Decoding pseudouridine: an emerging target for therapeutic development
Authors: Jonas Cerneckis, Qi Cui, Chuan He, Chengqi Yi, Yanhong Shi
Journal: Trends in Pharmacological Sciences (2022)

Pseudouridine (Ψ) is the most common post-transcriptional RNA modification that is dynamically deposited throughout the transcriptome.RNA pseudouridylation can influence various cellular processes, such as translation and response to environmental stress, and becomes dysregulated in disease.Mutations in Ψ synthases lead to aberrant pseudouridylation of their RNA targets and contribute to cancer progression and genetic disease.The level of excreted Ψ and the expression profile of pseudouridylation machinery may have diagnostic and prognostic value as biomarkers for cancer.The dynamic Ψ epitranscriptome is a potential target for therapeutic development. Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification and is widespread in multiple RNA species. Ψ impacts various aspects of RNA biology, conferring distinct structural and functional properties to the RNA molecules that it decorates. However, aberrant pseudouridylation contributes to a variety of human diseases, including cancer and genetic disorders. Dysregulation of the Ψ epitranscriptome can arise from mutations and abnormal expression of pseudouridylation machinery, impacting protein translation and other cellular processes. With advancing understanding of Ψ roles in health and disease, efforts are now invested in developing therapeutic and diagnostic approaches targeting Ψ. Emerging reports indicate that Ψ and its installation machinery could be potential pharmacological targets for therapeutic development and may serve as biomarkers for human diseases. Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification and is widespread in multiple RNA species. Ψ impacts various aspects of RNA biology, conferring distinct structural and functional properties to the RNA molecules that it decorates. However, aberrant pseudouridylation contributes to a variety of human diseases, including cancer and genetic disorders. Dysregulation of the Ψ epitranscriptome can arise from mutations and abnormal expression of pseudouridylation machinery, impacting protein translation and other cellular processes. With advancing understanding of Ψ roles in health and disease, efforts are now invested in developing therapeutic and diagnostic approaches targeting Ψ. Emerging reports indicate that Ψ and its installation machinery could be potential pharmacological targets for therapeutic development and may serve as biomarkers for human diseases. The presence of modified nucleosides (see Glossary) in RNA was discovered in the 1950s [1.Lane B.G. Historical perspectives on RNA nucleoside modifications.in: Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, 1998: 1-20Crossref Google Scholar]. 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Pseudouridine (Ψ), known as the 'fifth nucleotide' of RNA, was first discovered in 1951 [1.Lane B.G. Historical perspectives on RNA nucleoside modifications.in: Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, 1998: 1-20Crossref Google Scholar,8.Cohn W.E. Volkin E. Nucleoside-5-phosphates from ribonucleic acid.Nature. 1951; 167: 483-484Crossref Scopus (124) Google Scholar]. It is the most abundant post-transcriptional modification and is widespread in most RNA species [9.Li X. et al.Pseudouridine: the fifth RNA nucleotide with renewed interests.Curr. Opin. Chem. Biol. 2016; 33: 108-116Crossref PubMed Scopus (79) Google Scholar,10.Ge J. Yu Y.T. RNA pseudouridylation: new insights into an old modification.Trends Biochem. Sci. 2013; 38: 210-218Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar]. Ψ is a C–C glycosidic isomer of uridine (U) which incorporates the C5 atom of the nucleobase into the glycosidic bond such that Ψ has an extra hydrogen bond donor on the non-Watson–Crick edge and