The Dihydrouridine landscape from tRNA to mRNA: a perspective on synthesis, structural impact and function

Figures & data

Figure 1. Biochemistry of dihydrouridine.

A Reduction of uridine into dihydrouridine. B Dihydrouracil is a nonplanar nucleobase (carbon in gray, nitrogen in blue, oxygen in red and hydrogen in white). C First published structure of a ribonucleic acid (yeast tRNA^Ala) where D (red arrows) are shown in a loop at the 5’-end [12]. D Schematic representation of ribose pucker. C5’ (orange dot) is considered as being above the C4’-O’-C1’ plane (red dashed line and red dots). Left panel: C3’-endo has the C3’ (green dot) above the plane. Right panel: C2’-endo has the C2’ (blue dot) above the plane. E C2’-endo pucker produces a longer 5’-phosphate/3’-phosphate distance and therefore spans the polynucleotide [147]. F Schematic representation of ribose gauche-gauche and gauche-trans conformations. C5’ (purple dot) adopts different torsion angles (γ) that modulate the positioning of C5’-bound atoms (H_5ʹ1,H_5ʹ2 and O_5’).

Table 1. Seminal studies on optical and structural properties of dihydro-uracil/-uridine

Structural and optical studies	Comments	References
dihydrouracil	C5-C6 and N1 out of the base plane expectation for Watson-Crick interaction impairment	[140]
dihydrouridine	deviation from planarity anti orientation C2’-endo conformation	[143]
	anti orientation favoured gauche-trans and trans-gauche conformations	[149]
	deviation from planarity anti orientation preference for C3’-exo and C2’-endo conformations preference for gauche-gauche conformation	[85]
	deviation from planarity anti orientation C2’-endo conformation favoured gauche-trans and trans-gauche conformations	[142]
	C6 out of the base plane no base stacking and anti orientation C2’-endo conformation favoured gauche-trans and trans-gauche conformations	[141]
dinucleoside phosphate: DpA, ApD, GpD	DpA stacking ApD and GpD low stacking	[137]
yeast or E. coli tRNAs	formation of a D-loop with increased hydrophobicity D provides extra flexibility interaction of D-loop and TψC-loop	[135,138,139,144–146]
trinucleoside phosphate: Dp(acp^3U)pA	C2’-endo conformation and upstream propagation	150
trinucleoside phosphate: ApDpA	C6 out of the base plane no base stacking enhanced C2’-endo conformation at low temperature (5°C) and downstream propagation	[134,, 136]

The table is divided into three columns; (I) studied chemical entities, (II) main conclusions relative to the nucleobase (green writing) or to the ribose (blue writing) and (III) references. C (carbon), N (nitrogen), A (adenosine), D (dihydrouridine), G (guanosine), p (5’-3’ phosphodiester bond), acp³U (3-(3-amino-3-carboxypropyl) uridine).

Figure 2. Molecular fate of D upon sodium borohydride (NaBH₄) or alkaline (OH⁻) treatments.

R stands for ribose attached to the RNA chain. Red circles highlight ureido-groups.H⁺ stands for acid conditions. Details in the text.

Table 2. Chemical reactions and techniques specifically applicable to D

Principles	tRNA	Comments	References
alkaline hydrolysis	/	sodium hydroxide → cleavage at the N3-C4 linkage of dihydrouracil	[19]
β-alanine detection	Y	sodium hydroxide → D-ring opening → partial β-alanine formation → ninhydrin colorimectric assay	[32]
sodium borohydride reduction	Y	sodium borohydride → cleavage at the N3-C4 linkage of dihydrouridine	[20]
hydrochloric acid hydrolysis	B, Y, M	sodium borohydride → D-ring opening → hydrochloric acid → cleavage of the glycosodic bond	[24,and b; 26]
loss of absorbance	Y	loss of absorbance at 265 nm upon mild sodium hydroxide treatment	[132]
cleavage at D position	Y	sodium borohydride → D-ring opening → aniline treatment → RNA chain cleavage	[34]
ureido-group detection	B, Y, M	sodium hydroxide → D-ring opening → solution neutralization → colorimetric assay after iron chloride addition (550 nm)	[22]
replacement of D by proflavine or EtBr	Y	sodium borohydride → D-ring opening → incubation with the dye	[29]
microarray	Y	differential fluorescent labeling of tRNA from WT and dus strains → annealing on a microchip → quantification of A-U vs A-D interaction	151
primer extension	B, Y	sodium hydroxide → D-ring opening → assessment of RT termination by primer extension	[23,33]
fluorescent labeling with rhodamine 110	B	in vitro tRNA → in vitro dihydrouridylation → sodium borohydride → incubation with the dye	[23]
replacement of D by Cy3 or 5	B, Y	sodium borohydride → D-ring opening → incubation with the dye	[27]
benzoyhydrazide addition	B	sodium borohydride → THU formation → incubation with benzohydrazide	[42]
predictive modelling	B, Y	jackknife-based test to predict modified sites	[113,115]
nanopore sequencing	B	RNA through a nanopore scale → specific ionic signature	[116]

