The molecular basis of tRNA selectivity by human pseudouridine synthase 3.

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

Long-lasting, biochemically modified mRNA, and its frameshifted recombinant spike proteins in human tissues and circulation after COVID-19 vaccination.

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.

Structural and mechanistic insights into the function of Leishmania ribosome lacking a single pseudouridine modification.

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.

A bisulfite-assisted and ligation-based qPCR amplification technology for locus-specific pseudouridine detection at base resolution.

Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE. pseU-TRACE relied on the fact that RNA containing Ψ underwent a base deletion after treatment of bisulfite (BS) during reverse transcription, which enabled efficient ligation of two probes complementary to the cDNA sequence on either side of the Ψ site and successful amplification in subsequent real-time quantitative PCR (qPCR), thereby achieving selective and accurate Ψ detection. Our method accurately and sensitively detected several known Ψ sites in 28S, 18S, 5.8S, and even mRNA. Moreover, pseU-TRACE could be employed to measure the Ψ fraction in RNA and explore the Ψ metabolism of different pseudouridine synthases (PUSs), providing valuable insights into the function of Ψ. Overall, pseU-TRACE represents a reliable, time-efficient and sensitive Ψ detection method.

Modified Nucleotides and RNA Structure Prediction.

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).

Identifying Individual Pseudouridine (Ψ) Sites Across Transcripts from HIV-1 Infected Cells.

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.

NanoMUD: Profiling of pseudouridine and N1-methylpseudouridine using Oxford Nanopore direct RNA sequencing.

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.

Efficient Synthetic Access to Stable Isotope Labelled Pseudouridine Phosphoramidites for RNA NMR Spectroscopy.

Here we report the efficient synthetic access to 13C/15N-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 tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis. 13C/15N 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 13C and a 15N 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 tRNAGly CCmnm5U, 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.

Review: N1-methyl-pseudouridine (m1Ψ): Friend or foe of cancer?

Due to the health emergency created by SARS-CoV-2, the virus that causes the COVID-19 disease, the rapid implementation of a new vaccine technology was necessary. mRNA vaccines, being one of the cutting-edge new technologies, attracted significant interest and offered a lot of hope. The potential of these vaccines in preventing admission to hospitals and serious illness in people with comorbidities has recently been called into question due to the vaccines' rapidly waning immunity. Mounting evidence indicates that these vaccines, like many others, do not generate sterilizing immunity, leaving people vulnerable to recurrent infections. Additionally, it has been discovered that the mRNA vaccines inhibit essential immunological pathways, thus impairing early interferon signaling. Within the framework of COVID-19 vaccination, this inhibition ensures an appropriate spike protein synthesis and a reduced immune activation. Evidence is provided that adding 100 % of N1-methyl-pseudouridine (m1Ψ) to the mRNA vaccine in a melanoma model stimulated cancer growth and metastasis, while non-modified mRNA vaccines induced opposite results, thus suggesting that COVID-19 mRNA vaccines could aid cancer development. Based on this compelling evidence, we suggest that future clinical trials for cancers or infectious diseases should not use mRNA vaccines with a 100 % m1Ψ modification, but rather ones with the lower percentage of m1Ψ modification to avoid immune suppression.

Fuzzy kernel evidence Random Forest for identifying pseudouridine sites.

Pseudouridine is an RNA modification that is widely distributed in both prokaryotes and eukaryotes, and plays a critical role in numerous biological activities. Despite its importance, the precise identification of pseudouridine sites through experimental approaches poses significant challenges, requiring substantial time and resources.Therefore, there is a growing need for computational techniques that can reliably and quickly identify pseudouridine sites from vast amounts of RNA sequencing data. In this study, we propose fuzzy kernel evidence Random Forest (FKeERF) to identify pseudouridine sites. This method is called PseU-FKeERF, which demonstrates high accuracy in identifying pseudouridine sites from RNA sequencing data. The PseU-FKeERF model selected four RNA feature coding schemes with relatively good performance for feature combination, and then input them into the newly proposed FKeERF method for category prediction. FKeERF not only uses fuzzy logic to expand the original feature space, but also combines kernel methods that are easy to interpret in general for category prediction. Both cross-validation tests and independent tests on benchmark datasets have shown that PseU-FKeERF has better predictive performance than several state-of-the-art methods. This new method not only improves the accuracy of pseudouridine site identification, but also provides a certain reference for disease control and related drug development in the future.

Systematic Engineering of Escherichia coli for Efficient Production of Pseudouridine from Glucose and Uracil.