contributes to its host RNA stability [11.Kierzek E. et al.The contribution of pseudouridine to stabilities and structure of RNAs.Nucleic Acids Res. 2014; 42: 3492-3501Crossref PubMed Scopus (121) Google Scholar]. The dynamic nature of pseudouridylation and its importance in multiple cellular processes have prompted investigators to study the roles of Ψ in cancer and other human diseases so that novel therapeutics targeting Ψ and its processing machinery can be developed. In the present review we discuss the roles of Ψ in basic RNA biology, the emerging contributions of Ψ to cancer and genetic disorders, and the potential of pharmacological targeting of the Ψ epitranscriptome for therapeutic development. To better understand the contributions of Ψ to human diseases, it is important to first consider how Ψ is installed and how it affects structural and functional properties of RNA molecules. Dyskerin (DKC1) and stand-alone pseudouridine synthases (PUSs) catalyze pseudouridylation in an RNA-dependent and in RNA-independent mechanisms, respectively (Box 1) [12.Borchardt E.K. et al.Regulation and function of RNA pseudouridylation in human cells.Annu. Rev. Genet. 2020; 54: 309-336Crossref PubMed Scopus (23) Google Scholar, 13.Penzo M. Montanaro L. Turning uridines around: role of rRNA pseudouridylation in ribosome biogenesis and ribosomal function.Biomolecules. 2018; 8: 38Crossref Scopus (38) Google Scholar, 14.Rintala-Dempsey A.C. Kothe U. Eukaryotic stand-alone pseudouridine synthases – RNA modifying enzymes and emerging regulators of gene expression?.RNA Biol. 2017; 14: 1185-1196Crossref PubMed Scopus (73) Google Scholar]. In a complex with DKC1, small-nucleolar RNAs (snoRNAs) guide rRNA pseudouridylation, whereas small Cajal body-specific RNAs (scaRNAs) guide small nuclear RNA (snRNA) pseudouridylation. RNA-independent PUS enzymes decorate a variety of RNA substrates, including tRNAs, mRNAs, and others. When installed, Ψ confers distinct functional properties to its host RNAs, affecting protein translation, mRNA biogenesis, and other cellular processes.Box 1The mechanisms of pseudouridylationPseudouridylation is catalyzed by Ψ synthases (PUSs) in an RNA-independent or an RNA-dependent mechanism. In the RNA-independent mechanism, pseudouridylation is carried out by a single PUS (Figure IA). Human PUS enzymes share a core with a common fold, a conserved active-site cleft, and a catalytically essential aspartate residue [14.Rintala-Dempsey A.C. Kothe U. Eukaryotic stand-alone pseudouridine synthases – RNA modifying enzymes and emerging regulators of gene expression?.RNA Biol. 2017; 14: 1185-1196Crossref PubMed Scopus (73) Google Scholar]. Individual PUS proteins have both unique and overlapping substrates and can target diverse RNA species, including tRNAs, mRNAs, pre-mRNAs, and others. Moreover, PUS localization in the nucleus, cytoplasm, or mitochondria, as well as relocalization upon stimuli, can influence the RNA substrate pool available for pseudouridylation [21.Schwartz S. et al.Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.Cell. 2014; 159: 148-162Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar].The RNA-dependent mechanism relies on RNA–protein complexes known as the box H/ACA small ribonucleoproteins, which consist of a box H/ACA noncoding RNA and four core proteins, DKC1, GAR1, NOP10, and NHP2 (Figure IB) [100.Hamma T. Ferre-D'Amare A.R. The box H/ACA ribonucleoprotein complex: interplay of RNA and protein structures in post-transcriptional RNA modification.J. Biol. Chem. 2010; 285: 805-809Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. Box H/ACA noncoding RNAs adopt a hairpin–hinge–hairpin–tail structure, in which the hinge and tail part work as a sequence-specific pairing guide RNA, whereas the hairpin part forms a large internal loop referred to as the pseudouridylation pocket [56.McMahon M. et al.Small RNAs with big implications: new