The table is divided into four columns; (I) list of principles, (II) phylogenetic origin of the studied tRNA; B (bacterial), Y (yeast), M (mammalian), (III) general comments on the principle. EtBr (ethidium bromide), N (nitrogen), C (carbon), RT (reverse transcription), WT (wild type), dus (dihydrouridine synthase gene), A-U (adenine-uridine Watson-Crick interaction), A-D (adenine-dihydrouridine Watson-Crick interaction), Cy3/5 (cyanines 3 or 5), THU (tetrahydrouridine) and (IV) references. Only the seminal works are cited although most of the techniques were implemented in other studies. Chromatographic applications are not listed here.

Figure 3. Proposed mechanism of D rhodamine labeling following sodium borohydride (NaBH₄) reduction.

Left panel, dihydrouridine is reduced to tetrahydrouridine that is characterized by an electrophilic carbonilamine (green circle), including the C4 hydroxyl group (C-OH). Upon addition of the nucleophilic rhodamine 110 in acid conditions, the tetrahydrocytidine intermediate is formed with its reactive Schiff base (blue circle). Covalent binding of rhodamine 110 occurs by substitution of the C4 hydroxyl group. Right panel, ureidopropanol generated by D-ring opening is replaced by rhodamine 110. R stands for ribose attached to the RNA chain.

Figure 4. The biology of dihydrouridine.

A Specificity of yeast Dus enzymes for cytoplasmic tRNAs.B Specificity of E. coli Dus enzymes for tRNAs.C Putative enzymatic mechanism of Dus enzymes. Details in the text.D Currently available structures of Dus enzymes in different species (Sp.) from different domains (dm; B for Bacteria and E for Eukarya). The references are indicated in the right column.

Figure 5. Molecular strategies of bacterial Dus enzymes for substrate specificities.

Left panel, interaction of the U nucleobase (in the context of the RNA chain (R)) with Dus amino acids (orange: F for Phe, N for Asn, K for Lys and Y for Tyr) through ionic (blue lines) or hydrophobic (purple lines) interactions. In the DusB catalytic pocket, the rotation of the U nucleobase (180°) is observed in comparison to the DusA and C counterparts. Right panel, schematic representations of the Dus enzymes (orange line) with the N-terminal nucleobase-containing catalytic domain (round) and the C-terminal RNA binding domain (rectangle). The tRNA (gray line) docking is similar in DusB and C but differs by a rotation (160°) in DusA. These strategies (tRNA or nucleobase rotations) allow the unvariable targeting of a specific uridine.

Holley RW, Apgar J, Everett GA, et al. Structure of a ribonucleic acid. Science. 1965;147:1462–1465.

 PubMed Web of Science ®Google Scholar

Moreau C, Ashamu GA, Bailey VC, et al. Synthesis of cyclic adenosine 5’-diphosphate ribose analogues: a C2ʹendo/syn “southern” ribose conformation underlies activity at the sea urchin cADPR receptor. Org Biomol Chem. 2011;9:278–290.

 PubMed Web of Science ®Google Scholar

Rohrer DC, Sundaralingam M. Stereochemistry of nucleic acids and their constituents. VI. The crystal structure and conformation of dihydrouracil: a minor base of transfer-ribonucleic acid. Acta Crystallogr B. 1970;26:546–553.

 PubMedGoogle Scholar

Sundaralingam M, Rao ST, Abola J. Molecular conformation of dihydrouridine: puckered base nucleoside of transfer RNA. Science. 1971a;172:725–727.

 PubMed Web of Science ®Google Scholar

Sundaralingam M, Rao ST, Abola J. Stereochemistry of nucleic acids and their constituents. 23. Crystal and molecular structure of dihydrouridine “hemihydrate”, a rare nucleoside with a saturated base occurring in the dihydrouridine loop of transfer ribonucleic acids. J Am Chem Soc. 1971b;93:7055–7062.

 PubMed Web of Science ®Google Scholar

Deslauriers R, Lapper RD, Smith IC. A proton magnetic resonance study of the molecular conformation of a modified nucleoside from transfer RNA. Dihydrouridine. Can J Biochem. 1971;49:1279–1284.

 PubMedGoogle Scholar

Suck D, Saenger W, Zechmeister K. Conformation of the tRNA minor constituent dihydrouridine. FEBS Lett. 1971;12:257–259.

 PubMed Web of Science ®Google Scholar

Suck D, Saenger W, Hobbs J. Molecular and crystal structure of 2’-chloro-2’-deoxyuridine. Biochim Biophys Acta. 1972;259:157–163.

