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Background: Pseudouridine (Ψ), a C5-glycoside isomer of uridine, stands as one of the most prevalent RNA modifications in all RNA types. Distinguishing from the C-N bond linking uridine to ribose, the link between Ψ and ribose is a C-C bond, endowing Ψ modified RNA distinct properties and functions in various biological processes. The conversion of uridine to Ψ is governed by pseudouridine synthases (PUSs). RNA pseudouridylation is implicated in cancer biology and therapeutics., Objectives: In this review, we will summarize the methods for detecting Ψ, the process of Ψ generation, the impact of Ψ modification on RNA metabolism and gene expression, the roles of dysregulated Ψ and pseudouridine synthases in cancers, and the underlying mechanism., Methods: We conducted a comprehensive search of PubMed from its inception through February 2024. The search terms included "pseudouridine"; "pseudouridine synthase"; "PUS"; "dyskerin"; "cancer"; "tumor"; "carcinoma"; "malignancy"; "tumorigenesis"; "biomarker"; "prognosis" and "therapy". We included studies published in peer-reviewed journals that focused on Ψ detection, specific mechanisms involving Ψ and PUSs, and prognosis in cancer patients with high Ψ expression. We excluded studies lacking sufficient methodological details or appropriate controls., Results: Ψ has been recognized as a significant biomarker in cancer diagnosis and prognosis. Abnormal Ψ modifications mediated by various PUSs result in dysregulated RNA metabolism and impaired RNA function, promoting the development of various cancers. Overexpression of PUSs is common in cancer cells and predicts poor prognosis. PUSs inhibition arrests cell proliferation and enhances apoptosis in cancer cells, suggesting PUS-targeting cancer therapy may be a potential strategy in cancer treatment., Discussion: High Ψ levels in serum, urine, and saliva may suggest cancer, but do not specify the type, requiring additional lab markers and imaging for accurate diagnosis. Standardized detection methods are also crucial for reliable results. PUSs are linked to cancer, but more researches are needed to understand their mechanisms in different cancers. Anticancer treatments targeting PUSs are still under developed., (© 2024. The Author(s).)

Pseudouridine (Ψ) is an abundant RNA chemical modification that can play critical roles in the biological functions of RNA, and RNA-therapeutic applications. Current Ψ detection methods are limited in identifying Ψs at base-resolution in U-rich sequence contexts, where Ψ occurs frequently. The N-cyclohexyl N'-(2-morpholinoethyl)carbodiimide (CMC) can selectively label Ψ in RNA by forming the CMC-Ψ adduct. Here we report that an evolved reverse transcriptase ("RT-1306") shows promoted read-through and mutation against the CMC-Ψ. The mutation signature can resolve the occurrence of Ψs within UU-containing sequences. We developed "Mut-Ψ-seq" utilizing CMC and RT-1306 for transcriptome-wide mapping of Ψ at base-resolution. The mutation signatures robustly identify reported Ψs in human rRNAs via the ROC analysis, and elongated CMC reaction duration increases the detection sensitivity of Ψ. We report a high-confidence list of Ψ sites in polyA-enriched RNAs from HEK-293T cells identified by orthogonal chemical treatments (CMC and bisulfite). The mutation signatures resolve the position of Ψ in UU-containing sequences, revealing diverse occurrence of Ψs in such sequences. This work provides new methods and datasets for biological research of Ψ, and demonstrates the potential of combining the reverse transcriptase engineering and selective chemical labeling to expand the toolkit for RNA chemical modifications studies.

Here we report the efficient synthetic access to 13 C/ 15 N-labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75 nt E. coli tRNA Gly . The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis. 13 C/ 15 N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5'-monophosphate (ΨMP) by using YeiN, a Ψ-5'-monophosphate C-glycosidase. Removal of the 5'-phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ -building block carrying both a 13 C and a 15 N label was incorporated into a product RNA and the complex formation with a 63 nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75 nt E. coli tRNA Gly CCmnm 5 U, identifying the TΨC-loop 5-methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI-labelled Ψ building blocks will allow the solution and solid-state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ-modified nucleic acids., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Almost all elongator tRNAs (Transfer RNAs) harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, generated by the enzymes TrmA and TruB, respectively, in Escherichia coli. TrmA and TruB both act as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wild type. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of trmA and truB in E. coli causes a global decrease in aminoacylation and alters other tRNA modifications such as acp 3 U47. While overall protein synthesis is not affected in Δ trmA and Δ truB strains, the translation of a subset of codons is significantly impaired. As a consequence, we observe translationally reduced expression of many specific proteins, that are either encoded with a high frequency of these codons or that are large proteins. The resulting proteome changes are not related to a specific growth phenotype, but overall cellular fitness is impaired upon deleting trmA and truB in accordance with a general protein synthesis impact. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing tRNA maturation, tRNA aminoacylation, and translation, thereby improving cellular fitness irrespective of the growth conditions which explains the conservation of trmA and truB ., Competing Interests: Competing interests statement:The authors declare no competing interest.

Therapeutic modified mRNAs are being developed for a broad range of human diseases. However, the impact of potential miscoding of modified mRNAs on self-tolerance remains unknown. Additionally, more studies are needed to explore the effects of nucleoside alkylation on translation. While all six tested modifications are tolerated as substrates by T7 RNA polymerase and inhibited mRNA immunogenicity, the translation efficiency varied significantly depending on the type of modification. In contrast to methylation, ethylation at the N1 position of pseudouridine (Ψ) hindered translation, suggesting that the C5-C1' glycosidic bond alone is not a critical element for high translation. Inhibition of mRNA translation was also observed with 5-methoxyuridine modification. However, this inhibition was partially alleviated through the optimization of mRNA coding sequences. BALB/c mice immunized with syngeneic ψ-modified mRNA encoding for Wilms' tumor antigen-1 (WT1) developed a low but significant level of anti-WT1 IgG antibodies compared to those immunized with either unmodified or N1-methyl ψ-modified mRNA. Overall, the data indicate that adding a simple ethyl group (-CH 2 CH 3 ) at the N1 position of ψ has a major negative effect on translation despite its reduced immunogenicity. Additionally, mRNA containing Ψ may alter translation fidelity at certain codons, which could lead to a breakdown of immune tolerance to self-antigens. This concern should be taken into account during gene replacement therapies, although it could benefit mRNA-based vaccines by generating a diverse repertoire of antigens.