insights into H/ACA snoRNA function and their role in human disease.Wiley Interdiscip. Rev. RNA. 2015; 6: 173-189Crossref PubMed Scopus (78) Google Scholar]. DKC1 interacts with the box H/ACA noncoding RNA and installs Ψ on a substrate RNA within the pseudouridylation pocket. Box H/ACA noncoding RNAs are found in the nucleolus and the Cajal bodies and are thus classified as snoRNAs and scaRNAs, respectively. Whereas snoRNAs guide pseudouridylation of rRNAs, scaRNAs are required for snRNA pseudouridylation. Both rRNA and snRNA are pseudouridylated at functionally important regions, while the lack of pseudouridylation can affect rRNA and snRNA functionality [13.Penzo M. Montanaro L. Turning uridines around: role of rRNA pseudouridylation in ribosome biogenesis and ribosomal function.Biomolecules. 2018; 8: 38Crossref Scopus (38) Google Scholar,28.Deryusheva S. Gall J.G. Orchestrated positioning of post-transcriptional modifications at the branch point recognition region of U2 snRNA.RNA. 2018; 24: 30-42Crossref PubMed Scopus (11) Google Scholar,29.Bohnsack M.T. Sloan K.E. Modifications in small nuclear RNAs and their roles in spliceosome assembly and function.Biol. Chem. 2018; 399: 1265-1276Crossref PubMed Scopus (52) Google Scholar]. Apart from its role in catalyzing U isomerization into Ψ, DKC1 also interacts with the telomerase RNA component (TERC), leading to its stabilization [41.Nagpal N. Agarwal S. Telomerase RNA processing: Implications for human health and disease.Stem Cells. 2020; 38: 1532-1543Crossref Scopus (12) Google Scholar]. Pseudouridylation is catalyzed by Ψ synthases (PUSs) in an RNA-independent or an RNA-dependent mechanism. In the RNA-independent mechanism, pseudouridylation is carried out by a single PUS (Figure IA). Human PUS enzymes share a core with a common fold, a conserved active-site cleft, and a catalytically essential aspartate residue [14.Rintala-Dempsey A.C. Kothe U. Eukaryotic stand-alone pseudouridine synthases – RNA modifying enzymes and emerging regulators of gene expression?.RNA Biol. 2017; 14: 1185-1196Crossref PubMed Scopus (73) Google Scholar]. Individual PUS proteins have both unique and overlapping substrates and can target diverse RNA species, including tRNAs, mRNAs, pre-mRNAs, and others. Moreover, PUS localization in the nucleus, cytoplasm, or mitochondria, as well as relocalization upon stimuli, can influence the RNA substrate pool available for pseudouridylation [21.Schwartz S. et al.Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.Cell. 2014; 159: 148-162Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar]. The RNA-dependent mechanism relies on RNA–protein complexes known as the box H/ACA small ribonucleoproteins, which consist of a box H/ACA noncoding RNA and four core proteins, DKC1, GAR1, NOP10, and NHP2 (Figure IB) [100.Hamma T. Ferre-D'Amare A.R. The box H/ACA ribonucleoprotein complex: interplay of RNA and protein structures in post-transcriptional RNA modification.J. Biol. Chem. 2010; 285: 805-809Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. Box H/ACA noncoding RNAs adopt a hairpin–hinge–hairpin–tail structure, in which the hinge and tail part work as a sequence-specific pairing guide RNA, whereas the hairpin part forms a large internal loop referred to as the pseudouridylation pocket [56.McMahon M. et al.Small RNAs with big implications: new insights into H/ACA snoRNA function and their role in human disease.Wiley Interdiscip. Rev. RNA. 2015; 6: 173-189Crossref PubMed Scopus (78) Google Scholar]. DKC1 interacts with the box H/ACA noncoding RNA and installs Ψ on a substrate RNA within the pseudouridylation pocket. Box H/ACA noncoding RNAs are found in the nucleolus and the Cajal bodies and are thus classified as snoRNAs and scaRNAs, respectively. Whereas snoRNAs guide pseudouridylation of rRNAs, scaRNAs are required for snRNA pseudouridylation. Both rRNA and snRNA are pseudouridylated at functionally important regions, while the lack of pseudouridylation can affect rRNA and snRNA functionality [13.Penzo M. Montanaro L. Turning uridines around: role of rRNA pseudouridylation in ribosome biogenesis and ribosomal function.Biomolecules. 