5′-Bromouridine IP Chase (BRIC)-Seq to Determine RNA Half-Lives

Yamada, Toshimichi; Imamachi, Naoto; Onoguchi‐Mizutani, Rena; Imamura, Katsutoshi; Suzuki, Yutaka; Akimitsu, Nobuyoshi

doi:10.1007/978-1-4939-7540-2_1

Cited by 13 publications

(12 citation statements)

References 21 publications

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“…In the following sections, we briefly describe each method and discuss the advantages and disadvantages of these methods. This review does not cover the detailed experimental designs of each method, which have been covered elsewhere (Russo, Heck, Wilusz, & Wilusz, ; Yamada et al, ).…”

Section: Methods For Measurement Of the Kinetics Of Mrna Dynamicsmentioning

confidence: 99%

“…Bromo‐uridine (BrU) immunoprecipitation chase‐deep sequencing analysis (BRIC‐seq) uses BrU for the metabolic labeling of endogenous transcripts (Imamachi, Salam, Suzuki, & Akimitsu, ; Tani et al, ; Yamada et al, ) (Figure a). After removal of BrU from the medium, total RNAs are isolated from cells at sequential time points and BrUs‐labeled RNAs are immunopurified using BrU antibody.…”

Section: Methods For Measurement Of the Kinetics Of Mrna Dynamicsmentioning

confidence: 99%

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Contributions of regulated transcription and mRNA decay to the dynamics of gene expression

Yamada

Akimitsu

2018

WIREs RNA

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Organisms have acquired sophisticated regulatory networks that control gene expression in response to cellular perturbations. Understanding of the mechanisms underlying the coordinated changes in gene expression in response to external and internal stimuli is a fundamental issue in biology. Recent advances in highthroughput technologies have enabled the measurement of diverse biological information, including gene expression levels, kinetics of gene expression, and interactions among gene expression regulatory molecules. By coupling these technologies with quantitative modeling, we can now uncover the biological roles and mechanisms of gene regulation at the system level. This review consists of two parts. First, we focus on the methods using uridine analogs that measure synthesis and decay rates of RNAs, which demonstrate how cells dynamically change the regulation of gene expression in response to both internal and external cues. Second, we discuss the underlying mechanisms of these changes in kinetics, including the functions of transcription factors and RNA-binding proteins. Overall, this review will help to clarify a system-level view of gene expression programs in cells.

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Section: Methods For Measurement Of the Kinetics Of Mrna Dynamicsmentioning

confidence: 99%

Section: Methods For Measurement Of the Kinetics Of Mrna Dynamicsmentioning

confidence: 99%

Contributions of regulated transcription and mRNA decay to the dynamics of gene expression

Yamada

Akimitsu

2018

WIREs RNA

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“…A more recent (and more global) approach to mRNA half-life is to "pulse-label" cells with a modified nucleoside (e.g. thiouridine [22,23] or bromouridine [24,25] and then chase with uridine). The RNA containing the modified nucleotide is purified and subjected to RNA-Seq analysis.…”

Section: Studying Mrna Degradationmentioning

confidence: 99%

Determining degradation intermediates and the pathway of 3′ to 5′ degradation of histone mRNA using high-throughput sequencing

Holmquist

Marzluff

2019

Methods

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The half-life of an mRNA is an important parameter contributing to the steady-state level of the mRNA. Rapid changes in mRNA levels can result from decreasing the half-life of an mRNA. Establishing the detailed pathway of mRNA degradation for a particular class of mRNAs requires the ability to isolate mRNA degradation intermediates. High-throughput sequencing provides a method for detecting these intermediates. Here we describe a method for determining the intermediates in 3' to 5' degradation. Characterizing these intermediates requires not only determining the precise 3' end of the molecule to a single nucleotide resolution, but also the ability to detect and characterize any untemplated nucleotides present on the intermediates. We achieve this by ligating a known sequence to all the 3' termini in the cell, and then sequence the 3' termini and the ligated linker to identify any alterations to the genomic reference sequence. We have applied this method to characterize the intermediates in histone mRNA metabolism, allowing us to deduce the pathway of 3' to 5' degradation. This method can potentially be applied to any RNA, and we discuss possible strategies for extending the method to include simultaneous determination of the 3' and 5' end of the same RNA molecule.

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“…One criterion is the mRNA binds to PUMs, which we determined by RNA immunoprecipitation sequencing (RIP-seq) 36 . The other criterion is the mRNA stability is decreased by PUMs, which we determined by 5′-bromo-uridine (BrU) immunoprecipitation chase sequencing (BRIC-seq) 37,38 . BRIC-seq enables the determination of genome-wide RNA stability by monitoring the decrease in BrU-labeled RNA abundance over time from which the RNA half-life of each transcript is calculated.…”

Section: Identification Of Target Mrnas Of Human Pum1 and Pum2 By Ripmentioning

confidence: 99%

“…BRIC was performed as previously described 38 . In brief, cells were incubated at 37 °C in the presence of 150 μM bromo-uridine (BrU) (Wako Chemical, Tokyo, Japan) for 24-hours in a humidified incubator with 5% CO2.…”

Section: Bromo-uridine (Bru) Immunoprecipitation Chase-deep Sequencinmentioning

confidence: 99%

Systematic Analysis of Targets of Pumilio (PUM)-Mediated mRNA Decay Identifies a Role of PUM1 in Regulating DNA Damage Response Pathway

Yamada

Imamachi

Imamura

et al. 2018

Preprint

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RNA-binding proteins (RBPs) play a pivotal role in gene expression by modulating the stability of transcripts; however, the identification of degradation targets of RBPs remains difficult. Here, we identified 48 target mRNAs of human Pumilio 1 (PUM1), an evolutionally conserved RBP, by combined analysis of transcriptome-wide mRNA stabilities and the binding of mRNAs to PUM1. Here, we developed an approach to identify mRNA targets of Pumilio 1 (PUM1), an evolutionally conserved RBP. By combined analysis of transcriptome-wide mRNA stabilities and the binding of mRNAs to PUM1, we identified 48 mRNAs that both bound to PUM1 and exhibited PUM1-dependent degradation. Analysis of changes in the abundance of PUM1 and its targets in RNA-seq data indicated that DNA-damaging agents negatively regulated PUM1-mediated mRNA decay. Cells exposed to cisplatin had reduced PUM1 abundance and increased PCNA and UBE2A mRNAs, encoding proteins involved in DNA repair by translesion synthesis (TLS). Cells overexpressing PUM1 exhibited impaired DNA synthesis and TLS and increased sensitivity to the cytotoxic effect of cisplatin. Thus, our method identified targets of PUM1-mediated decay and revealed that cells respond to DNA damage by inhibiting PUM1-mediated mRNA decay to activate TLS.

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5′-Bromouridine IP Chase (BRIC)-Seq to Determine RNA Half-Lives

Cited by 13 publications

References 21 publications

Contributions of regulated transcription and mRNA decay to the dynamics of gene expression

Contributions of regulated transcription and mRNA decay to the dynamics of gene expression

Determining degradation intermediates and the pathway of 3′ to 5′ degradation of histone mRNA using high-throughput sequencing

Systematic Analysis of Targets of Pumilio (PUM)-Mediated mRNA Decay Identifies a Role of PUM1 in Regulating DNA Damage Response Pathway

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