modified nucleosides' chemistries prestructure tRNA to read the two codons of a specific amino acid that shares a twofold degenerate codon box, and other chemistries allow a different tRNA to respond to all four codons of a fourfold degenerate codon box. Thus, tRNA ASL modifications are critical and mutations in genes for the modification enzymes and tRNA, the consequences of which is a lack of modification, lead to mistranslation and human disease. By optimizing tRNA anticodon chemistries, structure, and dynamics in all organisms, modifications ensure translational fidelity of mRNA transcripts.

Keywords: Codon degeneracy and decoding; Human disease and tRNA modifications; Modification chemistry and structure; Modifications 3′-adjacent to the anticodon; Modifications prestructure tRNA; Wobble position tRNA modifications.

FIG. 1

The Universal Genetic Code. Twofold degenerate codons are highlighted in tan; threefold (Ile) in gray, fourfold in yellow, and sixfold in blue. The single codons of Met and Trp are highlighted in green and orange, respectively, and the three stop codons are highlighted in red. The figure is annotated with the abbreviations for those modified nucleosides found in the anticodon domain of tRNAs responding to the codons and discussed in this review. The chemical structures and full names of the modifications are found in Fig. 3.

FIG. 2

tRNA and its anticodon stem and loop (ASL) domain. Left: General cloverleaf structure of a 76-nucleotide tRNA with the aminoacyl-accepting stem in green, dihydrouridine (D) stem and loop in black, ASL in red, extra loop in blue, and ribothymidine (T) stem and loop in plum. Right: An enlargement of the ASL domain, with the abbreviations of the important modifications discussed in this review listed. A darker color hue in plum represents more important modification sites.

FIG. 3

Chemical structures of the major and modified nucleosides of tRNA. Top row: General structures of purine and pyrimidine nucleosides with numbering of the atoms. R represents ribose and the asterisk (*) marks the most important and/or frequent sites of modification. Second row: Conventional RNA nucleosides and their letter abbreviations. Third through fifth rows: The modified nucleosides discussed in this review with their shorthand notions [14,15].

FIG. 4

Nucleoside sequence and cloverleaf structures of tRNA^Met. (A) Human mitochondrial tRNA^Met displaying the condensed variable loop and the shortened D- and T-arms. Nucleotides affected by disease-causing point mutations are boxed. (B) Saccharomyces cerevisiae initiator tRNA^Met. Ar(p) at position-64 refers to the 2′-O-ribosyl phosphate modification, which is an identity element of initiator tRNA^Met in yeast [60]. (C) S. cerevisiae elongator tRNA^Met; 5-methyluridine at position-54, indicated by T, is an important determinant of elongator tRNA^Met. (D) Chemical structures of the modifications f⁵C₃₄, Ψ at various noted positions, and t⁶A₃₇ found in the three methionyl tRNA [61].

FIG. 5

The tautomeric forms of 5-formylcytidine, f⁵C₃₄. Tautomerism of f⁵C₃₄ allows for the stereochemistry of the Watson–Crick base pairing between tRNA's f⁵C₃₄ and A3 of the AUA codon to approximate that of a canonical U●A pair. (A) Steric repulsion between the common amino-oxo form of f⁵C₃₄ N4H and A₃N⁶H marked by an X. (B) Favorable interactions between the imino-oxo form of f⁵C₃₄ and A₃[70]. Dotted lines indicate hydrogen bonding between nucleoside residues [70].

FIG. 6

tRNA^Lys ASL sequences and the chemical structures of their modified. Top: Sequences and secondary structures of the anticodon stem and loop (ASL) domains of human tRNA^Lys1,2_CUU, tRNA^Lys3, human mitochondrial tRNA^Lys_UUU, and E. coli tRNA^Lys_UUU. Nucleosides in parenthesis indicate differences between tRNA^Lys1 and tRNA^Lys2 sequences. Bottom: Chemical structures of the modified nucleosides in the ASLs above and their shorthand notations [92,93].

FIG. 7

N⁶-Threonylcarbamoyladenosine and its cyclic derivative, t⁶A and ct⁶A. Left: Chemical structure of t⁶A with the atoms of the modification numbered. The hydrogen bonding that produces a pseudocyclic conformation of the modified nucleoside is shown as a dashed line. Right: The cyclic form of t⁶A, ct⁶A, with the corresponding atoms numbered [88,133].

FIG. 8

The primary sequences and nucleoside modifications of the E. coli tRNA^Val isoacceptors and the structures of ASL^Val_UAC and ASL^Val_UAC-cmo⁵U₃₄;m⁶A₃₇ compared. (A) The anticodon domain primary sequences of the three tRNA^Val isoacceptors from E. coli and their modifications: uridine-5-oxyacetic acid at position-34, red and N⁶-methyladenosine at position-37, green. The two tRNA^Val species containing the GAC anticodon have identical ASLs with variations in sequence in other regions of the tRNA molecule. (B) The chemical structures, names, and abbreviations of the modifications that appear in the E. coli tRNA^Val_UAC-cmo⁵U₃₄;m⁶A₃₇. (C) Structure of unmodified ASL^Val_UAC, pink. (D) Fully modified ASL^Val_UAC containing the modifications cmo⁵U₃₄ and m⁶A₃₇, ASL^Val_UAC-cmo⁵U₃₄;m⁶A₃₇, red, showing the base stacking of cmo⁵U₃₄ and an open loop structure. The structures of the unmodified and modified ASL^Val_UAC were determined by NMR [45] (Protein Data Bank 2JR4 for unmodified ASL^Val3_UAC and 2JRG for ASL^Val3_UAC-cmo⁵U₃₄;m⁶A₃₇).

FIG. 9

cmo⁵U₃₄○U3 wobble pair. Base pairing between cmo⁵U₃₄ of the ASL^Val_UAC and U3 of the GUU codon [46] (Protein Data Bank 2UUB). A hydrogen bond occurs between the 2′-OH of the invariant U₃₃ (background) of the ASL^Val_UAC and O5 of cmo⁵U₃₄ (black dashed line), structuring cmo⁵U₃₄ for base pairing with U3. The hydrogen bond that allows for the base pair between O2 of cmo⁵U₃₄ and N2 of U3 (red dashed line).

FIG. 10

The primary sequences and nucleoside modifications of the E. coli tRNA^Arg isoacceptors. (A) The anticodon domain primary sequences of the five tRNA^Arg isoacceptors from E. coli and their modifications: 2-thiocytidine at position-32 (blue); inosine and 5-methylaminomethyluridine at position-34 (red); 2-methyladenosine, N⁶-threonylcarbamoyladenosine, and 1-methylguanosine at position-37 (green); and pseudouridine at position-40 (orange). (B) The chemical structures, names, and abbreviations of the modifications that appear in the five E. coli tRNA^Arg isoacceptors.