A Radiolabeling‐Free, qPCR‐Based Method for Locus‐Specific Pseudouridine Detection

Lei, Zhi‐Xin; Yi, Chengqi

doi:10.1002/anie.201708276

Cited by 45 publications

(60 citation statements)

References 23 publications

(9 reference statements)

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“…However, profiling methods that provide sensitive and true single-base resolution are currently available only for m 5 C (9, 14, 15) and m 1 A (16); three of these (m 6 A, m 1 A, and Ψ) have involved initial enrichment or detection via antibodies (for m 6 A or m 1 A) (5, 6, 8, 10) or by techniques involving polymerase pausing/termination during reverse transcription (for m 1 A and Ψ) (7, 11–13, 17, 18). Recent single-base techniques for Ψ (19) rely on a bulky adduct formation before detection. Furthermore, although the current methods for Ψ profiling are useful, most lack the sensitivity, resolution, and technical ease needed for widespread adoption or straightforward candidate site validation (7, 11–13, 20).…”

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confidence: 99%

Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution

Khoddami

Yerra

Mosbruger

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

195

226

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The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1–2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.

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mentioning

confidence: 99%

Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution

Khoddami

Yerra

Mosbruger

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

195

226

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“…Although these sequencingbased methods can map m 6 A candidate sites at the transcriptomic level, they cannot provide the fraction of modification at each site, due to factors such as antibody binding efficiency, specificity and cross-linking reactivity (15). Realtime quantitative PCR (qPCR) was previously applied for locus specific detection of pseudouridine (y) modification though chemical labelling of y residue, causing a shift in the melting peak of the resulting qPCR amplicons (16). Similar quantitative methods were recently developed for detection of m 6 A.…”

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confidence: 99%

Deoxyribozyme-based method for absolute quantification of N6-methyladenosine fractions at specific sites of RNA

Bujnowska

Zhang

Dai

et al. 2020

Journal of Biological Chemistry

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N 6 -methyladenosine (m 6 A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA (lncRNA). Although candidate sites for the m 6 A modification are identified at the transcriptomic level, methods for site-specific quantification of absolute m 6 A modification levels are still limited. Herein, we present a facile method implementing a deoxyribozyme, VMC10, which preferentially cleaves the unmodified RNA. We leveraged reverse transcription and real-time quantitative PCR along with key control experiments to quantify the methylation fraction of specific m 6 A sites. We validated the accuracy of this method with synthetic RNA in which methylation fractions ranged from 0% to 100% and applied our method to several endogenous sites that were previously identified in sequencing-based studies. This method provides a time-and cost-effective approach for absolute quantification of the m 6 A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications. AUTHOR CONTRIBUTIONSM.B. J.Z. and J.F. designed the experiments. M.B., J.Z., and E.H. performed the experiments. M.B. analyzed the data. Q.D. synthesized the 35-40 mer synthetic oligonucleotides. M.B., J.Z. and J.F. wrote the manuscript.

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“…In fact, the ratio of Ψ/U in mammalian mRNA as measured by LC-MS/MS (about 0.2%–0.6%) is comparable to the content of m 6 A (Li et al, 2015 ), which further supports the existence of thousands of Ψ sites in mRNA. The CMC chemistry can also be coupled to high resolution qPCR analysis to conveniently detect locus-specific Ψ sites in mRNA and lncRNA (Lei and Yi, 2017 ). Moreover, bisulfite treatment can have Ψ nucleotide to form a monobisulfite adduct, which causes a deletion signature at the Ψ sites during RT.…”

Section: The Biogenesis and Sequencing Approaches For Rna Modificatiomentioning

confidence: 99%

Mapping the epigenetic modifications of DNA and RNA

et al. 2020

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Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

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A Radiolabeling‐Free, qPCR‐Based Method for Locus‐Specific Pseudouridine Detection

Cited by 45 publications

References 23 publications

Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution

Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution

Deoxyribozyme-based method for absolute quantification of N6-methyladenosine fractions at specific sites of RNA

Mapping the epigenetic modifications of DNA and RNA

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