2018; 8: 38Crossref Scopus (38) Google Scholar,28.Deryusheva S. Gall J.G. Orchestrated positioning of post-transcriptional modifications at the branch point recognition region of U2 snRNA.RNA. 2018; 24: 30-42Crossref PubMed Scopus (11) Google Scholar,29.Bohnsack M.T. Sloan K.E. Modifications in small nuclear RNAs and their roles in spliceosome assembly and function.Biol. Chem. 2018; 399: 1265-1276Crossref PubMed Scopus (52) Google Scholar]. Apart from its role in catalyzing U isomerization into Ψ, DKC1 also interacts with the telomerase RNA component (TERC), leading to its stabilization [41.Nagpal N. Agarwal S. Telomerase RNA processing: Implications for human health and disease.Stem Cells. 2020; 38: 1532-1543Crossref Scopus (12) Google Scholar]. The regulation of gene expression at the level of translation is a critical component in steering cellular programs both in development and disease. The importance of Ψ in translation is reflected by its widespread distribution, influencing structural and functional properties of key RNA modalities involved in translation: rRNA, tRNA, and mRNA (Table 1). Ψ accounts for about 1.4% of all bases in human rRNAs and has been shown to modulate rRNA conformational dynamics [13.Penzo M. Montanaro L. Turning uridines around: role of rRNA pseudouridylation in ribosome biogenesis and ribosomal function.Biomolecules. 2018; 8: 38Crossref Scopus (38) Google Scholar,15.Jiang J. et al.Modulation of conformational changes in helix 69 mutants by pseudouridine modifications.Biophys. Chem. 2015; 200-201: 48-55Crossref PubMed Scopus (5) Google Scholar]. Moreover, Ψ is clustered at functionally important regions of rRNA, including the binding sites of tRNAs and mRNAs [10.Ge J. Yu Y.T. RNA pseudouridylation: new insights into an old modification.Trends Biochem. Sci. 2013; 38: 210-218Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar]. rRNA Ψ increases the affinity for tRNA binding to the A and P sites, whereas defects of rRNA pseudouridylation decrease ribosome–ligand interactions and translational fidelity [16.Jack K. et al.rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells.Mol. Cell. 2011; 44: 660-666Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar]. Ψ is found in nearly all tRNAs within the TΨC stem–loop at Ψ55, while other Ψ sites are found less frequently at many other tRNA positions, such as Ψ13 and Ψ39, and can stabilize the specific structure of tRNAs [17.Gray M. Charette Michael W. Pseudouridine in RNA: what, where, how, and why.IUBMB Life. 2000; 49: 341-351Crossref PubMed Scopus (376) Google Scholar,18.Motorin Y. Helm M. tRNA stabilization by modified nucleotides.Biochemistry. 2010; 49: 4934-4944Crossref PubMed Scopus (300) Google Scholar]. Ψ also influences protein translation by altering the properties of tRNA-derived fragments (tRFs); Guzzi et al. found that one type of tRF – termed mTOGs – inhibited cap-dependent protein translation only when decorated at Ψ8 by PUS7 [19.Guzzi N. et al.Pseudouridylation of tRNA-derived fragments steers translational control in stem cells.Cell. 2018; 173: 1204-1216. e26Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar]. It is worth noting that Ψ8 has not been detected in tRNAs and thus must have been installed in tRFs [2.Boccaletto P. et al.MODOMICS: a database of RNA modification pathways. 2021 update.Nucleic Acids Res. 2022; 50: D231-D235Crossref PubMed Scopus (22) Google Scholar].Table 1The roles Ψ in RNA biologyProcessRNA classFunctionStudied modelDetection methodRefsTranslationrRNAAffects translational fidelityVariousHPLC[16.Jack K. et al.rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells.Mol. Cell. 2011; 44: 660-666Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar]TranslationrRNAAlters mRNA decodingVariousSCARLET[37.McMahon M. et al.A single H/ACA small nucleolar RNA mediates tumor suppression downstream of oncogenic RAS.elife. 