Pseudouridinylation of mRNA coding sequences alters translation

Abstract

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNA^Phe bound to a ΨUU codon in the A site supports these findings. We also find that the presence of Ψ can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNA^Val reacting on a ΨUU codon. The vast majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ can be to modestly alter both translation speed and mRNA decoding.

Keywords: mRNA modification; pseudouridine; ribosome; translation.

Fig. 1.

Ψ changes amino acid incorporation by the ribosome. (A) Coding sequences for the Ψ-containing mRNA constructs. (B and C) Time courses displaying the formation of ^fMet-Phe peptide on an unmodified and modified UUU codon [UUU (black circles), ΨUU (blue squares), UΨU (green diamonds), UUΨ (red triangles)]. Time courses were collected under single-turnover conditions (70–100 nM 70S ribosome initiation complexes, with either [B] near-saturating [1 μM] or [C] high [5 μM] levels of Phe-tRNA^Phe).

Fig. 2.

Ψ alters GTP hydrolysis during ternary complex binding to the ribosome. (A) Time courses displaying the formation of GDP when 1.6 μM ³H-fMet-labeled complexes were mixed with 100 nM of γ-³²P-GTP labeled ternary complex formed with Phe-tRNA^Phe and nucleotide-free EF-Tu. Single-exponential curves were fitted to data collected in 3 independent experiments. (B) Observed rate constants for data fit in A. Error bars are the SE of the fitted value of k_obs. (C and D) 2Fo-Fc electron difference Fourier maps (blue mesh) for the ribosome-bound A site (green) and the P site (dark blue) tRNAs interacting with unmodified (C) or Ψ-containing mRNA (D). In C, both the map and the model are from PDB entry 4Y4P. The direction of the view for both panels is indicated on the Upper Right Inset in C. The refined models of mRNA (magenta) and tRNA (green) are displayed in their respective electron densities contoured at 1.2σ. Close-up views of the CCA-ends of the A-site tRNAs are shown by Lower Right Insets in each of the panels. The electron density corresponding to the CCA-end of the tRNA interacting with the Ψ-containing mRNA is much weaker compared to the CCA-end of the tRNA interacting with the unmodified mRNA, while the electron density corresponding to the bodies of the A-site tRNAs are comparable between the 2 complexes.

Fig. 3.

Ψ promotes incorporation of alternative amino acids by the ribosome at limiting concentrations of aa-tRNA. (A) Electrophoretic TLC displaying the translation products a mixture of mRNAs containing a single randomized codon (NNN), and unmodified and Ψ-containing UUU messages in the presence of no tRNA (null), Phe-tRNA^Phe tRNA (phe TC), and total aa-tRNA (total TC). Translation of the NNN pool of mRNAs with random codons in the A site demonstrates the presence of multiple aa-tRNA^aa species in the total tRNA preparation. (B) Percent of amino acid substituted dipeptides, relative to the correct fMet-Phe product, on unmodified and modified UUU codons (e.g., % not forming expected MF peptide). (C) Percent of ribosomes that react with 2 μM Lys-tRNA^Lys ternary complex on UAA and ΨAA stop codons to form a MK peptide after 10 min. The near-cognate Lys-tRNA^Lys reacts to produce twice as much peptide on ΨAA than on UAA. All of the data displayed in plots reflect the averages and SEs of at least 3 experiments.

Fig. 4.

Amino acids from near-cognate and noncognate tRNAs are incorporated on Ψ-containing codons. (A) Electrophoretic TLC displaying the translation products of NNN and Ψ-containing messages in the presence of total aa-tRNA (total), total tRNA aminoacylated with valine (val TC), total tRNA aminoacylated with isoleucine (ile TC), and total aa-tRNA aminoacylated with leucine (leu TC). (B) Percent of MV/ML/MI products generated on UUU and Ψ-containing codons relative to NNN. The values plotted are the mean of 4 experiments and the error bars reflect the SE. (C) Summary of amino acid substitutions observed by mass spectrometry in a luciferase peptide incorporated on Ψ-containing mRNAs translated in 293H cells.

Fig. 5.

Ψ changes how codons are read. (A) k_app values for ^fMet-Val and formation on UUU and ΨUU codons in the presence of 10 nM EF-Tu and 10 µM Val-tRNA^Val. (B) Position of the codons and peptidyl-tRNA in the purified ribosome elongation complexes prior to addition of RF2 and RF2/RF3 P-site mismatch surveillance assay. (C) Rate constants for premature hydrolysis of ^fMet-Lys from fMet-Lys-tRNA^lys bound to UAA or ΨAA in the P site catalyzed by RF2 (white) and RF2/RF3 (gray). (D and E) ^fMet release on UAA (squares), ΨAA (circles) stop codons catalyzed by 500 nM RF1 (D) or RF2 (E).