Pseudouridinylation of mRNA coding sequences alters translation Daniel E. Eylera,1, Monika K. Francob,1, Zahra Batoolc, Monica Z. Wud, Michelle L. Dubukee,f, Malgorzata Dobosz-Bartoszekc, Joshua D. Jonesa, Yury S. Polikanovc,g,2, Bijoyita Royd,2, and Kristin S. Koutmoua,b,2 aDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109; bProgram in Chemical Biology, University of Michigan, Ann Arbor, MI 48109; cDepartment of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607; dRNA and Genome Editing, New England Biolabs Inc., Ipswich, MA 01938; eProteomics and Mass Spectrometry Facility, University of Massachusetts, Worcester, MA 01545; fDepartment of Biochemistry and Molecular Pharmacology, University of Massachusetts, Worcester, MA 01605; and gDepartment of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL 60612 Edited by Joseph D. Puglisi, Stanford University School of Medicine, Stanford, CA, and approved September 30, 2019 (received for review January 18, 2019) Chemical modifications of RNAs have long been established as key cellular component to read Ψ in mRNAs (2). How, or even if, modulators of nonprotein-coding RNA structure and function in mRNA pseudouridinylation contributes to gene expression is not cells. There is a growing appreciation that messenger RNA (mRNA) yet apparent. Reporter-based studies in human cells and bacterial sequences responsible for directing protein synthesis can also be lysates come to conflicting conclusions regarding the role of Ψ,with posttranscriptionally modified. The enzymatic incorporation of mRNA some studies suggesting that the presence of Ψ in mRNA codons modifications has many potential outcomes, including changing mRNA increases protein production (15) and others reporting a reduction stability, protein recruitment, and translation. We tested how one in protein synthesis (16, 17). The clearest evidence of a biological of the most common modifications present in mRNA coding regions, Ψ Ψ role for in mRNAs comes from studies in the parasite Toxoplasma pseudouridine ( ), impacts protein synthesis using a fully reconsti- gondii where Ψ increases mRNA stability and facilitates parasite tuted bacterial translation system and human cells. Our work reveals Ψ differentiation (12, 13). Regardless of whether or not further that replacing a single uridine nucleotide with in an mRNA codon Ψ impedes amino acid addition and EF-Tu GTPase activation. A crystal studies reveal a significant role for in gene regulation, the ribosome Thermus thermophilus Phe surely translates Ψ-containing codons in cells and it is important structure of the 70S ribosome with a tRNA BIOCHEMISTRY bound to a ΨUU codon in the A site supports these findings. We also to establish the possible outcomes of these events. Ψ find that the presence of Ψ can promote the low-level synthesis of Since can alter the fundamental properties of RNAs, including – multiple peptide products from a single mRNA sequence in the recon- their secondary structures and base-pairing abilities (18 20), it has stituted translation system as well as human cells, and increases the been proposed that one consequence of Ψ couldbetopromotethe rate of near-cognate Val-tRNAVal reacting on a ΨUU codon. The vast incorporation of multiple amino acids on a single codon (2, 8). Indeed, majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ Significance can be to modestly alter both translation speed and mRNA decoding. The posttranscriptional modification of messenger RNAs (mRNAs) translation | pseudouridine | mRNA modification | ribosome is an emerging frontier in gene regulation. Understanding the biological implications of one of the most common mRNA ucleosides in messenger RNAs (mRNAs) can be enzymatically modifications, pseudouridine, in cells is complicated by the Nmodified posttranscriptionally (1, 2) to expand the chemical and substoichiometric occurrence of mRNA modifications and the topological properties of these essential biomolecules. Transcriptome- difficulty of decoupling the effects on translation from mRNA wide mapping of individual modifications revealed the presence of stability. Here we used in vitro biochemical and structural studies modifications in both the untranslated and protein-coding regions of together with cell-based assays to demonstrate that pseudouridine mRNAs (2, 3). The localization of modifications throughout mRNAs impedes translation elongation and increases the occurrence of suggests that modifications could potentially alter protein production amino acid substitutions. Our work supports the idea that mRNA by multiple mechanisms, including affecting interactions of the modifications can modulate mRNA translatability and provides translating ribosomal complex with the mRNA, mRNA structure, evidence that pseudouridine can alter tRNA selection by the ri- and mRNA stability. Among the mRNA modifications identified to bosome. This study presents a biochemical foundation for better date, N6-methyladenonsine (m6A) and pseudouridine (Ψ)arethe understanding of the consequences of modifications in mRNA most prevalent (2, 4). m6A modifications are estimated to occur in coding regions. half of the human mRNAs and cells contain a complement of proteins reported to write, read, and erase the modification (5, 6). Author contributions: D.E.E., M.K.F., Z.B., M.Z.W., M.L.D., Y.S.P., B.R., and K.S.K. designed Ψ has been mapped to hundreds of mRNA sequences (7–9), research; D.E.E., M.K.F., Z.B., M.Z.W., M.L.D., M.D.