RESUMO
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.
Assuntos
Microscopia Crioeletrônica , Hidroliases , Transferases Intramoleculares , Pseudouridina , RNA de Transferência , Humanos , Domínio Catalítico , Células HEK293 , Hidroliases/metabolismo , Hidroliases/genética , Hidroliases/química , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/enzimologia , Modelos Moleculares , Mutação , Ligação Proteica , Pseudouridina/metabolismo , Pseudouridina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , Especificidade por SubstratoRESUMO
Pseudouridine (Ψ), the most abundant RNA modification, plays a role in pre-mRNA splicing, RNA stability, protein translation efficiency, and cellular responses to environmental stress. Dysregulation of pseudouridylation is linked to human diseases. This review explores recent insights into the role of RNA pseudouridylation alterations in human disorders and the therapeutic potential of Ψ. We discuss the impact of the reduction of Ψ levels in ribosomal, messenger, and transfer RNA in RNA processing, protein translation, and consequently its role in neurodevelopmental diseases and cancer. Furthermore, we review the success of N1-methyl-Ψ messenger RNA vaccines against COVID-19 and the development of RNA-guided pseudouridylation enzymes for treating genetic diseases caused by premature stop codons.
Assuntos
COVID-19 , Pseudouridina , Humanos , Pseudouridina/metabolismo , Pseudouridina/genética , COVID-19/genética , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Neoplasias/patologia , SARS-CoV-2/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/terapia , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Vacinas contra COVID-19 , Biossíntese de Proteínas , Splicing de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismoRESUMO
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.
Assuntos
HIV-1 , Pseudouridina , RNA Mensageiro , RNA Viral , Pseudouridina/metabolismo , Pseudouridina/genética , HIV-1/genética , Humanos , RNA Viral/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Infecções por HIV/virologia , Infecções por HIV/genética , Processamento Pós-Transcricional do RNA , Linhagem CelularRESUMO
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.
Assuntos
Pseudouridina , Reação em Cadeia da Polimerase em Tempo Real , Sulfitos , Humanos , Pseudouridina/química , Pseudouridina/genética , Pseudouridina/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , RNA/química , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Sulfitos/químicaRESUMO
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.
Assuntos
Pseudouridina , Algoritmo Florestas Aleatórias , Pseudouridina/genética , RNA/genética , Sequência de BasesRESUMO
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.
Assuntos
Escherichia coli , Pseudouridina , Pseudouridina/genética , Pseudouridina/química , Pseudouridina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Uridina/genética , Uridina/química , Uridina/metabolismo , Catálise , Hidrolases/metabolismoRESUMO
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.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Pseudouridina , Pseudouridina/genética , RNA Nuclear Pequeno/genética , RNA Ribossômico , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Pseudouridina , Precursores de RNA , Pseudouridina/genética , Pseudouridina/metabolismo , Precursores de RNA/metabolismo , RNA Guia de Sistemas CRISPR-Cas , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Biossíntese de ProteínasRESUMO
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.
Assuntos
Acrilonitrila , Pseudouridina , Humanos , Pseudouridina/genética , Espectrometria de Massas em Tandem , Escherichia coli/genética , Escherichia coli/metabolismo , RNA , RNA Bacteriano/metabolismo , Nucleotídeos , Processamento Pós-Transcricional do RNA , RNA de Transferência/genéticaRESUMO
Pseudouridine (Ψ) is an abundant RNA modification that is present in and affects the functions of diverse non-coding RNA species, including rRNA, tRNA and small nuclear RNA. Ψ also exists in mammalian mRNA and probably exhibits functional roles; however, functional investigations of mRNA Ψ modifications in mammals have been hampered by the lack of a quantitative method that detects Ψ at base precision. We have recently developed bisulfite-induced deletion sequencing (BID-seq), which provides the community with a quantitative method to map RNA Ψ distribution transcriptome-wide at single-base resolution. Here, we describe an optimized BID-seq protocol for mapping Ψ distribution across cellular mRNAs, which includes fast steps in both library preparation and data analysis. This protocol generates highly reproducible results by inducing high deletion ratios at Ψ modification within diverse sequence contexts, and meanwhile displayed almost zero background deletions at unmodified uridines. When used for transcriptome-wide Ψ profiling in mouse embryonic stem cells, the current protocol uncovered 8,407 Ψ sites from as little as 10 ng of polyA+ RNA input. This optimized BID-seq workflow takes 5 days to complete and includes four main sections: RNA preparation, library construction, next-generation sequencing (NGS) and data analysis. Library construction can be completed by researchers who have basic knowledge and skills in molecular biology and genetics. In addition to the experimental protocol, we provide BID-pipe ( https://github.com/y9c/pseudoU-BIDseq ), a user-friendly data analysis pipeline for Ψ site detection and modification stoichiometry quantification, requiring only basic bioinformatic and computational skills to uncover Ψ signatures from BID-seq data.
