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外切核酸酶辅助的单链RNA中假尿苷的富集及碱基分辨率分析

Exonuclease-assisted enrichment and base resolution analysis of pseudouridine in single-stranded RNA.

作者信息

Fang Xin, Lu Ziang, Wang Yafen, Zhao Ruiqi, Mo Jing, Yang Wei, Sun Mei, Zhou Xiang, Weng Xiaocheng

机构信息

College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers-Ministry of Education, Wuhan University Wuhan Hubei 430072 P. R. China

School of Public Health, Wuhan University Wuhan Hubei 430071 P. R. China.

出版信息

Chem Sci. 2024 Oct 21;15(45):19022-8. doi: 10.1039/d4sc03576c.

DOI:10.1039/d4sc03576c
PMID:39479159
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11515940/
Abstract

Pseudouridine (Ψ) is one of the most abundant RNA modifications, playing crucial roles in various biological processes. Identifying Ψ sites is vital for understanding their functions. In this study, we proposed a novel method for identifying Ψ sites with an improved signal-to-noise ratio. This method, called RNA exonuclease-assisted identification of pseudouridine sites (RIPS), combines specific CMC-labeling of Ψ sites with an exonuclease-assisted digestion strategy for the detection of Ψ sites. Utilizing exonuclease XRN1 to digest RNA strands not labeled by CMC, RIPS significantly reduces the background signal from unlabeled strands and enhances the positive signal of Ψ sites labeled by CMC, which terminates exonuclease digestion. As a result, we can enrich Ψ sites and identify them at single-base resolution. Considering the unique functions of single-stranded RNA (ssRNA), we employed RIPS to distinguish Ψ sites in single-stranded and double-stranded regions of RNA. Our results indicated that CMC could specifically label Ψ sites in ssRNA under natural conditions, enabling RIPS to selectively identify Ψ sites in ssRNA, which may facilitate the study on the functions of Ψ sites.

摘要

假尿苷（Ψ）是最丰富的RNA修饰之一，在各种生物学过程中发挥着关键作用。识别Ψ位点对于理解它们的功能至关重要。在本研究中，我们提出了一种用于识别Ψ位点的新方法，该方法具有更高的信噪比。这种方法称为RNA外切核酸酶辅助假尿苷位点鉴定（RIPS），它将Ψ位点的特异性CMC标记与用于检测Ψ位点的外切核酸酶辅助消化策略相结合。利用外切核酸酶XRN1消化未被CMC标记的RNA链，RIPS显著降低了未标记链的背景信号，并增强了被CMC标记的Ψ位点的阳性信号，而CMC标记会终止外切核酸酶的消化。结果，我们可以富集Ψ位点并以单碱基分辨率识别它们。考虑到单链RNA（ssRNA）的独特功能，我们使用RIPS来区分RNA单链和双链区域中的Ψ位点。我们的结果表明，在自然条件下，CMC可以特异性地标记ssRNA中的Ψ位点，使RIPS能够选择性地识别ssRNA中的Ψ位点，这可能有助于对Ψ位点功能的研究。

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本文引用的文献

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Quantitative profiling of pseudouridylation landscape in the human transcriptome.定量分析人类转录组中天假尿嘧啶化修饰谱。
Nat Chem Biol. 2023 Oct;19(10):1185-1195. doi: 10.1038/s41589-023-01304-7. Epub 2023 Mar 30.
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Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing.使用直接长读测序对半定量检测人 mRNA 中的假尿嘧啶修饰和 I/II 型超修饰。
Nat Commun. 2023 Jan 19;14(1):334. doi: 10.1038/s41467-023-35858-w.
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Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution.
BID-seq 定量测序技术在碱基分辨率水平上揭示了哺乳动物 mRNA 中的大量假尿嘧啶核苷。
Nat Biotechnol. 2023 Mar;41(3):344-354. doi: 10.1038/s41587-022-01505-w. Epub 2022 Oct 27.
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Pseudouridine-modified tRNA fragments repress aberrant protein synthesis and predict leukaemic progression in myelodysplastic syndrome.假尿嘧啶核苷修饰的 tRNA 片段抑制异常蛋白质合成，并预测骨髓增生异常综合征中的白血病进展。
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