meCLICK-Seq, a Substrate-Hijacking and RNA Degradation Strategy for the Study of RNA Methylation.
meCLICK-Seq, a Substrate-Hijacking and RNA Degradation Strategy for the Study of RNA Methylation.
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meCLICK-Seq：一种用于RNA甲基化研究的底物劫持和RNA降解策略。
DOI：
10.1021/acscentsci.0c01094
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发表时间：
2020-12-23
影响因子：
18.2
通讯作者：
Bernardes GJL
中科院分区：
文献类型：
--
作者：
Mikutis S;Gu M;Sendinc E;Hazemi ME;Kiely-Collins H;Aspris D;Vassiliou GS;Shi Y;Tzelepis K;Bernardes GJL
细胞中RNA种类的命运由核糖核酸酶控制，核糖核酸酶通过利用通用的结构2′-OH基团来降解它们。这种现象在许多变革性技术中起着关键作用，例如RNA干扰和基于CRISPR/Cas 13的RNA编辑系统。然而，这些方法是基于遗传或寡聚体的，因此具有固有的局限性。这引起了对能够以靶向方式降解核酸的小分子的开发的兴趣。在这里，我们描述了点击降解剂，可以通过点击化学共价连接到RNA种类并可以降解它们的小分子，类似于核糖核酸酶。通过使用这些分子，我们开发了meCLICK-Seq（甲基化CLICK-degradation Sequencing），这是一种在内含子和基因间区域以高分辨率鉴定RNA修饰底物的方法。该方法劫持RNA甲基转移酶活性以在RNA上引入炔而不是甲基部分。随后的铜（I）催化的叠氮化物-炔环加成反应与点击降解剂导致RNA切割和降解，利用内源性核糖核酸酶使用的机制。专注于N6-甲基腺苷（m6 A），meCLICK-Seq识别甲基化转录本，确定RNA甲基化酶特异性，并可靠地映射内含子和基因间区域的修饰位点。重要的是，我们发现，胃L16沉积m6 A的内含子多聚腺苷酸化（IPA）的网站，这表明胃L16在IPA的潜在作用，反过来，剪接。与其他方法不同，meCLICK-Seq的读数是修饰的RNA种类的耗尽，而不是富集，这允许对整个转录组（包括低丰度区域）的RNA修饰进行全面和动态的研究。点击降解器是高度模块化的，因此可以用于研究任何RNA修饰和设计依赖于RNA降解的新技术。RNA甲基化控制着许多生物学过程。我们描述了一个基于小分子的平台，通过引导降解来劫持RNA甲基化，用于修饰底物的高分辨率分析。
The fates of RNA species in a cell are controlled by ribonucleases, which degrade them by exploiting the universal structural 2′-OH group. This phenomenon plays a key role in numerous transformative technologies, for example, RNA interference and CRISPR/Cas13-based RNA editing systems. These approaches, however, are genetic or oligomer-based and so have inherent limitations. This has led to interest in the development of small molecules capable of degrading nucleic acids in a targeted manner. Here we describe click-degraders, small molecules that can be covalently attached to RNA species through click-chemistry and can degrade them, that are akin to ribonucleases. By using these molecules, we have developed the meCLICK-Seq (methylation CLICK-degradation Sequencing) a method to identify RNA modification substrates with high resolution at intronic and intergenic regions. The method hijacks RNA methyltransferase activity to introduce an alkyne, instead of a methyl, moiety on RNA. Subsequent copper(I)-catalyzed azide–alkyne cycloaddition reaction with the click-degrader leads to RNA cleavage and degradation exploiting a mechanism used by endogenous ribonucleases. Focusing on N6-methyladenosine (m6A), meCLICK-Seq identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions. Importantly, we show that METTL16 deposits m6A to intronic polyadenylation (IPA) sites, which suggests a potential role for METTL16 in IPA and, in turn, splicing. Unlike other methods, the readout of meCLICK-Seq is depletion, not enrichment, of modified RNA species, which allows a comprehensive and dynamic study of RNA modifications throughout the transcriptome, including regions of low abundance. The click-degraders are highly modular and so may be exploited to study any RNA modification and design new technologies that rely on RNA degradation. RNA methylation controls many biological processes. We describe a small molecule-based platform to hijack RNA methylation by guided degradation for high resolution profiling of modified substrates.
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影响因子：
64.8
作者：
Lee SH;Singh I;Tisdale S;Abdel-Wahab O;Leslie CS;Mayr C
通讯作者：
Mayr C
影响因子：
48
作者：
Linder, Bastian;Grozhik, Anya V.;Olarerin-George, Anthony O.;Meydan, Cem;Mason, Christopher E.;Jaffrey, Samie R.
通讯作者：
Jaffrey, Samie R.
影响因子：
48
作者：
Meyer, Kate D.
通讯作者：
Meyer, Kate D.
赖氨酸 36 处的组蛋白 H3 三甲基化引导 m(6)A RNA 共转录修饰
影响因子：
64.8
作者：
Huang, Huilin;Weng, Hengyou;Chen, Jianjun
通讯作者：
Chen, Jianjun
影响因子：
64.5
作者：
Meyer KD;Saletore Y;Zumbo P;Elemento O;Mason CE;Jaffrey SR
通讯作者：
Jaffrey SR