Assuntos
Pseudouridina , Transcriptoma , Animais , Camundongos , Pseudouridina/análise , Pseudouridina/genética , RNA Mensageiro/genética , Perfilação da Expressão Gênica/métodos , RNA Ribossômico/genética , Mamíferos/genéticaRESUMO
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.
Assuntos
Pseudouridina , RNA , Humanos , Pseudouridina/genética , Pseudouridina/metabolismo , RNA Mensageiro/metabolismo , RNA/metabolismo , Uridina/química , Uridina/metabolismo , Processamento Pós-Transcricional do RNA , Precursores de RNA/genéticaRESUMO
Nucleophilic addition of bisulfite to pyrimidine bases has been known for a half century, and the reaction has been in use for at least a quarter of a century for identifying 5-methylcytidine in DNA. This account focuses on the chemistry of bisulfite with pseudouridine, an isomer of the RNA nucleoside uridine in which the uracil base is connected to C1' of ribose via C5 instead of N1. Pseudouridine, Ψ, is the most common nucleotide modification found in cellular RNA overall, in part due to its abundance in rRNAs and tRNAs. It has a stabilizing influence on RNA structure because N1 is now available for additional hydrogen bonding and because the heterocycle is slightly better at π stacking. The isomerization of U to Ψ in RNA strands is catalyzed by 13 different enzymes in humans and 11 in E. coli; some of these enzymes are implicated in disease states which is testament to the biological importance of pseudouridine in cells. Recently, pseudouridine came into the limelight as the key modification that, after N1 methylation, enables mRNA vaccines to be delivered efficiently into human tissue with minimal generation of a deleterious immunogenic response. Here we describe the bisulfite reaction with pseudouridine which gives rise to a chemical sequencing method to map the modified base in the epitranscriptome. Unlike the reaction with cytidine, the addition of bisulfite to Ψ leads irreversibly to form an adduct that is bypassed during cDNA synthesis by reverse transcriptases yielding a characteristic deletion signature. Although there were hints to the structure of the bisulfite adduct(s) 30 to 50 years ago, it took modern spectroscopic and computational methods to solve the mystery. Raman spectroscopy along with extensive NMR, ECD, and computational work led to the assignment of the major product as the (R) diastereomer of an oxygen adduct at C1' of a ring-opened pseudouridine. Mechanistically, this arose from a succession of conjugate addition, E2 elimination, and a [2,3] sigmatropic rearrangement, all of which are stereodefined reactions. A minor reaction with excess bisulfite led to the (S) isomer of a S-adducted SO3- group. Understanding structure and mechanism aided the design of a Ψ-specific sequencing reaction and guided attempts to improve the utility and specificity of the method. Separately, we have been investigating the use of nanopore direct RNA sequencing, a single-molecule method that directly analyzes RNA strands isolated from cells after end-ligation of adaptor sequences. By combining the electrical current and base-calling data from the nanopore with dwell-time analysis from the helicase employed to deliver RNA to the nanopore, we were able to map Ψ sites in nearly all sequence contexts. This analysis was employed to find Ψ residues in the SARS-CoV-2 vRNA, to analyze the sequence context effects of mRNA vaccine synthesis via in vitro transcription, and to evaluate the impact of stress on chemical modifications in the E. coli ribosome. Most recently, we found that bisulfite treatment of RNA leading to Ψ adducts could modulate the nanopore signal to help in mapping modifications of low occupancy.