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.

[Whole-cell catalytic production of pseudouridine by recombinant Escherichia coli].

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.

Locus-specific detection of pseudouridine with CRISPR-Cas13a.

We combined the CRISPR-Cas13a system with CMC chemical labeling, developing an approach that enables precise identification of pseudouridine (Ψ) sites at specific loci within ribosomal RNA (rRNA), messenger RNA (mRNA) and small nuclear RNAs (snRNA). This method, with good efficiency and simplicity, detects Ψ sites through fluorescence measurement, providing a straightforward and fast validation for targeted Ψ sites of interest.

Landscape of RNA pseudouridylation in archaeon Sulfolobus islandicus.

Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.

In-Gel Cyanoethylation for Pseudouridines Mass Spectrometry Detection of Bacterial Regulatory RNA.

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.

Pseudouridylation-mediated gene expression modulation.

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.

Pseudouridine-modified RNA probe for label-free electrochemical detection of nucleic acids on 2D MoS2 nanosheets.

RNA modification, particularly pseudouridine (Ψ), has played an important role in the development of the mRNA-based COVID-19 vaccine. This is because Ψ enhances RNA stability against nuclease activity and decreases the anti-RNA immune response. Ψ also provides structural flexibility to RNA by enhancing base stacking compared with canonical nucleobases. In this report, we demonstrate the first application of pseudouridine-modified RNA as a probe (Ψ-RNA) for label-free nucleic acid biosensing. It is known that MoS2 has a differential affinity for nucleic acids, which may be translated into a unique electronic signal. Herein, the Ψ-RNA probe interacts with the pristine MoS2 surface and causes a change in interfacial electrochemical charge transfer in the MoS2 nanosheets. Compared with an unmodified RNA probe, Ψ-RNA exhibited faster adsorption and higher affinity for MoS2. Moreover, Ψ-RNA could bind to complementary RNA and DNA targets with almost equal affinity when engaged with the MoS2 surface. Ψ-RNA maintained robust interactions with the MoS2 surface following the hybridization event, perhaps through its extra amino group. The detection sensitivity of the Ψ-RNA/MoS2 platform was as low as 500 attomoles, while the results also indicate that the probe can distinguish between complementary targets, single mismatches, and non-complementary nucleic acid sequences with statistical significance. This proof-of-concept study shows that the Ψ-RNA probe may solve numerous problems of adsorption-based biosensing platforms due to its stability and structural flexibility.

RNA oligomers at atomic resolution containing 1-methylpseudouridine, an essential building block of mRNA vaccines.

All widely used mRNA vaccines against COVID-19 contain in their sequence 1-methylpseudouridine (m1Ψ) instead of uridine. In this publication, we report two high resolution crystal structures (at up to 1.01 and 1.32 Å, respectively) of one such double-stranded 12-mer RNA sequence crystallized in two crystal forms. The structures are compared with similar structures which do not contain this modification. Additionally, the X-ray structure of 1-methyl-pseudouridine itself was determined.

Global RNA modifications to the MALAT1 triple helix differentially affect thermostability and weaken binding to METTL16.

Therapeutic mRNAs are generated using modified nucleotides, namely N1-methylpseudouridine (m1Ψ) 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 N6-methyladenosine (m6A), pseudouridine (Ψ), or m1Ψ. UV thermal denaturation assays showed that m6A 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 m1Ψ 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 m6A, Ψ, or m1Ψ. 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.

Predicting nearest neighbor free energies of modified RNA with LIE: results for pseudouridine and N1-methylpseudouridine within RNA duplexes.

Pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) are among the key modifications in the field of mRNA therapeutics and vaccine research. The accuracy of the design and development of therapeutic RNAs containing such modifications depends on the accuracy of the secondary structure prediction, which in turn depends on the nearest neighbor (NN) thermodynamic parameters for the standard and modified residues. Here, we propose a simple approach based on molecular dynamics simulations and linear interaction energy (LIE) approximation that is able to predict the NN free energy parameters for U-A, Ψ-A and m1Ψ-A pairs in reasonable agreement with the recent experimental reports. We report the NN thermodynamic parameters for different U, Ψ and m1Ψ base pairs, which might be helpful for a deeper understanding of the effect of these modifications in RNA. The predicted NN free energy parameters in this study are able to closely reproduce the folding free energies of duplexes containing internal Ψ for which the thermodynamic data were available. Additionally, we report the predicted folding free energies for the duplexes containing internal m1Ψ.