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固相化学共价且可逆地捕获巯基化 RNA。

Solid phase chemistry to covalently and reversibly capture thiolated RNA.

机构信息

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06511, USA.

Chemical Biology Institute, Yale University, West Haven, CT 06516, USA.

出版信息

Nucleic Acids Res. 2018 Aug 21;46(14):6996-7005. doi: 10.1093/nar/gky556.


DOI:10.1093/nar/gky556
PMID:29986098
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6101502/
Abstract

Here, we describe an approach to enrich newly transcribed RNAs from primary mouse neurons using 4-thiouridine (s4U) metabolic labeling and solid phase chemistry. This one-step enrichment procedure captures s4U-RNA by using highly efficient methane thiosulfonate (MTS) chemistry in an immobilized format. Like solution-based methods, this solid-phase enrichment can distinguish mature RNAs (mRNA) with differential stability, and can be used to reveal transient RNAs such as enhancer RNAs (eRNAs) and primary microRNAs (pri-miRNAs) from short metabolic labeling. Most importantly, the efficiency of this solid-phase chemistry made possible the first large scale measurements of RNA polymerase II (RNAPII) elongation rates in mouse cortical neurons. Thus, our approach provides the means to study regulation of RNA metabolism in specific tissue contexts as a means to better understand gene expression in vivo.

摘要

在这里,我们描述了一种从原代小鼠神经元中富集新转录 RNA 的方法,该方法使用 4-硫代尿嘧啶 (s4U) 代谢标记和固相化学。这种一步富集程序通过在固定化形式中使用高效的甲烷硫代磺酸酯 (MTS) 化学来捕获 s4U-RNA。与基于溶液的方法一样,这种固相富集可以区分具有不同稳定性的成熟 RNA(mRNA),并且可用于从短期代谢标记中揭示瞬时 RNA,如增强子 RNA (eRNA) 和初级 microRNA (pri-miRNA)。最重要的是,这种固相化学的效率使得首次能够在小鼠皮质神经元中大规模测量 RNA 聚合酶 II (RNAPII) 延伸率。因此,我们的方法为在特定组织背景下研究 RNA 代谢的调控提供了手段,以便更好地理解体内基因表达。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d22/6101502/11fc2be3d5c1/gky556fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d22/6101502/1167b3c0339f/gky556fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d22/6101502/8ff7057c059a/gky556fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d22/6101502/39e9f6f82e35/gky556fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d22/6101502/11fc2be3d5c1/gky556fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d22/6101502/1167b3c0339f/gky556fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d22/6101502/8ff7057c059a/gky556fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d22/6101502/39e9f6f82e35/gky556fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d22/6101502/11fc2be3d5c1/gky556fig4.jpg

相似文献

[1]
Solid phase chemistry to covalently and reversibly capture thiolated RNA.

Nucleic Acids Res. 2018-8-21

[2]
Enriching s U-RNA Using Methane Thiosulfonate (MTS) Chemistry.

Curr Protoc Chem Biol. 2016-12-7

[3]
Metabolic Labeling of Newly Synthesized RNA with 4sU to in Parallel Assess RNA Transcription and Decay.

Methods Mol Biol. 2018

[4]
Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry.

Mol Cell. 2015-9-3

[5]
Isolation of Newly Transcribed RNA Using the Metabolic Label 4-Thiouridine.

Methods Mol Biol. 2017

[6]
Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability.

Methods. 2017-5-1

[7]
Uncovering the Stability of Mature miRNAs by 4-Thio-Uridine Metabolic Labeling.

Methods Mol Biol. 2018

[8]
High resolution gene expression profiling of RNA synthesis, processing, and decay by metabolic labeling of newly transcribed RNA using 4-thiouridine.

Methods Mol Biol. 2013

[9]
Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates.

Methods Mol Biol. 2020

[10]
Osmium-Mediated Transformation of 4-Thiouridine to Cytidine as Key To Study RNA Dynamics by Sequencing.

Angew Chem Int Ed Engl. 2017-9-18

引用本文的文献

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Nat Cell Biol. 2024-4

[2]
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Stem Cell Reports. 2023-12-12

[3]
DOT1L is a barrier to histone acetylation during reprogramming to pluripotency.

Sci Adv. 2023-11-15

[4]
Improving the study of RNA dynamics through advances in RNA-seq with metabolic labeling and nucleotide-recoding chemistry.

bioRxiv. 2023-5-24

[5]
A selective and sensitive detection system for 4-thiouridine modification in RNA.

RNA. 2023-2

[6]
Connecting the DOTs on Cell Identity.

Front Cell Dev Biol. 2022-6-6

[7]
Estimating RNA dynamics using one time point for one sample in a single-pulse metabolic labeling experiment.

BMC Bioinformatics. 2022-4-22

[8]
Synthesis of 4-thiouridines with prodrug functionalization for RNA metabolic labeling.

RSC Chem Biol. 2022-2-25

[9]
DOT1L inhibition enhances pluripotency beyond acquisition of epithelial identity and without immediate suppression of the somatic transcriptome.

Stem Cell Reports. 2022-2-8

[10]
HP1γ regulates H3K36 methylation and pluripotency in embryonic stem cells.

Nucleic Acids Res. 2020-12-16

本文引用的文献

[1]
TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding.

Nat Methods. 2018-1-22

[2]
Understanding Tissue-Specific Gene Regulation.

Cell Rep. 2017-10-24

[3]
Early-Life Gene Expression in Neurons Modulates Lasting Epigenetic States.

Cell. 2017-11-16

[4]
mA mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.

Nature. 2017-8-17

[5]
Identification of active miRNA promoters from nuclear run-on RNA sequencing.

Nucleic Acids Res. 2017-7-27

[6]
TT-seq captures enhancer landscapes immediately after T-cell stimulation.

Mol Syst Biol. 2017-3-7

[7]
Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability.

Methods. 2017-5-1

[8]
Enriching s U-RNA Using Methane Thiosulfonate (MTS) Chemistry.

Curr Protoc Chem Biol. 2016-12-7

[9]
C-terminal domain (CTD) phosphatase links Rho GTPase signaling to Pol II CTD phosphorylation in Arabidopsis and yeast.

Proc Natl Acad Sci U S A. 2016-12-13

[10]
TT-seq maps the human transient transcriptome.

Science. 2016-6-3

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