AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA,Analytical Chemistry

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AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-12-30 , DOI: 10.1021/acs.analchem.3c04457
Xia Guo ₁ , Neng-Bin Xie ₂ , Wei Chen ₃ , Tong-Tong Ji ₁ , Jun Xiong ₂ , Tian Feng ₂ , Min Wang ₁ , Shan Zhang ₁ , Shu-Yi Gu ₂ , Yu-Qi Feng _{1,

2} , Bi-Feng Yuan _{1,

2,

4}

Affiliation

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N⁶,N⁶-Dimethyladenosine (m^6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m^6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m^6,6A) method for the site-specific detection and quantification of m^6,6A in RNA. The N⁶,N⁶-dimethyl groups in m^6,6A can cause reverse transcription to stall at the m^6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m^6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m^6,6A in RNA. Using the AD-m^6,6A method, we successfully detected and quantified m^6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m^6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m^6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m^6,6A in RNA, which can aid in uncovering the functions of m^6,6A in human diseases.

中文翻译：

AlkB 促进的去甲基化能够定量和位点特异性检测 RNA 中腺苷的双甲基化

RNA 分子经历各种化学修饰，在广泛的生物过程中发挥着关键作用。 N ⁶ , N ⁶ -二甲基腺苷 (m ^6,6 A) 是一种保守的 RNA 修饰，对于 rRNA 的加工至关重要。为了更深入地了解 m ^6,6 A 的功能，对 RNA 中的这种修饰进行位点特异性和准确定量是必不可少的。在本研究中，我们开发了一种 AlkB 促进去甲基化 (AD-m ^6,6 A) 方法，用于 RNA 中 m ^6,6 A 的位点特异性检测和定量。 m ^6,6 A 中的N ⁶ 、 N ⁶ -二甲基基团可导致逆转录在 m ^6,6 A 位点停滞，从而产生截短的 cDNA。然而，我们发现大肠杆菌AlkB去甲基化酶可以有效地将RNA中的m ^6,6 A去甲基化，从AlkB处理的RNA中生成全长cDNA。通过使用定量实时 PCR 定量产生的全长 cDNA 的量，我们能够实现 RNA 中 m ^6,6 A 的位点特异性检测和定量。利用 AD-m ^6,6 A 方法，我们成功检测并定量了人类细胞中 18S rRNA 1851 位和线粒体 12S rRNA 937 位的 m ^6,6 A。此外，我们发现与对照小鼠相比，睡眠剥夺小鼠肺组织中线粒体 12S rRNA 1007 位的 m ^6,6 A 水平显着降低。总体而言，AD-m ^6,6 A 方法为简单、准确、定量和位点特异性检测 RNA 中的 m ^6,6 A 提供了一个有价值的工具，这有助于揭示 m ^6,6 A 在 RNA 中的功能。人类疾病。

更新日期：2023-12-30

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