Quantitative profiling of pseudouridylation landscape in the human transcriptome

Zhang, Meiling; Jiang, Zhe; Ma, Yichen; Liu, Wenqing; Zhuang, Yuan; Lu, Bo; Li, Kai; Ji, Peng; Yi, Chengqi

doi:10.1038/s41589-023-01304-7

Cited by 66 publications

(58 citation statements)

References 51 publications

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“…coli. , The occupancy of Ψ across the transcriptome is organism, cell-type, and stress-dependent, suggesting that this U isomer has a significant role in cellular processes. ,,,, Determination of these roles will require accurate and quantitative sequencing of RNA, in which nanopore direct RNA sequencing is a candidate method to achieve this goal. , Our work identified that nanopore signatures for Ψ are highly sequence context dependent and that using a consensus of nanopore current, helicase dwell time, and base miscalling is an approach to sequence this U isomer . The pH 7 bisulfite reaction to yield Ψ adducts results in detection of adducts via a deletion signature in traditional sequencing after reverse transcription, ,, and it is found as an indel in nanopore direct RNA sequencing . In the nanopore data, the indel results from the current levels being poorly recognized by the software.…”

Section: Discussionmentioning

confidence: 97%

“…Nanopore direct RNA sequencing is conducted without the introduction of biases during reverse transcription of the RNA to a cDNA and PCR amplification of the cDNA to yield amplicons for NGS. Direct RNA sequencing allows sequencing modifications in their native form without the application of chemicals or antibodies selective for the modifications to raise a signal from the background. ,, The commercial nanopore sequencer is comprised of a lipid-bilayer embedded protein nanopore sensor with a central constriction zone slightly larger than single-stranded RNA for recording current levels as the RNA passes the pore (Figure a) . Within the central constriction zone resides ∼5 nts of RNA that contribute to the current levels, referred to as a k-mer.…”

Section: Nanopore Direct Rna Sequencing For Pseudouridine Using Dwell...mentioning

confidence: 99%

“…Using nucleosides, we monitored bisulfite adduction using 2 M NaHSO 3 at 50 °C and found that the rate of reaction for C was nearly undetectable at pH 7, while the rate for Ψ was slowed to about half at pH 7 compared to pH 5 . Nevertheless, there is sufficient reactivity of Ψ at pH 7 to develop highly efficient sequencing protocols for Ψ alone, and precisely that was done recently in both the He laboratory with BID-seq and shortly thereafter in the Yi laboratory where the PRAISE protocol was developed (Figure b) …”

Section: Chemical Sequencing For Pseudouridine In Rnamentioning

confidence: 99%

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Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

Burrows,

Fleming

2023

Acc. Chem. Res.

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Section: Discussionmentioning

confidence: 97%

Section: Nanopore Direct Rna Sequencing For Pseudouridine Using Dwell...mentioning

confidence: 99%

Section: Chemical Sequencing For Pseudouridine In Rnamentioning

confidence: 99%

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Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

Burrows,

Fleming

2023

Acc. Chem. Res.

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“…Recently, we and others have independently developed two approaches, namely, BID-seq and PRAISE, for the absolute quantification of transcriptome-wide mammalian Ψs , (Figure c). BID-seq utilizes an elevated neutral pH (∼7.5), whereas PRAISE leverages augmented effective ion concentrations during the bisulfite reaction, resulting in the inhibition of C-to-T conversion and a greatly improved reaction efficiency to Ψ.…”

Section: Pseudouridine: the Most Abundant Rna Modificationmentioning

confidence: 99%

Quantifying m⁶A and Ψ Modifications in the Transcriptome via Chemical-Assisted Approaches

Zhang,

Xiao,

Jiang

et al. 2023

Acc. Chem. Res.