 PubMedGoogle Scholar

Formoso C, Tinoco I Jr. Minor nucleosides in RNA: optical studies of dinucleoside phosphates containing dihydrouridine. Biopolymers. 1971;10:1533–1541.

 PubMed Web of Science ®Google Scholar

Davis DR, Griffey RH, Yamaizumi Z, et al. 15N-labeled tRNA. Identification of dihydrouridine in Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR. J Biol Chem. 1986;261:3584–3587.

 PubMed Web of Science ®Google Scholar

Jack A, Ladner JE, Klug A. Crystallographic refinement of yeast phenylalanine transfer RNA at 2-5A resolution. J Mol Biol. 1976;108:619–649.

 PubMed Web of Science ®Google Scholar

Quigley GJ, Rich A. Structural domains of transfer RNA molecules. Science. 1976;194:796–806.

 PubMed Web of Science ®Google Scholar

Stuart JW, Basti MM, Smith WS, et al. Structure of the trinucleotide D-acp3u-A with coordinated Mg2+ demonstrates that modified nucleosides contribute to regional conformations of RNA. Nucl Nucl. 1996;15:1009–1028.

 Web of Science ®Google Scholar

Dalluge JJ, Hashizume T, McCloskey JA. Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS). Nucleic Acids Res. 1996a;24:3242–3245.

 PubMed Web of Science ®Google Scholar

Batt RD, J.k M, J.m P, et al. Chemistry of dihydropyrimidines. Ultraviolet spectra and alkaline decomposition. J Am Chem Soc. 1954;76:3663–3665.

 Web of Science ®Google Scholar

Magrath DI, Shaw DC. The occurrence and source of beta-alanine in alkaline hydrolysates of sRNA: a sensitive method for the detection and assay of 5,6-dihydrouracil residues in RNA. Biochem Biophys Res Commun. 1967;26:32–37.

 PubMedGoogle Scholar

Cerutti P, Miller N. Selective reduction of yeast transfer ribonucleic acid with sodium borohydride. J Mol Biol. 1967;26:55–66.

 PubMed Web of Science ®Google Scholar

Molinaro M, Sheiner LB, Neelon FA, et al. Effect of chemical modification of dihydrouridine in yeast transfer ribonucleic acid on amino acid acceptor activity and ribosomal binding. J Biol Chem. 1968;243:1277–1282.

 PubMed Web of Science ®Google Scholar

Beltchev B, Grunberg-Manago M. Preparation of a pG-fragment from tRNA(Phe)(yeast) by chemical scission at the dihydrouracil, and inhibition of tRNA(Phe)(yeast) charging by this fragment when combined with the -CCA half of this tRNA. FEBS Lett. 1970;12:24–26.

 PubMed Web of Science ®Google Scholar

Jacobson M, Hedgcoth C. Determination of 5,6-dihydrouridine in ribonucleic acid. Anal Biochem. 1970;34:459–469.

 PubMed Web of Science ®Google Scholar

Wintermeyer W, Zachau HG. Replacement of Y base, dihydrouracil, and 7-methylguanine in tRNA by artificial odd bases. FEBS Lett. 1971;18:214–218.

 PubMed Web of Science ®Google Scholar

Peng WT, Robinson MD, Mnaimneh Set al. A panoramic view of yeast noncoding RNA processing. Cell. 2003;113:919–933.

 PubMed Web of Science ®Google Scholar

Betteridge T, Liu H, Gamper H, et al. Fluorescent labeling of tRNAs for dynamics experiments. RNA. 2007;13:1594–1601.

 PubMed Web of Science ®Google Scholar

Xing F, Hiley SL, Hughes TR, et al. The specificities of four yeast dihydrouridine synthases for cytoplasmic tRNAs. J Biol Chem. 2004;279:17850–17860.

 PubMed Web of Science ®Google Scholar

Pan D, Qin H, Cooperman BS. Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop. RNA. 2009;15:346–354.

 PubMed Web of Science ®Google Scholar

Kaur J, Raj M, Cooperman BS. Fluorescent labeling of tRNA dihydrouridine residues: mechanism and distribution. RNA. 2011;17:1393–1400.

 PubMed Web of Science ®Google Scholar

Feng P, Xu Z, Yang H, et al. Identification of D modification sites by integrating heterogeneous features in saccharomyces cerevisiae. Molecules. 2019;24(3):380.

 PubMed Web of Science ®Google Scholar

Xu ZC, Feng PM, Yang H, et al. iRNAD: a computational tool for identifying D modification sites in RNA sequence. Bioinformatics. 2019;35:4922–4929.

 PubMed Web of Science ®Google Scholar

Thomas NK, Poodari VC, Jain M, et al. Direct nanopore sequencing of individual full length tRNA strands. ACS Nano. 2021;15:16642–16653.

 PubMed Web of Science ®Google Scholar