2019; 8e48847Crossref Scopus (54) Google Scholar]TranslationrRNAModulates conformational dynamicsThermodynamic analysisUsed chemically synthesized RNA[15.Jiang J. et al.Modulation of conformational changes in helix 69 mutants by pseudouridine modifications.Biophys. Chem. 2015; 200-201: 48-55Crossref PubMed Scopus (5) Google Scholar]TranslationtRNARegulates tRF functionhESCsSCARLET and CMC-based RT-qPCR[19.Guzzi N. et al.Pseudouridylation of tRNA-derived fragments steers translational control in stem cells.Cell. 2018; 173: 1204-1216. e26Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar]TranslationtRNAModulates codon-biased translationGSCsPseudo-seq and DM-Ψ-seq[38.Cui Q. et al.Targeting PUS7 suppresses tRNA pseudouridylation and glioblastoma tumorigenesis.Nat. Cancer. 2021; 2: 932-949Crossref PubMed Scopus (9) Google Scholar]TranslationmRNASuppresses translation termination and expands the genetic codeS. cerevisiaeUsed chemically synthesized RNA[24.Karijolich J. Yu Y.T. Converting nonsense codons into sense codons by targeted pseudouridylation.Nature. 2011; 474: 395Crossref PubMed Scopus (209) Google Scholar]TranslationmRNAAccommodates noncanonical base pairsCrystallographic analysisUsed chemically synthesized RNA[25.Fernandez I.S. et al.Unusual base pairing during the decoding of a stop codon by the ribosome.Nature. 2013; 500: 107-110Crossref PubMed Scopus (108) Google Scholar]TranslationmRNAAffects kinetic parameters of ribosomesVariousUsed chemically synthesized RNA[26.Eyler D.E. et al.Pseudouridinylation of mRNA coding sequences alters translation.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 23068-23074Crossref PubMed Scopus (50) Google Scholar]SplicingsnRNAModulates pre-mRNA splicingS. cerevisiaeCMC-based primer extension and TLC[32.Basak A. Query C.C. A pseudouridine residue in the spliceosome core is part of the filamentous growth program in yeast.Cell Rep. 2014; 8: 966-973Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar,33.Wu G. et al.U2 snRNA is inducibly pseudouridylated at novel sites by Pus7p and snR81 RNP.EMBO J. 2011; 30: 79-89Crossref PubMed Scopus (109) Google Scholar]Splicingpre-mRNAModulates pre-mRNA splicingX. laevisUsed in vitro synthesized RNA[34.Chen C. et al.A flexible RNA backbone within the polypyrimidine tract is required for U2AF65 binding and pre-mRNA splicing in vivo.Mol. Cell. Biol. 2010; 30: 4108-4119Crossref PubMed Scopus (30) Google Scholar]Splicingpre-mRNAModulates alternative splicingHepG2 cell linePseudo-seq[35.Martinez N.M. et al.Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect pre-mRNA processing.Mol. Cell. 2022; 82: 645-659 e9Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar]RNA stabilityVariousStabilizes RNA duplexesThermodynamic analysisUsed chemically synthesized RNA[11.Kierzek E. et al.The contribution of pseudouridine to stabilities and structure of RNAs.Nucleic Acids Res. 2014; 42: 3492-3501Crossref PubMed Scopus (121) Google Scholar]Protein–RNA interactionVariousModulates protein bindingVariousUsed chemically synthesized RNA[97.Vaidyanathan P.P. et al.Pseudouridine and N(6)-methyladenosine modifications weaken PUF protein/RNA interactions.RNA. 2017; 23: 611-618Crossref PubMed Scopus (32) Google Scholar, 98.Delorimier E. et al.Pseudouridine modification inhibits muscleblind-like 1 (MBNL1) binding to CCUG repeats and minimally structured RNA through reduced RNA flexibility.J. Biol. Chem. 2017; 292: 4350-4357Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 99.Levi O. Arava Y.S. Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase.Nucleic Acids Res. 2021; 49: 432-443Crossref PubMed Scopus (10) Google Scholar]Response to environmentVariousModulates response to heat shock, nutrient deprivation, and othersVariousVarious[20.Carlile T.M. et al.Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.Nature. 2014; 515: 143-146Crossref PubMed Scopus (560) Google Scholar, 21.Schwartz S. et al.Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.Cell. 2014; 159: 148-162Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 22.Li X. et al.Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome.Nat. Chem. Biol. 2015; 11: 592-597Crossref PubMed Scopus (292) Google Scholar,27.Song D. et al.HSP90-dependent PUS7 overexpression facilitates the metastasis of colorectal cancer cells by regulating LASP1 abundance.J. Exp. Clin. Cancer Res. 2021; 40: 170Crossref PubMed Scopus (2) Google Scholar,31.Wu G. et al.The TOR signaling pathway regulates starvation-induced pseudouridylation of yeast U2 snRNA.RNA. 2016; 22: 1146-1152Crossref PubMed Scopus (12) Google Scholar,33.Wu G. et al.U2 snRNA is inducibly pseudouridylated at novel sites by Pus7p and snR81 RNP.EMBO J. 2011; 30: 79-89Crossref PubMed Scopus (109) Google Scholar]HPLC, high-performance liquid chromatography; SCARLET, site-specific cleavage and radioactive labeling followed by ligation-assisted extraction and thin-layer chromatography; tRF, tRNA-derived fragment; hESCs, human embryonic stem cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CMC, N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide p-tosylate; GSCs, glioblastoma stem cells; Pseudo-seq and DM-Ψ-seq, high-throughput Ψ detection methods; S. cerevisiae, Saccharomyces cerevisiae; TLC, thin-layer chromatography; X. laevis, Xenopus laevis. Open table in a new tab HPLC, high-performance liquid chromatography; SCARLET, site-specific cleavage and radioactive labeling followed by ligation-assisted extraction and thin-layer chromatography; tRF, tRNA-derived fragment; hESCs, human embryonic stem cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CMC, N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide p-tosylate; GSCs, glioblastoma stem cells; Pseudo-seq and DM-Ψ-seq, high-throughput Ψ detection methods; S. cerevisiae, Saccharomyces cerevisiae; TLC, thin-layer chromatography; X. laevis, Xenopus laevis. Until recently, mapping of Ψ in mRNA has been technically challenging due to a lower abundance of mRNA transcripts as compared to other classes of RNA, limiting our understanding of Ψ roles in mRNA biology. With the development of high-throughput Ψ detection methods, thousands of Ψ sites have been identified on mRNA, revealing ubiquitous and dynamic distribution with a tendency to be enriched in the coding sequence (CDS) and the 3′-untranslated region (3′UTR) [20.Carlile T.M. et al.Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.Nature. 2014; 515: 143-146Crossref PubMed Scopus (560) Google Scholar, 21.Schwartz S. et al.Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.Cell. 2014; 159: 148-162Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 22.Li X. et al.Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome.Nat. Chem. Biol. 2015; 11: 592-597Crossref PubMed Scopus (292) Google Scholar, 23.Karijolich J. et al.Transcriptome-wide dynamics of RNA pseudouridylation.Nat. Rev. Mol. Cell Biol. 2015; 16: 581-585Crossref PubMed Scopus (73) Google Scholar]. Although its context-dependent regulation is only beginning to be understood, Ψ is involved in multiple steps of mRNA processing (Figure 1) and has been mostly studied in protein translation. Karijolich and Yu demonstrated that Ψ-modified termination codons (ΨAA, ΨAG, and ΨGA) suppressed translation termination in yeast, leading to stop codon readthrough in premature termination codon (PTC)-bearing mRNAs [24.Karijolich J. Yu Y.T. Converting nonsense codons into sense codons by targeted pseudouridylation.Nature. 