-B., J.D.J., and B.R. performed research; Ψ J.D.J. contributed new reagents/analytic tools; D.E.E., M.K.F., Z.B., M.Z.W., M.L.D., Y.S.P., and mass spectrometry studies report the /U ratio in human B.R., and K.S.K. analyzed data; and D.E.E., M.K.F., Z.B., M.Z.W., Y.S.P., B.R., and K.S.K. 6 cell lines to be comparable to that of m A/A (∼0.3% for Ψ/U vs. wrote the paper. 6 ∼0.5% m A/A) (10, 11). While the frequency of Ψ at most mapped The authors declare no competing interest. Ψ sites has not been established, estimates of -frequency based on This article is a PNAS Direct Submission. Ψ Ψ -seq experiments, and the direct measurement of occupancy Published under the PNAS license. at a discrete site (in EEF1A1) indicate that Ψ can be incorpo- > 6 Data deposition: Coordinates and structure factors were deposited in the RCSB Protein rated at frequencies ( 50%) comparable to well-occupied m A Data Bank with accession code 6OU1 for the Thermus thermophilus 70S ribosome in sites (8, 10). The preponderance of Ψ moieties in mRNA are in complex with ΨUU-mRNA, A-, P-, and E-site tRNAs. coding regions (>60%), and while a host of pseudouridinylating 1D.E.E. and M.K.F. contributed equally to this work. Ψ enzymes have been identified that incorporate into both mRNAs 2To whom correspondence may be addressed. Email: [email protected], yuryp@uic. and noncoding RNAs in a reproducible, specific, and inducible edu, or [email protected]. manner (7–10, 12–14), no proteins that read or erase Ψ have This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. been discovered. Consequently, the ribosome surely encounters 1073/pnas.1821754116/-/DCSupplemental. Ψ in cells and it has been hypothesized that it could serve as a key www.pnas.org/cgi/doi/10.1073/pnas.1821754116 PNAS Latest Articles | 1of7 Downloaded by guest on September 24, 2021 Ψ-containing stop codons have been observed to direct the nonsense incorporation on UUU and ΨUU and found that the value is suppression of translation termination (14, 21), though the effect of increased by 2-fold on ΨUU. Consistent with this, we find that Ψ in stop codons remains an unresolved question (22). Thus far, the production of a full-length luciferase peptide in the recon- differential decoding of Ψ-containing sense codons has not yet stituted in vitro translation system (NEB PURExpress) is 3-fold been reported (16, 17). Establishing if Ψ can alter tRNA selection on slower on luciferase reporter mRNA with every U substituted for the ribosome is a timely question given that a wide range of modified Ψ (SI Appendix, Fig. S2). nucleosides (Ψ, N1-methyl-Ψ, 2-thiouridine, 5-methyl-cytosine) The decreased observed rate constants for amino acid incor- are being routinely inserted into synthetic mRNAs at high stoi- poration on pseudouridinylated codons under sub- and near- chiometric ratios for therapeutic applications (15). saturating concentrations of Phe-tRNAPhe could reflect changes Identifying the consequences of Ψ mRNA modification is com- in the rate constants for one or more of the multiple upstream plicated in cells because the enzymes that incorporate Ψ into kinetic steps (23) (SI Appendix,Fig.S3). To gain further insight into mRNA also catalyze Ψ addition to noncoding RNA species. which steps are affected by Ψ, we measured the rate constants for Furthermore, the impact of mRNA and protein stability on protein GTP hydrolysis by EF-Tu after binding of the aa-tRNA•EF-Tu•GTP output can be difficult to deconvolute from effects on translation in ternary complex to the A site. In these assays, 1.8 μM 3H-fMet- cells. Here, we directly investigate the mechanistic effects of mRNA labeled complexes were mixed with 100 nM of α-32P-GTP labeled pseudouridinylation on translation using in vitro enzymology as well ternary complex. The observed rate constant for GTP hydro- −1 as X-ray crystallography, and support our in vitro conclusions with lysis on the unmodified UUU codon (kGTP = 78 ± 10 s )was cell-based approaches. Our results demonstrate that the insertion consistent with previously reported values (23), while the rate Ψ −1 of a single perturbs ribosome function and promotes the low- constantwasslowerontheΨUU codon (kGTP = 42 ± 6s )(Fig.2 level synthesis of multiple peptide products from a single mRNA A and B, SI Appendix,TableS1).