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.

HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA.

Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed 'HydraPsiSeq', a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10-50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20-25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.

Chemical constituents from the fruiting bodies of Cryptoporus volvatus.

New drimane-type sesquiterpene cryptoporol A (1), cryptoporic acid derivative 6'-cryptoporic acid E methyl ester (2), and pseudouridine derivative cryptoporine A (3), as well as a known ergosterol 5α,8α-epidioxy-22E-ergosta-6,22-dien-3ß-ol (4), were isolated from a 90 % alcohol extract of the fruiting bodies of Cryptoporus volvatus. The structures of these compounds were established by spectroscopic analysis and circular dichroism. 5α,8α-epidioxy-22E-ergosta-6,22-dien-3ß-ol (4) exhibited antiviral activity against porcine reproductive and respiratory syndrome virus, and all compounds showed weak antioxidant activities.

Pseudouridylation meets next-generation sequencing.

The isomerization of uridine to pseudouridine (Ψ), known as pseudouridylation, is the most abundant post-transcriptional modification of stable RNAs. Due to technical limitations in pseudouridine detection methods, studies on pseudouridylation have historically focused on ribosomal RNAs, transfer RNAs, and spliceosomal small nuclear RNAs, where Ψs play a critical role in RNA biogenesis and function. Recently, however, a series of deep sequencing methods-Pseudo-seq, Ψ-seq, PSI-seq, and CeU-seq-has been published to map Ψ positions across the entire transcriptome with single nucleotide resolution. These data have greatly expanded the catalogue of pseudouridylated transcripts, which include messenger RNAs and noncoding RNAs. Furthermore, these methods have revealed conditionally-dependent sites of pseudouridylation that appear in response to cellular stress, suggesting that pseudouridylation may play a role in dynamically modulating RNA function. Collectively, these methods have opened the door to further study of the biological relevance of naturally occurring Ψs. However, an in-depth comparison of these techniques and their results has not yet been undertaken despite all four methods relying on the same basic principle: Ψ detection through selective chemical labeling by the carbodiimide known as CMC. In this article, we will outline the currently available high-throughput Ψ-detection methods and present a comparative analysis of their results. We will then discuss the merits and limitations of these approaches, including those inherent in CMC conjugation, and their potential to further elucidate the function of this ubiquitous and dynamic modification.

Probing RNA Modification Status at Single-Nucleotide Resolution in Total RNA.

RNA modifications, with over one hundred known so far, are commonly proposed to fine-tune the structure and function of RNA. While modifications in rRNA and tRNA are used to modulate RNA folding and decoding properties, little is known about the function of internal modifications in mRNA/lncRNA, which includes N(6)-methyl adenosine (m(6)A), 5-methyl cytosine (m(5)C), 2'-O-methylated nucleotides (Nm), pseudouridine (Ψ), and possible others. Functional studies of mRNA/lncRNA modifications have been hindered by the lack of methods for their identification at single-nucleotide resolution. Challenges for the determination of mRNA/lncRNA modifications at single-nucleotide resolution are mainly due to the low abundance of mRNA/lncRNA. Traditional deep sequencing methods cannot identify mRNA/lncRNA modifications, such as m(6)A, m(5)C, Nm, and Ψ, because reverse transcriptase is insensitive to their presence in cDNA synthesis. Antibody-based approach enables the identification of m(6)A regions in mRNA/lncRNA, but currently at ~100 nucleotide resolution. Here, we describe a method that accurately identifies m(6)A position and modification fraction in human mRNA and lncRNAs at single-nucleotide resolution, termed "Site-specific Cleavage And Radioactive-labeling followed by Ligation-assisted Extraction and Thin-layer chromatography (SCARLET)." This method combines two previously established techniques, site-specific cleavage and splint ligation, to probe the RNA modification status at any mRNA/lncRNA site in the total RNA pool. SCARLET can potentially analyze any nucleotide that maintains Watson-Crick base pairing in the transcriptome and determine whether it contains m(6)A, m(5)C, Nm, Ψ, or other modifications yet to be discovered. Precise determination of the position and modification fraction of RNA modifications reveals crucial parameters for functional investigation of RNA modifications.

Pseudo-Seq: Genome-Wide Detection of Pseudouridine Modifications in RNA.

RNA molecules contain a variety of chemically diverse, posttranscriptionally modified bases. The most abundant modified base found in cellular RNAs, pseudouridine (Ψ), has recently been mapped to hundreds of sites in mRNAs, many of which are dynamically regulated. Though the pseudouridine landscape has been determined in only a few cell types and growth conditions, the enzymes responsible for mRNA pseudouridylation are universally conserved, suggesting many novel pseudouridylated sites remain to be discovered. Here, we present Pseudo-seq, a technique that allows the identification of sites of pseudouridylation genome-wide with single-nucleotide resolution. In this chapter, we provide a detailed description of Pseudo-seq. We include protocols for RNA isolation from Saccharomyces cerevisiae, Pseudo-seq library preparation, and data analysis, including descriptions of processing and mapping of sequencing reads, computational identification of sites of pseudouridylation, and assignment of sites to specific pseudouridine synthases. The approach presented here is readily adaptable to any cell or tissue type from which high-quality mRNA can be isolated. Identification of novel pseudouridylation sites is an important first step in elucidating the regulation and functions of these modifications.

Pseudouridine in mRNA: Incorporation, Detection, and Recoding.

