Distinct domains and proposed function of ABH8. a) ABH8 contains three domains: the N-terminal RNA recognition motif (RRM; blue), the middle iron/αKG-dependent AlkB domain (red), and the C-terminal methyltransferase domain (green). The C-terminal domain is homologous to a tRNA modification methyltransferase Trm9 in yeast. b) The anticodon wobble-base U*34 in higher eukaryotes tRNA^Gly(U*CC) can be hypermodified. c) Examples of hypermodification on U*34: 5-carboxymethyluridine (cm⁵U), 5-methoxycarbonylmethyluridine (mcm⁵U), and 5-methoxycarbonylhydroxymethyluridine (mchm⁵U). d) The proposed sequential modification of the wobble U*34 in a 17-mer RNA that contains the anticodon stem–loop of tRNA^Gly(U*CC) by the two catalytic domains of ABH8.

Synthesis of mcm⁵U phosphoramidite (5), and incorporation of mcm⁵U and cm⁵U into a tRNA^Gly(U*CC) anticodon stem–loop oligomer sequence (RNA-I and RNA-II). Reaction conditions: a) Ac₂O, Py, RT, overnight; b) BzCl, DMAP, NEt₃, 0.5 h; c) CH₂(COMe)₂, DBU, THF, RT, overnight; d) NaOMe, MeOH, 50°C, 1 h; e) DMTr, Py, RT, overnight; f) TBDMSCl, Im, Py, RT, 10 h; g) (iPr₂N)P(Cl)OCH₂CH₂CN, iPr₂NEt, THF, RT, 4 h; h) standard solid-phase RNA synthesis and subsequent deprotection under ultramild conditions (see the Supporting Information). Bz=benzoyl, DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, DMAP=4-dimethylaminopyridine, Im=imidazole, Py=pyridine, TBDMS=tert-butyldimethylsilyl, THF=tetrahydrofuran.

Hydroxylation of mcm⁵U in a 17-mer tRNA^Gly(U*CC) anticodon stem-loop. a) The chemically synthesized 17-mer RNA oligonucleotide resembling the tRNA^Gly(U*CC) anticodon stem-loop was treated with mABH8₁₋₃₄₀ in the presence of iron(II) and α-KG. The HPLC traces show the appearance of a peak for the product (S)-mchm⁵U when mcm⁵U is converted in the presence of the enzyme. b) HRMS data of the product (S)-mchm⁵U confirms the addition of a hydroxy group to mcm⁵U. MES = 2-(N-morpholino)ethanesulfonic acid.