Nanopore direct RNA sequencing provided a promising solution for unraveling the landscapes of modifications on single RNA molecules. Here, we proposed NanoMUD, a computational framework for predicting the RNA pseudouridine modification (Ψ) and its methylated analog N1-methylpseudouridine (m1Ψ), which have critical application in mRNA vaccination, at single-base and single-molecule resolution from direct RNA sequencing data. Electric signal features were fed into a bidirectional LSTM neural network to achieve improved accuracy and predictive capabilities. Motif-specific models (NNUNN, N = A, C, U or G) were trained based on features extracted from designed dataset and achieved superior performance on molecule-level modification prediction (Ψ models: min AUC = 0.86, max AUC = 0.99; m1Ψ models: min AUC = 0.87, max AUC = 0.99). We then aggregated read-level predictions for site stoichiometry estimation. Given the observed sequence-dependent bias in model performance, we trained regression models based on the distribution of modification probabilities for sites with known stoichiometry. The distribution-based site stoichiometry estimation method allows unbiased comparison between different contexts. To demonstrate the feasibility of our work, three case studies on both in vitro and in vivo transcribed RNAs were presented. NanoMUD will make a powerful tool to facilitate the research on modified therapeutic IVT RNAs and provides useful insight to the landscape and stoichiometry of pseudouridine and N1-pseudouridine on in vivo transcribed RNA species., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

According to the CDC, both Pfizer and Moderna COVID-19 vaccines contain nucleoside-modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS-CoV-2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS-CoV-2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1-methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex-diverse people. The translation of 1-methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off-target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18-flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days). This review article focuses on medical biochemistry, proteomics and deutenomics principles that explain the persisting spike phenomenon in circulation with organ-related functional damage even in asymptomatic individuals. Proline and hydroxyproline residues emerge as prominent deuterium (heavy hydrogen) binding sites in structural proteins with robust isotopic stability that resists not only enzymatic breakdown, but virtually all (non)-enzymatic cleavage mechanisms known in chemistry., (© 2024 The Author(s). Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function. To decipher the molecular mechanism of this phenotype, we determine the structure of ribosomes lacking the single Ψ and its parental strain at ∼2.4-3 Å resolution using cryo-EM. Our findings demonstrate the significance of a single Ψ on H69 to its structure and the importance for its interactions with helix 44 and specific tRNAs. Our study suggests that rRNA modification affects translation of mRNAs carrying codon bias due to selective accommodation of tRNAs by the ribosome. Based on the high-resolution structures, we propose a mechanism explaining how the ribosome selects specific tRNAs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes ( snR34 , snR52, and snR65 ) and/or expressing enzymatically inactive variants of spb1(D52A/E679K) and nop2(C424A/C478A) were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification m 5 C2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.

In this study, we proposed a biological approach to efficiently produce pseudouridine (Ψ) from glucose and uracil in vivo using engineered Escherichia coli . By screening host strains and core enzymes, E. coli MG1655 overexpressing Ψ monophosphate (ΨMP) glycosidase and ΨMP phosphatase was obtained, which displayed the highest Ψ concentration. Then, optimization of the RBS sequences, enhancement of ribose 5-phosphate supply in the cells, and overexpression of the membrane transport protein UraA were investigated. Finally, fed-batch fermentation of Ψ in a 5 L fermentor can reach 27.5 g/L with a yield of 89.2 mol % toward uracil and 25.6 mol % toward glucose within 48 h, both of which are the highest to date. In addition, the Ψ product with a high purity of 99.8% can be purified from the fermentation broth after crystallization. This work provides an efficient and environmentally friendly protocol for allowing for the possibility of Ψ bioproduction on an industrial scale.

Pseudouridine is an abundant mRNA modification found in diverse organisms ranging from bacteria and viruses to multicellular plants and humans. New developments in pseudouridine profiling provide quantitative tools to map mRNA pseudouridylation sites. Sparse biochemical studies establish the potential for mRNA pseudouridylation to affect most stages of the mRNA life cycle from birth to death. This recent progress sets the stage for deeper investigations into the molecular and cellular functions of specific mRNA pseudouridines, including in disease., (© 2024 Gilbert; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Over the past decade, advancements in epitranscriptomics have significantly enhanced our understanding of mRNA metabolism and its role in human development and diseases. This period has witnessed breakthroughs in sequencing technologies and the identification of key proteins involved in RNA modification processes. Alongside the well-studied m 6 A, Ψ and m 1 A have emerged as key epitranscriptomic markers. Initially identified through transcriptome-wide profiling, these modifications are now recognized for their broad impact on RNA metabolism and gene expression. In this Perspective, we focus on the detections and functions of Ψ and m 1 A modifications in mRNA and discuss previous discrepancies and future challenges. We summarize recent advances and highlight the latest sequencing technologies for stoichiometric detection and their mechanistic investigations for functional unveiling in mRNA as the new research directions., (© 2024 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA pseudouridine modification exists in different RNA types of many species, and it has a significant role in regulating the expression of biological processes. To understand the functional mechanisms for RNA pseudouridine sites, the accurate identification of pseudouridine sites in RNA sequences is essential. Although several fast and inexpensive computational methods have been proposed, the challenge of improving recognition accuracy and generalization still exists. This study proposed a novel ensemble predictor called PseUpred-ELPSO for improved RNA pseudouridine site prediction. After analyzing the nucleotide composition preferences between RNA pseudouridine site sequences, two feature representations were determined and fed into the stacking ensemble framework. Then, using five tree-based machine learning classifiers as base classifiers, 30-dimensional RNA profiles are constructed to represent RNA sequences, and using the PSO algorithm, the weights of the RNA profiles were searched to further enhance the representation. A logistic regression classifier was used as a meta-classifier to complete the final predictions. Compared to the most advanced predictors, the performance of PseUpred-ELPSO is superior in both cross-validation and the independent test. Based on the PseUpred-ELPSO predictor, a free and easy-to-operate web server has been established, which will be a powerful tool for pseudouridine site identification.