Assuntos
COVID-19 , Sequenciamento por Nanoporos , Humanos , RNA/química , Pseudouridina/química , Pseudouridina/genética , Pseudouridina/metabolismo , Escherichia coli/metabolismo , COVID-19/genética , SARS-CoV-2/genética , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Processamento Pós-Transcricional do RNARESUMO
Pseudouridine (Ψ) is the most common chemical modification in RNA. In eukaryotes and archaea, pseudouridine synthases, mainly guided by box H/ACA snoRNAs, convert uridine to Ψ. Ψ stabilizes RNA structure and alters RNA-RNA and RNA-protein interactions, conferring important roles in gene expression. Notably, several Ψ-linked human diseases have been identified over the years. In addition, Ψ has also been extensively used in developing mRNA vaccines. Furthermore, it has been shown that pseudouridylation can be site-specifically directed to modify specific nonsense codons, leading to nonsense suppression. All of these, together with a need to better understand the specific functions of Ψs, have motivated the development of in vitro pseudouridylation assays using purified and reconstituted box H/ACA RNPs. Here, we describe an in vitro system for box H/ACA RNA-guided RNA pseudouridylation using human cell extracts. We show that a half guide RNA (only one hairpin) is just as functionally competent as the full-length guide RNA (two hairpins) in guiding site-specific pseudouridylation in the human cell extracts. This discovery offers the opportunity for direct delivery of a short guide RNA to human cells to promote site-specific nonsense suppression and therefore has potential clinical applications.
Assuntos
Pseudouridina , RNA Nucleolar Pequeno , Humanos , Extratos Celulares , Pseudouridina/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , CatáliseRESUMO
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.
Assuntos
Pseudouridina , RNA , RNA/genética , RNA/química , Pseudouridina/genética , Conformação de Ácido Nucleico , Pareamento de Bases , UridinaRESUMO
Pseudouridine (Ψ) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, stoichiometric measurement of individual Ψ sites in human transcriptome remains unaddressed. Here we develop 'PRAISE', via selective chemical labeling of Ψ by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the Ψ landscape in the human transcriptome. Unlike traditional bisulfite treatment, our approach is based on quaternary base mapping and revealed an ~10% median modification level for 2,209 confident Ψ sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7, TRUB1 and DKC1, with TRUB1 targets showing the highest modification stoichiometry. In addition, we quantified known and new Ψ sites in mitochondrial mRNA catalyzed by PUS1. Collectively, we provide a sensitive and convenient method to measure transcriptome-wide Ψ; we envision this quantitative approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.
Assuntos
Sulfitos , Transcriptoma , Humanos , Células HEK293 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Processamento Pós-Transcricional do RNA , Pseudouridina/genética , Pseudouridina/metabolismo , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/genéticaRESUMO
Pseudouridine (Ψ) is the first-discovered RNA modification abundantly present in many classes of RNAs, which plays a pivotal role in a series of biological processes. Accurately identifying the location of Ψ sites is helpful for relevant downstream researches. In this chapter, we introduce a website PIANO-for pseudouridine site (Ψ) identification and functional annotation, which enables researchers to predict human putative Ψ sites with a high-accuracy (average AUC of 0.955 under the full transcript model and 0.838 under the mature mRNA model when testing on six independent datasets). The posttranscriptional regulatory mechanisms of putative Ψ sites including miRNA-targets, RBP-binding regions, and splicing sites were also annotated. A comprehensive query database was also provided to deposit over 4300 human Ψ modifications, which is currently the most complete collection of experimental-derived Ψ sites. The PIANO website is freely accessible at: http://piano.rnamd.com or http://180.208.58.19/Ψ-WHISTLE .
Assuntos
MicroRNAs , Pseudouridina , Humanos , Pseudouridina/genética , RNA Mensageiro/genética , Análise de Sequência de RNA , Splicing de RNA , Processamento Pós-Transcricional do RNARESUMO
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.
Assuntos
RNA Longo não Codificante , RNA , RNA/química , Pseudouridina/genética , RNA de Transferência/genética , RNA Mensageiro/genética , RNA Ribossômico/genética , Processamento Pós-Transcricional do RNARESUMO
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.
Assuntos
Drosophila melanogaster , Transcriptoma , Feminino , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Pseudouridina/genética , Pseudouridina/análise , Pseudouridina/metabolismo , Perfilação da Expressão Gênica , RNA Ribossômico/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno , Processamento Pós-Transcricional do RNARESUMO
Quantitative and base-resolution sequencing methods are critical to investigations of the biological functions of diverse RNA modifications. These methods may also be employed for clinical studies and clinical applications in the future. In this In Focus article, we introduce and discuss the development of Bisulfite-Induced Deletion sequencing (BID-seq) for quantitatively detecting mRNA pseudouridine (Ψ) modifications at base resolution.
Assuntos
Pseudouridina , RNA , RNA/genética , Pseudouridina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Processamento Pós-Transcricional do RNARESUMO
Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.