Thioguanosine Conversion Enables mRNA‐Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC‐seq DUAL)

Gasser, Catherina; Delazer, Isabel; Neuner, Eva; Pascher, Katharina; Brillet, Karl; Klotz, Sarah; Trixl, Lukas; Himmelstoß, Maximilian; Ennifar, Eric; Rieder, Dietmar; Lusser, Alexandra; Micura, Ronald

doi:10.1002/ange.201916272

Cited by 11 publications

(12 citation statements)

References 43 publications

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“…A variation of the latter was recently proposed in which two modified nucleotides are used in subsequent pulses. The rationale is that differences in detecting the corresponding conversion signatures give information on both RNA synthesis and decay [ 39 ]. A couple of tools, GRAND-SLAM [ 40 ] and SLAM-DUNK [ 41 ], were developed that to be tailored to these data.…”

Section: Approaches That Quantify Rna Kinetic Rates Based On Nascent Rna Profilingmentioning

confidence: 99%

Dynamics of transcriptional and post-transcriptional regulation

Furlan

Pretis

Pelizzola

2020

Briefings in Bioinformatics

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Despite gene expression programs being notoriously complex, RNA abundance is usually assumed as a proxy for transcriptional activity. Recently developed approaches, able to disentangle transcriptional and post-transcriptional regulatory processes, have revealed a more complex scenario. It is now possible to work out how synthesis, processing and degradation kinetic rates collectively determine the abundance of each gene’s RNA. It has become clear that the same transcriptional output can correspond to different combinations of the kinetic rates. This underscores the fact that markedly different modes of gene expression regulation exist, each with profound effects on a gene’s ability to modulate its own expression. This review describes the development of the experimental and computational approaches, including RNA metabolic labeling and mathematical modeling, that have been disclosing the mechanisms underlying complex transcriptional programs. Current limitations and future perspectives in the field are also discussed.

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Section: Approaches That Quantify Rna Kinetic Rates Based On Nascent Rna Profilingmentioning

confidence: 99%

Dynamics of transcriptional and post-transcriptional regulation

Furlan

Pretis

Pelizzola

2020

Briefings in Bioinformatics

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“…在 RNA 中除了四种天然存在的碱基之外, 还存在着大量的修饰碱基, 它们对生命体正常的生长发育过程有着重要的影响. 在真核生物 mRNA 和长链非编码 RNA 中, m 6 A 是一种含量最丰富的转录后修饰 [1] . 其在调控 RNA 转录、加工、剪接、翻译及 RNA 稳定性方面都起着重要作用 [2] .…”

Section: 引言unclassified

“…mRNA 中的动态修饰对于基因表达的转录后调控起着至关重要的作用 [3][4] . 从发现 FTO (fat mass and obesity-associated)蛋白可以与 m 6 A 相互作用, 产生去甲基化作用之后 [5] , 对 m 6 A 相关蛋白的研究以及 m 6 A 检测技术的发展取得了很多重大的突破, 对 m 6 [7] . TimeLapse-seq 将 s 4 U 转化为胞嘧啶衍生物, 该策略使用过氧化物将 s 4 U 中的硫(S)为磺酸基 (SO 3 -)再使用氨基衍生物进行取代反应 [8] .…”

Section: 引言unclassified

Sodium Sulfite-Mediated s⁴U Conversion for m⁶A Profiling of Nascent Transcriptome-Wide RNA