Pseudouridine is the most abundant modified nucleoside found in non-coding RNA and is widely used in biological and pharmaceutical fields. However, current methods for pseudouridine production suffer from drawbacks such as complex procedures, low efficiency and high costs. This study presents a novel enzymatic cascade reaction route in Escherichia coli , enabling the whole-cell catalytic synthesis of pseudouridine from uridine. Initially, a metabolic pathway was established through plasmid-mediated overexpression of endogenous pseudouridine-5-phosphase glycosidase, ribokinase, and ribonucleoside hydrolase, resulting in the accumulation of pseudouridine. Subsequently, highly active endogenous ribonucleoside hydrolase was screened to enhance uridine hydrolysis and provide more precursors for pseudouridine synthesis. Furthermore, modifications were made to the substrates and products transport pathways to increase the pseudouridine yield while avoiding the accumulation of by-product uridine. The resulting recombinant strain Ψ-7 catalyzed the conversion of 30 g/L uridine into 27.24 g/L pseudouridine in 24 h, achieving a conversion rate of 90.8% and a production efficiency of 1.135 g/(L·h). These values represent the highest reported yield and production efficiency achieved by enzymatic catalysis methods to date.

The pseudouridine (ψ) synthase, RluD is responsible for three ψ modifications in the helix 69 (H69) of bacterial 23S rRNA. While normally dispensable, rluD becomes critical for rapid cell growth in bacteria that are defective in translation-termination. In slow-growing rluD - bacteria, suppressors affecting termination factors RF2 and RF3 arise frequently and restore normal termination and rapid cell growth. Here we describe two weaker suppressors, affecting rpsG, encoding ribosomal protein uS7 and ssrA, encoding tmRNA. In K-12 strains of E. coli, rpsG terminates at a TGA codon. In the suppressor strain, alteration of an upstream CAG to a TAG stop codon results in a shortened uS7 and partial alleviation of slow growth, likely by replacing an inefficient TGA stop codon with the more efficient TAG. Inefficient termination events, such as occurs in some rluD - strains, are targeted by trans-translation. Inactivation of the ssrA gene in slow-growing, termination-defective mutants lacking RluD and RF3, also partially restores robust growth, most probably by preventing destruction of completed polypeptides on ribosomes at slow-terminating stop codons. Finally, an additional role for RluD has been proposed, independent of its pseudouridine synthase activity. This is based on the observation that plasmids expressing catalytically dead (D139N or D139T) RluD proteins could nonetheless restore robust growth to an E. coli K-12 rluD - mutant. However, newly constructed D139N and D139T rluD plasmids do not have any growth-restoring activity and the original observations were likely due to the appearance of suppressors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nucleoside-modified messenger RNA (mRNA) technologies necessarily incorporate N1-methylpseudouridine into the mRNA molecules to prevent the over-stimulation of cytoplasmic RNA sensors. Despite this modification, mRNA concentrations remain mostly determined through the measurement of UV absorbance at 260 nm wavelength (A 260 ). Herein, we report that the N1-methylpseudouridine absorbs approximately 40% less UV light at 260 nm than uridine, and its incorporation into mRNAs leads to the under-estimation of nucleoside-modified mRNA concentrations, with 5%-15% error, in an mRNA-sequence-dependent manner. We therefore examined the RNA quantification methods and developed the mRNACalc webserver. It accounts for the molar absorption coefficient of modified nucleotides at 260 nm wavelength, the RNA composition of the mRNA, and the A 260 of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m 6 A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development., (© 2024 Wiley Periodicals LLC.)

