Pseudouridine: still mysterious, but never a fake (uridine)!

Abstract

Pseudouridine (Ψ) is the most abundant of >150 nucleoside modifications in RNA. Although Ψ was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of Ψ synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.

Keywords: E. coli, Escherichia coli; H. volcanii, Haloferax volcanii and/or Halobacterium volcanii.; Pus, pseudouridine synthase; RNA Modification; S. cerevisiae, Saccharomyces cerevisiae; S. typhimurium, Salmonella typhimurium; enzymatic mechanism; modified nucleoside; pseudouridine; rRNA; rRNA, ribosomal RNA; regulation; snRNA; snRNA, small nuclear RNA; tRNA; Ψ, Psi, pseudouridine.

Figure 1.

Isomerization of uridine into pseudouridine (Ψ). Post-isomerization several derivatives discovered to date can be formed by further modification at either position 1 (R₁), 3 (R₂) or 2’-O (R₃), while several modifications at once are possible.

Figure 2.

Structure of the archaeal ACA RNP (left) and the eukaryotic H/ACA RNP (right). Guide RNA in black, substrate RNA turquoise. Catalytically active component is light blue Cbf5 (NAP57)

Figure 3.

Distribution of Ψ and Ψ synthases in E. coli: Enzymes and their substrates positions color-coded: TruA in purple, RIuA and family members green, RsuA family members orange, TruB blue, all reviewed in, and TruD yellow. Substrate residues of RIuD are shown in a dashed box to indicate model helix H69.

Figure 4.

Distribution of Ψ and Ψ synthases in H. volcanii: TruA purple, TruD yellow, Pus10p brown, Cbf5 blue, positions with yet unknown enzyme in gray. Note that position 52 is only partially modified and that ribosomal Ψs are only available for 16S and not for 23S and 5S rRNA.

Figure 5.

Distribution of pseudouridine and pseudouridine synthases in yeast: Cellular location of enzyme and substrates as well as substrate position are given for TruA family members Pus1p, Pus2p and Pus3p (purple), RIuA family members Pus5p, Pus6p, Pus8p and Pus9p (green), TruD homolog Pus7p (yellow) and stand-alone TruB homolog Pus4p (blue), as well as the RNA-guided TruB homolog Cbf5 (blue) for U2 RNA und U5 snRNA. Modification sites without attributed enzymatic activity are indicated in gray. Mitochondrial LSU rRNA contains only one Ψ residue at position 2819 generated by Pus5. Note that for clarity the at least 44 ribosomal Ψs formed by Cbf5 are only suggested and that U2 snRNA positions 56 and 93 and U6 snRNA at position 28 have a dashed outline due to their inducibility. Pus7p is shown in the cytoplasm with dashed outline, since the enzymes changes its localization from nuclear to cytoplasmic upon heat shock.

Figure 6.

Distribution of Ψ and Ψ synthases in Homo sapiens: Cellular location of substrates and substrate position are given for TruA family members Pus1, Pus1L, Pus3 (UniProt Acc. number Q9BZE2) (purple), TruB family members TruB1, TruB2 and Cbf5 (blue), TruD family members Pus7 and Pus7L (UniProt Acc. number Q9H0K6), RIuA family members PusD1 (UniProt Acc. number Q9UJJ7.1), PusD3 (UniProt Acc. number Q6P087.3) and PusD4 (UniProt Acc. number Q96CM3.1) (green) and Pus10 (brown). In addition to tRNA and rRNA snRNA and snoRNA are modified. Note that, to current knowledge, Ψ-positions in snRNAs U2, U4 and U6, exclusively formed by H/ACA Box RNPs. Positions with known or putative guide RNAs are depicted in blue, while gray positions await guide RNA identification. Not shown are Ψ-containing SRA RNA and human telomerase RNA.

Figure 7.

The “Michael” addition-like mechanism of Ψ formation modified from Czudnochowski and coworkers. The substrate is either 5-fluorouridine (R = F) or uridine (R = H ). To account for the “generally accepted covalent adduct” of the substrate base’ C6 to the catalytic aspartate of the enzyme (if the substrate is 5FU), the aspartate would have to attack in an Michael addition-like manner. The protonation- and deprotonation steps proposed by Czudnochowski et al. would be carried out by yet unidentified bases (¹B, ²B, ³B). Please note that turnover of U and 5FU both result in compound 5. This final intermediate is either deprotonated to eventually result in pseudouridine or hydrated in case of 5FU (gray shaded reaction step) to generate 5S-6R-6-hydroxy-5-fluoro-pseudouridine.

Figure 8

(See previous page). The acylal mechanism and the glycal mechanism for Ψ formation in a version modified from ref. . (A) In case of 5FU the acylal intermediate can result in compound 5 to eventually yield the 5S-6R-6-hydroxy-5-fluoro-pseudouridine found in the crystal structures. However, an equilibrium of the 5FU-acylal intermediate with an oxocarbonium intermediate (compound 5b) might open an additional gray shaded reaction manifold exclusively to 5FU. This would account for the arabino-isomer as minor product of E. coli TruB action on 5FU RNA that was discovered by Miracco and Mueller. Pseuoduridine could be formed by the not-shaded acylal mechanism, the only difference would be the last step: The ‘F’ would be a proton that is abstracted to generate the product. (B) Miracco and Mueller proposed that pseudouridine could also be formed by a third glycal mechanism. This mechanism resembles the gray reaction manifold in a) but yield only one product in ribo conformation.