Wei¹,

Han²,

Chen³

et al. 2021

Acta Chimica Sinica

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RNA sequencing can profile gene expression at steady-state level but is weak in studying temporal RNA dynamics. A common method for nascent RNA analysis is metabolic labeling with nucleoside analog. We developed a method for nascent RNA sequencing based on sodium sulfite-mediated 4-thiouridine-to-cytidine (s 4 U-to-C) conversion. The RNA contained s 4 U is reacted in the buffer (10 mmol/L Na2SO3, 200 mmol/L NH4Cl and 200 mmol/L Na2HPO4/NaH2PO4, pH 7.4) at 70 ℃ for 4 h, the yield can reach more than 90%. This method can efficiently distinguish nascent RNA information from total RNAs. SUC-seq is used to investigate the m 6 A, which is the most prevalent modification in mammalian mRNA and has been shown to have an essential regulatory role in the control of gene expression. Here, we provide a time resolved high-resolution profiling of m 6 A on nascent RNA transcripts. 3×10 6 HEK293T cells were seeded per 10-cm cell dish and grown for 24 h. Then, s 4 U was added to the medium at a final concertation of 500 μmol/L chased for 0, 10, 15, 30, 60, 120 and 240 min. Total RNA was extracted using TRIzol reagent. 10 μg total RNA was fragmented using RNA fragmentation reagents at 70 ℃ for 10 min after gDNA was removed. Fragmented RNA was then subjected to s 4 U reaction to realize the conversion of s 4 U to C. Subsequently, 5 μg treated RNA were taken to perform m 6 A immunoprecipitation. After removing rRNA, library was constructed and perform next-generation sequencing. A large number of T to C mutations were observed in the mapping reads. The T to C mutation rate of the control RNA was kept at a low background level. The results show that our method can successfully distinguish nascent RNA from total RNA. Via analyzing the nascent RNA in m 6 A, the nascent m 6 A can be distinguished from the total m 6 A. This method can be a promising strategy to study the mechanism and function of m 6 A.

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“…TimeLapse-seq, thiol(SH)linked alkylation for the metabolic sequencing (SLAM seq), and thiouridine to cytidine conversion sequencing (TUC-seq) rely on chemical conversion of the metabolically incorporated analog. Modified positions are then identified in mutated cDNA in order to distinguish the metabolically labeled reads from pre-existing none-labeled reads [17][18][19] . One of the challenges of this group of methods is the low incorporation rate of 4SU that results in under-estimation of recently transcribed genes 20 , especially when using short-read sequencing, which is still a longstanding challenge in transcriptomics, especially when interrogating more complex transcriptomes with large dynamic range.…”

Section: Introductionmentioning

confidence: 99%

Long-TUC-seq is a robust method for quantification of metabolically labeled full-length isoforms

Rahmanian

Balderrama-Gutierrez

Wyman

et al. 2020

Preprint

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The steady state expression of each gene is the result of a dynamic transcription and degradation of that gene. While regular RNA-seq methods only measure steady state expression levels, RNA-seq of metabolically labeled RNA identifies transcripts that were transcribed during the window of metabolic labeling. Whereas short-read RNA sequencing can identify metabolically labeled RNA at the gene level, long-read sequencing provides much better resolution of isoform-level transcription. Here we combine thiouridine-to-cytosine conversion (TUC) with PacBio long-read sequencing to study the dynamics of mRNA transcription in the GM12878 cell line. We show that using long-TUC-seq, we can detect metabolically labeled mRNA of distinct isoforms more reliably than using short reads. Long-TUC-seq holds the promise of capturing isoform dynamics robustly and without the need for enrichment.

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Thioguanosine Conversion Enables mRNA‐Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC‐seq DUAL)

Cited by 11 publications

References 43 publications

Dynamics of transcriptional and post-transcriptional regulation

Dynamics of transcriptional and post-transcriptional regulation

Sodium Sulfite-Mediated s⁴U Conversion for m⁶A Profiling of Nascent Transcriptome-Wide RNA

Long-TUC-seq is a robust method for quantification of metabolically labeled full-length isoforms

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Thioguanosine Conversion Enables mRNA‐Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC‐seq DUAL)

Cited by 11 publications

References 43 publications

Dynamics of transcriptional and post-transcriptional regulation

Dynamics of transcriptional and post-transcriptional regulation

Sodium Sulfite-Mediated s4U Conversion for m6A Profiling of Nascent Transcriptome-Wide RNA

Long-TUC-seq is a robust method for quantification of metabolically labeled full-length isoforms

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Sodium Sulfite-Mediated s⁴U Conversion for m⁶A Profiling of Nascent Transcriptome-Wide RNA