RNA-guided pseudouridylation, a widespread post-transcriptional RNA modification, has recently gained recognition for its role in cellular processes such as pre-mRNA splicing and the modulation of premature termination codon (PTC) readthrough. This review provides insights into its mechanisms, functions, and potential therapeutic applications. It examines the mechanisms governing RNA-guided pseudouridylation, emphasizing the roles of guide RNAs and pseudouridine synthases in catalyzing uridine-to-pseudouridine conversion. A key focus is the impact of RNA-guided pseudouridylation of U2 small nuclear RNA on pre-mRNA splicing, encompassing its influence on branch site recognition and spliceosome assembly. Additionally, the review discusses the emerging role of RNA-guided pseudouridylation in regulating PTC readthrough, impacting translation termination and genetic disorders. Finally, it explores the therapeutic potential of pseudouridine modifications, offering insights into potential treatments for genetic diseases and cancer and the development of mRNA vaccine., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Nucleotide modifications are occurrent in all types of RNA and play an important role in RNA structure formation and stability. Modified bases not only possess the ability to shift the RNA structure ensemble towards desired functional confirmations. By changes in the base pairing partner preference, they may even enlarge or reduce the conformational space, i.e., the number and types of structures the RNA molecule can adopt. However, most methods to predict RNA secondary structure do not provide the means to include the effect of modifications on the result. With the help of a heavily modified transfer RNA (tRNA) molecule, this chapter demonstrates how to include the effect of different base modifications into secondary structure prediction using the ViennaRNA Package. The constructive approach demonstrated here allows for the calculation of minimum free energy structure and suboptimal structures at different levels of modified base support. In particular we, show how to incorporate the isomerization of uridine to pseudouridine ( Ψ ) and the reduction of uridine to dihydrouridine (D)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The identification of RNA modifications at single nucleotide resolution has become an emerging area of interest within biology and specifically among virologists seeking to ascertain how this untapped area of RNA regulation may be altered or hijacked upon viral infection. Herein, we describe a straightforward biochemical approach modified from two original published Ψ mapping protocols, BID-seq and PRAISE, to specifically identify pseudouridine modifications on mRNA transcripts from an HIV-1 infected T cell line. This protocol could readily be adapted for other viral infected cell types and additionally for populations of purified virions from infected cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Therapeutic mRNAs are generated using modified nucleotides, namely N 1 -methylpseudouridine (m 1 Ψ) triphosphate, so that the mRNA evades detection by the immune system. RNA modifications, even at a single-nucleotide position, perturb RNA structure, although it is not well understood how structure and function is impacted by globally modified RNAs. Therefore, we examined the metastasis-associated lung adenocarcinoma transcript 1 triple helix, a highly structured stability element that includes single-, double-, and triple-stranded RNA, globally modified with N 6 -methyladenosine (m 6 A), pseudouridine (Ψ), or m 1 Ψ. UV thermal denaturation assays showed that m 6 A destabilizes both the Hoogsteen and Watson-Crick faces of the RNA by ∼20 °C, Ψ stabilizes the Hoogsteen and Watson-Crick faces of the RNA by ∼12 °C, and m 1 Ψ has minimal effect on the stability of the Hoogsteen face of the RNA but increases the stability of the Watson-Crick face by ∼9 °C. Native gel-shift assays revealed that binding of the methyltransferase-like protein 16 to the metastasis-associated lung adenocarcinoma transcript 1 triple helix was weakened by at least 8-, 99-, and 23-fold, respectively, when RNA is globally modified with m 6 A, Ψ, or m 1 Ψ. These results demonstrate that a more thermostable RNA structure does not lead to tighter RNA-protein interactions, thereby highlighting the regulatory power of RNA modifications by multiple means., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The uridine modifications pseudouridine (Ψ), dihydrouridine, and 5-methyluridine are present in eukaryotic mRNAs. Many uridine-modifying enzymes are associated with human disease, underscoring the importance of uncovering the functions of uridine modifications in mRNAs. These modified uridines have chemical properties distinct from those of canonical uridines, which impact RNA structure and RNA-protein interactions. Ψ, the most abundant of these uridine modifications, is present across (pre-)mRNAs. Recent work has shown that many Ψs are present at intermediate to high stoichiometries that are likely conducive to function and at locations that are poised to influence pre-/mRNA processing. Technological innovations and mechanistic investigations are unveiling the functions of uridine modifications in pre-mRNA splicing, translation, and mRNA stability, which are discussed in this review., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The epitranscriptomics field has undergone tremendous growth since the discovery that the RNA N 6 -methyladenosine (m 6 A) modification is reversible and is distributed throughout the transcriptome. Efforts to map RNA modifications transcriptome-wide and reshape the epitranscriptome in disease settings have facilitated mechanistic understanding and drug discovery in the field. In this review we discuss recent advancements in RNA modification detection methods and consider how these developments can be applied to gain novel insights into the epitranscriptome. We also highlight drug discovery efforts aimed at developing epitranscriptomic therapeutics for cancer and other diseases. Finally, we consider engineering of the epitranscriptome as an emerging direction to investigate RNA modifications and their causal effects on RNA processing at high specificity., Competing Interests: Declaration of interests C.H. is a scientific founder, member of the scientific advisory board, and equity holder of Aferna Bio Inc. and AccuaDX Inc.; a scientific co-founder and equity holder of Accent Therapeutics Inc.; and a member of the scientific advisory board of Rona Therapeutics. No interests are declared by the remaining authors., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Regulatory RNAs, as well as many RNA families, contain chemically modified nucleotides, including pseudouridines (ψ). To map nucleotide modifications, approaches based on enzymatic digestion of RNA followed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis were implemented several years ago. However, detection of ψ by mass spectrometry (MS) is challenging as ψ exhibits the same mass as uridine. Thus, a chemical labeling strategy using acrylonitrile was developed to detect this mass-silent modification. Acrylonitrile reacts specifically to ψ to form 1-cyanoethylpseudouridine (Ceψ), resulting in a mass shift of ψ detectable by MS. Here, a protocol detailing the steps from the purification of RNA by polyacrylamide gel electrophoresis, including in-gel labeling of ψ, to MS data interpretation to map ψ and other modifications is proposed. To demonstrate its efficiency, the protocol was applied to bacterial regulatory RNAs from E. coli: 6S RNA and transfer-messenger RNA (tmRNA, also known as 10Sa RNA). Moreover, ribonuclease P (RNase P) was also mapped using this approach. This method enabled the detection of several ψ at single nucleotide resolution., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plasma nucleosides-pseudouridine (PU) and N 2 N 2 -dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes ( db/db mice) and control nondiabetic mice ( db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db Tubular dysfunction explains the association between the nucleosides pseudouridine and db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies. NEW & NOTEWORTHY Tubular dysfunction explains the association between the nucleosides pseudouridine and N 2 N 2 -dimethyl guanosine and diabetic kidney disease.

In the yeast Saccharomyces cerevisiae, the absence of the pseudouridine synthase Pus3/Deg1, which modifies tRNA positions 38 and 39, results in increased lipid droplet (LD) content and translational defects. In addition, starvation-like transcriptome alterations and induced protein aggregation were observed. In this study, we show that the deg1 mutant increases specific misreading errors. This could lead to altered expression of the main regulators of neutral lipid synthesis which are the acetyl-CoA carboxylase (Acc1), an enzyme that catalyzes a key step in fatty acid synthesis, and its regulator, the Snf1/AMPK kinase. We demonstrate that upregulation of the neutral lipid content of LD in the deg1 mutant is achieved by a mechanism operating in parallel to the known Snf1/AMPK kinase-dependent phosphoregulation of Acc1. While in wild-type cells removal of the regulatory phosphorylation site (Ser-1157) in Acc1 results in strong upregulation of triacylglycerol (TG), but not steryl esters (SE), the deg1 mutation more specifically upregulates SE levels. In order to elucidate if other lipid species are affected, we compared the lipidomes of wild type and deg1 mutants, revealing multiple altered lipid species. In particular, in the exponential phase of growth, the deg1 mutant shows a reduction in the pool of phospholipids, indicating a compromised capacity to mobilize acyl-CoA from storage lipids. We conclude that Deg1 plays a key role in the coordination of lipid storage and mobilization, which in turn influences lipid homeostasis. The lipidomic effects in the deg1 mutant may be indirect outcomes of the activation of various stress responses resulting from protein aggregation., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