2011; 474: 395Crossref PubMed Scopus (209) Google Scholar]. The unusual base-pairing geometry of Ψ-modified codons during translation was further confirmed by crystallographic analysis [25.Fernandez I.S. et al.Unusual base pairing during the decoding of a stop codon by the ribosome.Nature. 2013; 500: 107-110Crossref PubMed Scopus (108) Google Scholar]. These data suggest that targeted pseudouridylation could be used to regulate nonsense suppression or even expand the genetic code, with ΨAA and ΨAG found to encode serine or threonine and ΨGA – tyrosine or phenylalanine. Moreover, Eyler et al. demonstrated that various kinetic parameters of ribosomes were affected in the presence of pseudouridylated codons, which were associated with more frequent amino acid substitutions as compared to non-pseudouridylated codons [26.Eyler D.E. et al.Pseudouridinylation of mRNA coding sequences alters translation.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 23068-23074Crossref PubMed Scopus (50) Google Scholar]. Importantly, the authors argued that the effects of the mRNA Ψ modification on translation were context-dependent, indicating the complexity of Ψ-dependent regulation and the lack of generalized mechanisms. It is also becoming evident that the mRNA Ψ epitranscriptome is remodeled in response to environmental stimuli and cellular stress. Transcriptome-wide profiling revealed 265 Ψ sites that were induced in Saccharomyces cerevisiae upon heat shock, which could be attributed to the relocation of a PUS protein from the nucleus to the cytoplasm [21.Schwartz S. et al.Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.Cell. 2014; 159: 148-162Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar]. In our study, we found that stimuli, including heat shock and H2O2 treatment, contributed to stimuli-specific dynamic changes in the Ψ landscape of human cells [22.Li X. et al.Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome.Nat. Chem. Biol. 2015; 11: 592-597Crossref PubMed Scopus (292) Google Scholar]. Furthermore, a study by Song et al. implicated a heat-shock protein HSP90 as a critical binding partner of PUS7, suggesting a link between pseudouridylation and response to stress [27.Song D. et al.HSP90-dependent PUS7 overexpression facilitates the metastasis of colorectal cancer cells by regulating LASP1 abundance.J. Exp. Clin. Cancer Res. 2021; 40: 170Crossref PubMed Scopus (2) Google Scholar]. Biochemical assays confirmed that HSP90 stabilized PUS7 and prevented its proteasomal degradation. These data indicate that Ψ may provide an extra layer of gene expression regulation, which is dependent on changing cellular and environmental conditions. However, the consequences of dynamic and inducible pseudouridylation on individual mRNA molecules remain largely unknown due to limited understanding of protein factors that may interact with Ψ-decorated mRNA molecules and guide their processing (Figure 1). All five spliceosomal snRNAs are extensively pseudouridylated in a tightly regulated and orderly process, which is critical for correct assembly of the spliceosome [28.Deryusheva S. Gall J.G. Orchestrated positioning of post-transcriptional modifications at the branch point recognition region of U2 snRNA.RNA. 2018; 24: 30-42Crossref PubMed Scopus (11) Google Scholar, 29.Bohnsack M.T. Sloan K.E. Modifications in small nuclear RNAs and their roles in spliceosome assembly and function.Biol. Chem. 2018; 399: 1265-1276Crossref PubMed Scopus (52) Google Scholar, 30.Morais P. et al.Spliceosomal snRNA epitranscriptomics.Front. Genet. 2021; 12652129Crossref PubMed Scopus (12) Google Scholar]. Moreover, findings that yeast U2 and U6 snRNA pseudouridylation can be induced by distinct cellular states suggest that Ψ may influence the spliceosomal machinery on a global scale and in response to environmental cues (Table 1) [31.Wu G. et al.The TOR signaling pathway regulates starvation-induced pseudouridylation of yeast U2 snRNA.RNA. 2016; 22: 1146-1152Crossref PubMed Scopus (12) Google Scholar,32.Basak A. Query C.C. A p