It has long been known that pseudouridine (Ψ) is the most abundant modified nucleotide in stable RNAs, including tRNA, rRNA, and snRNA. Recent studies using massive parallel sequencing have uncovered the presence of hundreds of Ψs in mRNAs as well. In eukaryotes and archaea, RNA pseudouridylation is introduced predominantly by box H/ACA RNPs, RNA-protein complexes each consisting of a single RNA moiety and four core proteins. It has been well established that Ψ plays an essential role in regulating the structure and function of stable RNAs in several model organisms, including yeast, Xenopus laevis, and humans. However, the functional role of Ψ in mRNA remains to be elucidated. One possibility (and true for stop/termination codons) is that Ψ influences decoding during translation. It is imperative, therefore, to establish a system, in which one can site-specifically introduce pseudouridylation into target mRNA and biochemically test the impact of mRNA pseudouridylation on protein translation. Here, we present a method for (1) site-specific conversion of uridine into Ψ in mRNA by designer box H/ACA RNP, (2) detection of Ψ in target mRNA using site-specific labeling followed by nuclease digestion and thin layer chromatography, and (3) analysis of recoding of pseudouridylated premature termination codon in mRNA during translation.

Pseudouridine Chemical Labeling and Profiling.

Pseudouridine (Ψ) is the most abundant posttranscriptional RNA modification; yet little is known about its prevalence and function in messenger RNA, mostly due to the challenges in the transcriptome-wide detection of Ψ. Here, we report CeU-Seq-a selective chemical labeling and pull-down method for the comprehensive analysis of transcriptome-wide pseudouridylation; this sequencing method will hopefully pave the way for functional studies of Ψ-mediated biological regulation in the future.

Pseudouridine, an antimutagenic substance in beer towards N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Previously we reported that beer is antimutagenic against several food-derived mutagens including heterocyclic amines. We describe here the isolation and identification of pseudouridine from beer as an antimutagenic substance against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). All of the 17 samples of beer tested showed inhibition of the MNNG mutagenicity in Salmonella. Extensive fractionation through chromatography of the active components from a freeze-dried beer sample gave six antimutagenic fractions. One contained pseudouridine, as characterized by the UV spectra, nuclear magnetic resonance, and co-chromatography in HPLC. Pure pseudouridine inhibited the mutagenicity of MNNG in a dose-dependent manner. The amount of pseudouridine in the beer sample, estimated at about 0.4 mg/100 ml beer, can account for 3% of the total antimutagenicity of beer. Thus, the major active components in beer remain to be identified. The role of pseudouridine in inhibiting the mutagenicity of MNNG is to be studied further. Among analogs of pseudouridine, spongouridine, but not uridine, was antimutagenic against MNNG. The bacterial mutagenicity of another methylating agent N-methyl-N-nitrosourea was also inhibited by pseudouridine. Pseudouridine is the first example among nucleosides to be shown to possess an antimutagenic property.

Introduction of an intervening sequence into a human serine suppressor tRNA gene: effects on gene expression in vitro and in vivo.

The 13 nucleotide Xenopus laevis tyrosine tRNA gene intervening sequence was into a human serine suppressor tRNA gene which lacked an intron, by site-directed mutagenesis. Analysis of the products of in vitro transcription in a HeLa cell extract indicates that the intervening sequence is accurately removed to generate a mature sized RNA identical to that obtained from an intron-less gene. Analysis of the transcripts obtained in vitro and in vivo shows that the U in the CUA anticodon sequence is partially modified to psi. Total TRNA isolated from cells infected with recombinant SV40 viruses carrying the mutant tRNA genes is active in suppression of UAG codons in a reticulocyte cell-free system. Cotransfection of COS cells with the mutant tRNA genes and a mutant chloramphenicol acetyltransferase gene containing the termination codon UAG demonstrated that the tRNA functions as a UAG suppressor in vivo. Analysis of 32P-labeled RNA obtained from infected cells showed, however, that cells infected with the intron-containing gene accumulate less mature tRNA than cells infected with the intron-less tRNA genes.

Purification and properties of a mammalian tRNA pseudouridine synthase.

A tRNA pseudouridine synthase has been extensively purified from steer thymus extracts, using undermodified tRNA from hisT- mutants of Salmonella typhimurium as a substrate. The enzyme synthesizes a group of psi residues in the anticodon region of various hisT- isoacceptors and behaves like a eukaryotic homologue of Salmonella tRNA psi synthase I. The thymus enzyme requires a thiol and a monovalent cation (NH4+ or K+) for optimum activity; no energy sources or cofactors are required. The activity is inhibited by single tRNAs or bulk tRNA from all sources tested, and by ribosomal RNAs, various polyribonucleotides, and DNA. The enzyme modifies the two hisT- tRNAPhe isoacceptors, both tRNATyr acceptors and at least five of the tRNALeu isoacceptors to products that coelute with the respective wild type species on reverse-phase columns. With pure hisT- tRNA2Phe as substrate, the enzyme specifically converts residue U39 to psi. Interestingly, a psi residue is still present at position 32, in the anticodon loop of hisT- tRNA2Phe, indicating the existence of other uncharacterized pseudouridylation enzymes in S. typhimurium. These composite results show that the thymus enzyme can form psi at residues 38, 39, and 40 in the anticodon region of appropriate hisT- isoacceptors. During the enzyme purification, a second activity is partially resolved, which releases 3H from wild type S. typhimurium [pyrimidine-5-3H]tRNA. This activity may be associated with an enzyme that pseudouridylates sites that are uniquely modified in eukaryotic tRNAs, but not in Salmonella tRNAs. Our observations support the view that the psi residues in tRNA are synthesized by a family of enzymes, whose members act on uridine residues in specific regions of the molecule.