In this report, we present our studies on mRNA, which was modified by introducing various halogen substituents at the C(5) position of the pyrimidine base. Specifically, we synthesized C(5)-halogenated (F, Cl, Br, I) pyrimidine ribonucleoside triphosphates and incorporated them into mRNA during in-vitro transcription. The efficiency of the in-vitro transcription reaction of halogenated pyrimidine was observed to decrease as the size of the halogen substituent increased and the electronegativity thereof decreased (F > Cl > Br) except for iodine. Interestingly, we found that, among the C(5)-halogenated pyrimidine ribonucleotides, mRNA incorporating C(5)-halogenated cytidine (5-F rCTP and 5-Cl rCTP) exhibited more prominent protein expression than mRNA modified with C(5)-halogenated uridine and unmodified mRNA. In particular, in the case of mRNA to which fluorine (5-F rCTP) and chlorine (5-Cl rCTP) were introduced, the protein was dramatically expressed about 4 to 5 times more efficiently than the unmodified mRNA, which was similar to pseudouridine (ψ). More interestingly, when pseudouridine(ψ) and fluorocytidine nucleotides (5-F rCTP), were simultaneously introduced into mRNA for dual incorporation, the protein expression efficiency dramatically increased as much as tenfold. The efficiency of cap-dependent protein expression is much higher than the IRES-dependent (internal ribosome entry site) expression with mRNA incorporating C(5)-halogenated pyrimidine ribonucleotide. We expect these results to contribute meaningfully to the development of therapeutics based on modified mRNA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pseudouridine is one of the well-known chemical modifications in various RNA species. Current advances to detect pseudouridine show that the pseudouridine landscape is dynamic and affects multiple cellular processes. Although our understanding of this post-transcriptional modification mainly depends on yeast and human models, the recent findings provide strong evidence for the critical role of pseudouridine in plants. Here, we review the current knowledge of pseudouridine in plant RNAs, including its synthesis, degradation, regulatory mechanisms, and functions. Moreover, we propose future areas of research on pseudouridine modification in plants., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Glioblastoma is the most common type of glioma with a high incidence and poor prognosis, and effective medical treatment remains challenging. Pseudouridine (Ψ) is the first post-transcriptional modification discovered and one of the most abundant modifications to RNA. However, the prognostic value of Ψ-related lncRNAs (ΨrLs) for glioma patients has never been systematically evaluated. This study aims to construct a risk model based on ΨrLs signature and to validate the predictive efficiency of the model., Method: Transcriptomic data, genomic data, and relevant clinical data of glioma patients were extracted from the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA). ΨrLs with significant correlation with Ψ-related genes were identified, and univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and multivariate Cox regression were used to further select biomarkers and construct a ΨrLs signature risk model. Then, the expression of lncRNAs of ΨrLs signature in multiple glioma cell lines was detected by qPCR. Further, ROC analysis, stratification analysis, correlation analysis, survival analysis, nomogram, enrichment analysis, immune infiltration analysis, chemoradiotherapy sensitivity analysis, somatic mutation, and recurrent copy number variation (CNV) analysis were used to validate the predictive efficiency of ΨrLs signature in TCGA and CGGA datasets., Results: A four-lncRNA ΨrLs signature (DNAJC27-AS1, GDNF-AS1, ZBTB20-AS4, and DNMBP-AS1) risk model was constructed. By ROC analysis, stratified analysis, correlation analysis, survival analysis, and nomogram, the signature showed satisfactory predictive efficiency. Functional enrichment analysis revealed the differences in immune-related biological processes between high- and low-risk groups. Immune infiltration analysis showed that the high-risk group had lower tumor purity and higher stromal, immune and ESTIMATE scores. Mitoxantrone was identified as effective drug for low-risk group of glioma patients. Key genes in glioma development, including IDH1, EGFR, PTEN, etc., were differentially mutated between risk groups. The main recurrent CNVs in low-risk groups were 19q13.42 deletion and 7q34 amplification; 10q23.31 deletion and 12q14.1 in the high-risk group., Conclusions: Our study identified a four-lncRNA ΨrLs signature that effectively predicts the prognosis of glioma patients and may serve as a diagnostic tool. Risk scores of glioma patients generated by the signature is associated with immune-related biological processes and chemoradiotherapy sensitivity. These findings may inform the development of more targeted and effective therapies for glioma patients., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, engages in complex interactions with host cell proteins throughout its life cycle. While these interactions enable the host to recognize and inhibit viral replication, they also facilitate essential viral processes such as transcription, translation, and replication. Many aspects of these virus-host interactions remain poorly understood. Here, we employed the cat RAPID algorithm and utilized the RNA-protein interaction detection coupled with mass spectrometry technology to predict and validate the host proteins that specifically bind to the highly structured 5' and 3' terminal regions of the SARS-CoV-2 RNA. Among the interactions identified, we prioritized pseudouridine synthase PUS7, which binds to both ends of the viral RNA. Using nanopore direct RNA sequencing, we discovered that the viral RNA undergoes extensive post-transcriptional modifications. Modified consensus regions for PUS7 were identified at both terminal regions of the SARS-CoV-2 RNA, including one in the viral transcription regulatory sequence leader. Collectively, our findings offer insights into host protein interactions with the SARS-CoV-2 UTRs and highlight the likely significance of pseudouridine synthases and other post-transcriptional modifications in the viral life cycle. This new knowledge enhances our understanding of virus-host dynamics and could inform the development of targeted therapeutic strategies., Competing Interests: L.M. has received financial support from ONT for travel and accommodations to attend and present at ONT events. T.L. is a paid consultant to STORM therapeutics limited. This research was conducted in the absence of any commercial or financial relationships., (© 2023 The Authors.)

The post-transcriptional RNA modifications impact the dynamic regulation of gene expression in diverse biological and physiological processes. Host RNA modifications play an indispensable role in regulating innate immune responses against virus infection in mammals. Meanwhile, the viral RNAs can be deposited with RNA modifications to interfere with the host immune responses. The N 6 -methyladenosine (m 6 A) has boosted the recent emergence of RNA epigenetics, due to its high abundance and a transcriptome-wide widespread distribution in mammalian cells, proven to impact antiviral innate immunity. However, the other types of RNA modifications are also involved in regulating antiviral responses, and the functional roles of these non-m 6 A RNA modifications have not been comprehensively summarized. In this Review , we conclude the regulatory roles of 2'- O C), adenosine-inosine editing (A-to-I editing), pseudouridine (Ψ), 5 C), adenosine-inosine editing (A-to-I editing), pseudouridine (Ψ), N 1 -methyladenosine (m 1 A), N 7 -methylguanosine (m 7 G), N 6 ,2'- O -dimethyladenosine (m 6 Am), and N 4 -acetylcytidine (ac 4 C) in antiviral innate immunity. We provide a systematic introduction to the biogenesis and functions of these non-m 6 A RNA modifications in viral RNA, host RNA, and during virus-host interactions, emphasizing the biological functions of RNA modification regulators in antiviral responses. Furthermore, we discussed the recent research progress in the development of antiviral drugs through non-m 6 A RNA modifications. Collectively, this Review conveys knowledge and inspiration to researchers in multiple disciplines, highlighting the challenges and future directions in RNA epitranscriptome, immunology, and virology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shen and Zhang.)