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Metrics & MoreArticle Recommendations CONSPECTUS: Since the discovery of the first chemically modified RNA nucleotide in 1951, more than 170 types of chemical modifications have been characterized in RNA so far.Since the discovery of the reversible and dynamic nature of N 6methyladenosine (m 6 A) in mRNA modification, researchers have identified about ten modifications in eukaryotic mRNA; together with modifications on the noncoding RNAs, the term "epitranscriptome" has been coined to describe the ensemble of various chemical RNA modifications. The past decade has witnessed the discovery of many novel molecular functions of mRNA modifications, demonstrating their crucial roles in gene expression regulation. As the most abundant modifications in mRNA, the study of m 6 A and Ψ has been facilitated by innovative highthroughput sequencing technologies, which can be based on antibodies, enzymes, or novel chemistry. Among them, chemicalassisted methods utilize selective chemistry that can discriminate modified versus unmodified nucleotides, enabling the transcriptome-wide mapping of m 6 A and Ψ modifications and functional studies.Our group has developed several sequencing technologies to investigate these epitranscriptomic marks including m 6 A, Ψ, m 1 A, and m 6 Am. Among them, we have recently developed two methods for absolute quantification of m 6 A and Ψ in the transcriptome based on chemical reactivity to distinguish and measure the two modifications. In GLORI, we used glyoxal and nitrite to mediate efficient deamination of regular adenosine, while m 6 A remained unaffected, thereby enabling efficient and unbiased detection of single-base resolution and absolute quantification of m 6 A modification. In CeU-seq and PRAISE, we used different chemistry to achieve selective labeling and detection of transcriptome-wide Ψ. CeU-seq is based on an azido-derivatized carbodiimide compound, while PRAISE utilizes the unique activity of bisulfite to Ψ. PRAISE results in the formation of ring-opening Ψ-bisulfite adduct and quantitatively detects Ψ as 1−2 nt deletion signatures during sequencing. The resulting base-resolution and stoichiometric information expanded our understanding to the profiles of RNA modifications in the transcriptome. In particular, the quantitative information on RNA methylome is critical for characterizing the dynamic and reversible nature of RNA modifications, for instance, under environmental stress or during development. Additionally, base-resolution and stoichiometric information can greatly facilitate the analysis and characterization of functional modification sites that are important for gene expression regulation, especially when one modification type may have multiple or even opposing functions within a specific transcript. Together, the quantitative profiling methods provide the modification stoichiometry information, which is critical to study the regulatory roles of RNA modifications.In this Account, we will focus on the quantitative sequencing technologies of m 6 A and Ψ developed in our g...

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“…Further sophistication included the combination of stalling and misincorporation as important contributors to the RT-signature, which was then shown to vary, e.g., among different enzymes such that the combined RT-signature of two RT-enzymes was more informative than one alone . This inspired the directed evolution of enzymes whose RT-signature was specifically sensitive to modifications such as m 6 A, Nm, Q and Ψ, which were otherwise considered to be RT-silent. − Another sophistication included jumps in the RT-signature, a feature that seems to be fraught with less background. − However, it became clear that the number of RNA modifications whose altered base pairing properties allowed comprehensive detection was limited …”

Section: Introductionmentioning

confidence: 99%

General Principles and Limitations for Detection of RNA Modifications by Sequencing

Motorin,

Helm

2023

Acc. Chem. Res.

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Metrics & MoreArticle Recommendations * sı Supporting Information CONSPECTUS: Among the many analytical methods applied to RNA modifications, a particularly pronounced surge has occurred in the past decade in the field of modification mapping. The occurrence of modifications such as m 6 A in mRNA, albeit known since the 1980s, became amenable to transcriptome-wide analyses through the advent of next-generation sequencing techniques in a rather sudden manner. The term "mapping" here refers to detection of RNA modifications in a sequence context, which has a dramatic impact on the interpretation of biological functions. As a consequence, an impressive number of mapping techniques were published, most in the perspective of what now has become known as "epitranscriptomics". While more and more different modifications were reported to occur in mRNA, conflicting reports and controversial results pointed to a number of technical and theoretical problems rooted in analytics, statistics, and reagents. Rather than finding the proverbial needle in a haystack, the tasks were to determine how many needles of what color in what size of a haystack one was looking at.As the authors of this Account, we think it important to outline the limitations of different mapping methods since many life scientists freshly entering the field confuse the accuracy and precision of modification mapping with that of normal sequencing, which already features numerous caveats by itself. Indeed, we propose here to qualify a specific mapping method by the size of the transcriptome that can be meaningfully analyzed with it. We here focus on high throughput sequencing by Illumina technology, referred to as RNA-Seq. We noted with interest the development of methods for modification detection by other high throughput sequencing platforms that act directly on RNA, e.g., PacBio SMRT and nanopore sequencing, but those are not considered here.In contrast to approaches relying on direct RNA sequencing, current Illumina RNA-Seq protocols require prior conversion of RNA into DNA. This conversion relies on reverse transcription (RT) to create cDNA; thereafter, the cDNA undergoes a sequencing-bysynthesis type of analysis. Thus, a particular behavior of RNA modified nucleotides during the RT-step is a prerequisite for their detection (and quantification) by deep sequencing, and RT properties have great influence on the detection efficiency and reliability. Moreover, the RT-step requires annealing of a synthetic primer, a prerequisite with a crucial impact on library preparation. Thus, all RNA-Seq protocols must feature steps for the introduction of primers, primer landing sites, or adapters on both the RNA 3′-and 5′ends.

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Quantitative profiling of pseudouridylation landscape in the human transcriptome

Cited by 66 publications

References 51 publications

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

Quantifying m⁶A and Ψ Modifications in the Transcriptome via Chemical-Assisted Approaches

General Principles and Limitations for Detection of RNA Modifications by Sequencing

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Quantitative profiling of pseudouridylation landscape in the human transcriptome

Cited by 66 publications

References 51 publications

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

Quantifying m6A and Ψ Modifications in the Transcriptome via Chemical-Assisted Approaches

General Principles and Limitations for Detection of RNA Modifications by Sequencing

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Product

Resources

About

Quantifying m⁶A and Ψ Modifications in the Transcriptome via Chemical-Assisted Approaches