Endometrial cancer (EC) is a common gynaecological malignant tumour with unclear pathogenesis. Small nucleolar RNA (snoRNA) is involved in many biological processes, including those of cancers. Using the Cancer Genome Atlas (TCGA) database, the expression pattern of a snoRNA, SNORA73B, was analysed. The biological functions of SNORA73B were assessed by in vitro proliferation, apoptosis, migration, and invasion assays and in vivo by the xenograft model. RNA sequencing (RNA-seq) and RNA immunoprecipitation assays were performed to determine the relationship between SNORA73B and its target genes. High-performance liquid chromatography (HPLC) was performed to detect the pseudouridine content of the mindbomb E3 ubiquitin protein ligase 1 gene (MIB1). The stability of MIB1 mRNA was evaluated using a transcription inhibitor, actinomycin D. By performing co-immunoprecipitation assays, the change in the ubiquitin levels of the Jagged canonical Notch ligand 1 (Jag 1), caused by SNORA73B and MIB1, was identified. RNA-seq and qRT-PCR were performed to detect the alternative splicing of the regulator of the chromosome condensation 1 gene (RCC1). The TCGA database analysis showed that SNORA73B was highly expressed in EC. SNORA73B promoted cell proliferation, migration, and invasion and inhibited apoptosis. SNORA73B modified the pseudouridine content in MIB1 and increased the stability of MIB1 mRNA and protein; thus, it affected Jag 1 ubiquitination and further activated the Notch pathway. SNORA73B also affected the alternative splicing of RCC1, increasing the number of transcripts, RCC1-T2 and RCC1-T3, which promoted cell proliferation, migration, and invasion. SNORA73B can be a potential target for EC., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Bacterial ribosome small subunit rRNA (16S rRNA) contains 11 nucleotide modifications scattered throughout all its domains. The 16S rRNA pseudouridylation enzyme, RsuA, which modifies U516, is a survival protein essential for bacterial survival under stress conditions. A comparison of the growth curves of wildtype and RsuA knock-out E. coli strains illustrates that RsuA renders a survival advantage to bacteria under streptomycin stress. The RsuA-dependent growth advantage for bacteria was found to be dependent on its pseudouridylation activity. In addition, the role of RsuA as a trans-acting factor during ribosome biogenesis may also play a role in bacterial growth under streptomycin stress. Furthermore, circular dichroism spectroscopy measurements and RNase footprinting studies have demonstrated that pseudouridine at position 516 influences helix 18 structure, folding, and streptomycin binding. This study exemplifies the importance of bacterial rRNA modification enzymes during environmental stress.

It has been demonstrated that RNA modifications play essential roles in multiple biological processes. Accurate identification of RNA modifications in the transcriptome is critical for providing insights into the biological functions and mechanisms. Many tools have been developed for predicting RNA modifications at single-base resolution, which employ conventional feature engineering methods that focus on feature design and feature selection processes that require extensive biological expertise and may introduce redundant information. With the rapid development of artificial intelligence technologies, end-to-end methods are favorably received by researchers. Nevertheless, each well-trained model is only suitable for a specific RNA methylation modification type for nearly all of these approaches. In this study, we present MRM-BERT by feeding task-specific sequences into the powerful BERT (Bidirectional Encoder Representations from Transformers) model and implementing fine-tuning, which exhibits competitive performance to the state-of-the-art methods. MRM-BERT avoids repeated de novo training of the model and can predict multiple RNA modifications such as pseudouridine, m6A, m5C, and m1A in Mus musculus, Arabidopsis thaliana, and Saccharomyces cerevisiae. In addition, we analyse the attention heads to provide high attention regions for the prediction, and conduct saturated in silico mutagenesis of the input sequences to discover potential changes of RNA modifications, which can better assist researchers in their follow-up research.

Trypanosoma brucei and related kinetoplastid parasites possess unique RNA processing pathways, including in their mitochondria, that regulate metabolism and development. Altering RNA composition or conformation through nucleotide modifications is one such pathway, and modifications including pseudouridine regulate RNA fate and function in many organisms. We surveyed pseudouridine synthase (PUS) orthologs in trypanosomatids, with a particular interest in mitochondrial enzymes due to their potential importance for mitochondrial function and metabolism. Trypanosoma brucei mitochondrial (mt)-LAF3 is an ortholog of human and yeast mitochondrial PUS enzymes, and a mitoribosome assembly factor, but structural studies differ in their conclusion as to whether it has PUS catalytic activity. Here, we generated T. brucei cells that are conditionally null (CN) for mt-LAF3 expression and showed that mt-LAF3 loss is lethal and disrupts mitochondrial membrane potential (ΔΨm). Addition of a mutant gamma ATP synthase allele to the CN cells permitted ΔΨm maintenance and cell survival, allowing us to assess primary effects on mitochondrial RNAs. As expected, these studies showed that loss of mt-LAF3 dramatically decreases levels of mitochondrial 12S and 9S rRNAs. Notably, we also observed decreases in mitochondrial mRNA levels, including differential effects on edited vs. pre-edited mRNAs, indicating that mt-LAF3 is required for mitochondrial rRNA and mRNA processing, including of edited transcripts. To assess the importance of PUS catalytic activity in mt-LAF3 we mutated a conserved aspartate that is necessary for catalysis in other PUS enzymes and showed it is not essential for cell growth, or maintenance of ΔΨm and mitochondrial RNA levels. Together, these results indicate that mt-LAF3 is required for normal expression of mitochondrial mRNAs in addition to rRNAs, but that PUS catalytic activity is not required for these functions. Instead, our work, combined with previous structural studies, suggests that T. brucei mt-LAF3 acts as a mitochondrial RNA-stabilizing scaffold., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Pseudouridine is enriched in ribosomal, spliceosomal, transfer, and messenger RNA and thus integral to the central dogma. The chemical basis for how pseudouridine affects the molecular apparatus such as ribosome, however, remains elusive owing to the lack of structures without this natural modification. Here, we studied the translation of a hypopseudouridylated ribosome initiated by the internal ribosome entry site (IRES) elements. We analyzed eight cryo-electron microscopy structures of the ribosome bound with the Taura syndrome virus IRES in multiple functional states. We found widespread loss of pseudouridine-mediated interactions through water and long-range base pairings. In the presence of the translocase, eukaryotic elongation factor 2, and guanosine 5'-triphosphate hydrolysis, the hypopseudouridylated ribosome favors a rare unconducive conformation for decoding that is partially recouped in the ribosome population that remains modified at the P-site uridine. The structural principles learned establish the link between functional defects and modification loss and are likely applicable to other pseudouridine-associated processes.

Small RNAs (also referred to as small noncoding RNAs, sncRNA) are defined as polymeric ribonucleic acid molecules that are less than 200 nucleotides in length and serve a variety of essential functions within cells. Small RNA species include microRNA (miRNA), PIWI-interacting RNA (piRNA), small interfering RNA (siRNA), tRNA-derived small RNA (tsRNA), etc. Current evidence suggest that small RNAs can also have diverse modifications to their nucleotide composition that affect their stability as well as their capacity for nuclear export, and these modifications are relevant to their capacity to drive molecular signaling processes relevant to biogenesis, cell proliferation and differentiation. In this review, we highlight the molecular characteristics and cellular functions of small RNA and their modifications, as well as current techniques for their reliable detection. We also discuss how small RNA modifications may be relevant to the clinical applications for the diagnosis and treatment of human health conditions such as cancer., (© 2023. The Author(s).)

Pseudouridylation is one of the most abundant RNA modifications in eukaryotes, making pseudouridine known as the "fifth nucleoside." This highly conserved alteration affects all non-coding and coding RNA types. Its role and importance have been increasingly widely researched, especially considering that its absence or damage leads to serious hereditary diseases. Here, we summarize the human genetic disorders described to date that are related to the participants of the pseudouridylation process., (© 2023. The Author(s).)

Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. However, the effects of pseudouridine modifications on the structure and dynamics of RNAs have so far only been investigated in a limited number of different structural contexts. Here, we introduced pseudouridine modifications into the U-turn motif and the adjacent U:U closing base pair of the neomycin-sensing riboswitch (NSR)-an extensively characterized model system for RNA structure, ligand binding, and dynamics. We show that the effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, we rationalize the observed effects on a structural and dynamical level. Our results will help to better understand and predict the consequences of pseudouridine modifications on the structure and function of biologically important RNAs., (© 2023 Vögele et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Pseudouridine (Psi) is one of the most frequent post-transcriptional modification of RNA. Enzymatic Psi modification occurs on rRNA, snRNA, snoRNA, tRNA, and non-coding RNA and has recently been discovered on mRNA. Transcriptome-wide detection of Psi (Psi-seq) has yet to be performed for the widely studied model organism Drosophila melanogaster. Here, we optimized Psi-seq analysis for this species and have identified thousands of Psi modifications throughout the female fly head transcriptome. We find that Psi is widespread on both cellular and mitochondrial rRNAs. In addition, more than a thousand Psi sites were found on mRNAs. When pseudouridylated, mRNAs frequently had many Psi sites. Many mRNA Psi sites are present in genes encoding for ribosomal proteins, and many are found in mitochondrial encoded RNAs, further implicating the importance of pseudouridylation for ribosome and mitochondrial function. The 7SLRNA of the signal recognition particle is the non-coding RNA most enriched for Psi. The 3 mRNAs most enriched for Psi encode highly expressed yolk proteins (Yp1, Yp2, and Yp3). By comparing the pseudouridine profiles in the RluA-2 mutant and the w1118 control genotype, we identified Psi sites that were missing in the mutant RNA as potential RluA-2 targets. Finally, differential gene expression analysis of the mutant transcriptome indicates a major impact of loss of RluA-2 on the ribosome and translational machinery., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Genetics Society of America.)

Direct RNA sequencing with a commercial nanopore platform was used to sequence RNA containing uridine (U), pseudouridine (Ψ) or N1-methylpseudouridine (m1Ψ) in >100 different 5-nucleotide contexts. The base calling data for Ψ or m1Ψ were similar but different from U allowing their detection. Understanding the nanopore signatures for Ψ and m1Ψ enabled a running start T7 RNA polymerase assay to study the selection of UTP versus ΨTP or m1ΨTP competing mixtures in all possible adjacent sequence contexts. A significant sequence context dependency was observed for T7 RNA polymerase with insertion yields for ΨTP versus UTP spanning a range of 20-65%, and m1ΨTP versus UTP producing variable yields that differ by 15-70%. Experiments with SP6 RNA polymerase, as well as chemically-modified triphosphates and DNA templates provide insight to explain the observations. The SP6 polymerase introduced m1ΨTP when competed with UTP with a smaller window of yields (15-30%) across all sequence contexts studied. These results may aid in future efforts that employ RNA polymerases to make therapeutic mRNAs with sub-stoichiometric amounts of m1Ψ., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nonsense mutations create premature termination codons (PTCs), activating the nonsense-mediated mRNA decay (NMD) pathway to degrade most PTC-containing mRNAs. The undegraded mRNA is translated, but translation terminates at the PTC, leading to no production of the full-length protein. This work presents targeted PTC pseudouridylation, an approach for nonsense suppression in human cells. Specifically, an artificial box H/ACA guide RNA designed to target the mRNA PTC can suppress both NMD and premature translation termination in various sequence contexts. Targeted pseudouridylation exhibits a level of suppression comparable with that of aminoglycoside antibiotic treatments. When targeted pseudouridylation is combined with antibiotic treatment, a much higher level of suppression is observed. Transfection of a disease model cell line (carrying a chromosomal PTC) with a designer guide RNA gene targeting the PTC also leads to nonsense suppression. Thus, targeted pseudouridylation is an RNA-directed gene-specific approach that suppresses NMD and concurrently promotes PTC readthrough., Competing Interests: Declaration of interests A patent relating to this work was filed and granted. Y.-T.Y. serves as a member of the Scientific Advisory Board at ProQR Therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our workflow allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data and multiple modifications on the same strand., (© 2023. The Author(s).)

Post-transcriptional regulation of gene expression shapes the cell state both in health and disease. RNA modifications-especially N 6 -methyladenosine (m 6 A)-have recently emerged as key players in RNA processing that depends on a sophisticated interplay between proteins of the RNA modification machinery. Importantly, the RNA epitranscriptome becomes dysregulated in cancer and promotes cancer-associated gene expression programs as well as cancer cell adaptation to the tumor microenvironment. At the top of the tumor hierarchy, cancer stem cells (CSCs) are master regulators of tumorigenesis and resistance to therapeutic intervention. Therefore, defining how RNA modifications influence the CSC state is of great importance for cancer drug development. In this chapter, we summarize the current knowledge of the roles of RNA modifications in shaping the CSC state and driving gene expression programs that confer stem-like properties to CSCs, promote CSC adaptation to the local microenvironment, and endow CSCs with metastatic potential and drug resistance., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Synthesis of RNA standards that contain an internal site-specific modification is important for mapping and quantification of the modified nucleotide in sequencing analysis. While RNA containing a site-specific modification can be readily synthesized by solid-state coupling for less than 100-mer nucleotides, longer RNA must be synthesized by enzymatic ligation in the presence of a DNA splint. However, long RNAs have structural heterogeneity, and those generated by in vitro transcription have 3'-end sequence heterogeneity, which together substantially reduce the yield of ligation. Here we describe a method of 3-part splint ligation that joins an in vitro transcribed left-arm RNA, an in vitro transcribed right-arm RNA, and a chemically synthesized modification-containing middle RNA, with an efficiency higher than previously reported. We report that the improved efficiency is largely attributed to the inclusion of a pair of DNA disruptors proximal to the ligation sites, and to a lesser extent to the homogeneous processing of the 3'-end of the left-arm RNA. The yields of the ligated long RNA are sufficiently high to afford purification to homogeneity for practical RNA research. We also verify the sequence accuracy at each ligation junction by nanopore sequencing., (Copyright © 2023. Published by Elsevier Inc.)

Pseudouridine is a ubiquitous RNA modification and plays a crucial role in many biological processes. However, it remains a challenging task to identify pseudouridine sites using expensive and time-consuming experimental research. To this end, we present Porpoise, a computational approach to identify pseudouridine sites from RNA sequence data. Porpoise builds on a stacking ensemble learning framework with several informative features and achieves competitive performance compared with state-of-the-art approaches. This protocol elaborates on step-by-step use and execution of the local stand-alone version and the webserver of Porpoise. In addition, we also provide a general machine learning framework that can help identify the optimal stacking ensemble learning model using different combinations of feature-based features. This general machine learning framework can facilitate users to build their pseudouridine predictors using their in-house datasets., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Pseudouridine, a modified RNA residue formed by the isomerization of its parental U nucleotide, is prevalent in a majority of cellular RNAs; its presence was reported in tRNA, rRNA, and sn/snoRNA as well as in mRNA/lncRNA. Multiple analytical deep sequencing-based approaches have been proposed for pseudouridine detection and quantification, among which the most popular relies on the use of soluble carbodiimide (termed CMCT). Recently, we developed an alternative protocol for pseudouridine mapping and quantification. The principle is based on protection of pseudouridine against random RNA cleavage by hydrazine/aniline treatment (HydraPsiSeq protocol). This "negative" detection mode requires higher sequencing depth and provides a precise quantification of the pseudouridine content. All "wet-lab" technical details of the HydraPsiSeq protocol have been described in recent publications. Here, we describe all bioinformatics analysis steps required for data processing from raw reads to the pseudouridylation profile of known or unknown RNA., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Posttranscriptional RNA modifications occur in almost all types of RNA in all life forms. As an abundant RNA modification in mammals, pseudouridine (Ψ) regulates diverse biological functions of different RNA species such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), etc. However, the functional investigation of mRNA pseudouridine (Ψ) has been hampered by the lack of a quantitative method that can efficiently map Ψ transcriptome-wide. We developed bisulfite-induced deletion sequencing (BID-seq), with the optimized bisulfite-based chemical reaction to convert pseudouridine selectively and completely into Ψ-BS adduct without cytosine deamination. The Ψ-BS adduct can be further read out as deletion signatures during reverse transcription. The deletion ratios induced by Ψ sites were used for estimating the modification stoichiometry at each modified site. BID-seq starts with 10-20 ng polyA + RNA and detects thousands of mRNA Ψ sites with stoichiometry information in cell lines and tissues. We uncovered consensus motifs for Ψ in mammalian mRNA and assigned specific 'writer' proteins to individual Ψ deposition. BID-seq also confirmed the presence of Ψ within stop codons of mammalian mRNA. BID-seq set the stage for future investigations of Ψ functions in diverse biological processes., (Copyright © 2023. Published by Elsevier Inc.)

Ribonucleic acid (RNA) methylation is the most abundant modification in biological systems, accounting for 60% of all RNA modifications, and affects multiple aspects of RNA (including mRNAs, tRNAs, rRNAs, microRNAs, and long non-coding RNAs). Dysregulation of RNA methylation causes many developmental diseases through various mechanisms mediated by N 6 -methyladenosine (m 6 A), 5-methylcytosine (m 5 C), N 1 C), and pseudouridine (Ψ). The emerging tools of RNA methylation can be used as diagnostic, preventive, and therapeutic markers. Here, we review the accumulated discoveries to date regarding the biological function and dynamic regulation of RNA methylation/modification, as well as the most popularly used techniques applied for profiling RNA epitranscriptome, to provide new ideas for growth and development.1 A), 5-hydroxymethylcytosine (hm 5 C), and pseudouridine (Ψ). The emerging tools of RNA methylation can be used as diagnostic, preventive, and therapeutic markers. Here, we review the accumulated discoveries to date regarding the biological function and dynamic regulation of RNA methylation/modification, as well as the most popularly used techniques applied for profiling RNA epitranscriptome, to provide new ideas for growth and development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zou, Liu, Tan, Chen, Dong, Bai, Wu and Zeng.)