meCLICK-Seq, a Substrate-Hijacking and RNA Degradation Strategy for the Study of RNA Methylation

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.

细胞内RNA物种的命运由核糖核酸酶（ribonucleases）调控，这类酶通过利用普遍存在的2′-羟基结构基团实现RNA降解。这一现象在诸多变革性技术中发挥关键作用，例如RNA干扰（RNA interference）以及基于CRISPR/Cas13的RNA编辑系统。然而，此类方法均依托遗传物质或寡聚体，因而存在固有局限性。这促使学界关注可靶向降解核酸的小分子开发。本文报道了点击降解剂（click-degraders）：一类可通过点击化学（click-chemistry）与RNA物种共价结合，并可类似核糖核酸酶实现RNA降解的小分子。借助这类分子，我们开发出meCLICK-Seq（甲基化点击降解测序，methylation CLICK-degradation Sequencing）技术，可在内含子与基因间区以高分辨率鉴定RNA修饰底物。该技术利用RNA甲基转移酶的活性，将RNA上的甲基替换为炔基（alkyne）基团。随后通过铜(I)催化的叠氮-炔环加成反应，使点击降解剂与RNA结合，进而利用内源性核糖核酸酶的作用机制实现RNA的切割与降解。针对N6-甲基腺嘌呤（N6-methyladenosine，m6A），meCLICK-Seq可鉴定甲基化转录本，确定RNA甲基化酶的特异性，并可靠地定位内含子与基因间区的修饰位点。值得注意的是，我们证实METTL16可将m6A沉积至内含子多聚腺苷酸化（intronic polyadenylation，IPA）位点，这提示METTL16在IPA以及后续剪接过程中可能发挥潜在作用。与其他方法不同，meCLICK-Seq的检测信号为修饰RNA物种的耗竭而非富集，这使得我们能够对整个转录组（transcriptome），包括低丰度区域在内的RNA修饰开展全面且动态的研究。点击降解剂具有高度模块化的特性，因此可被用于研究任意RNA修饰，并开发依托RNA降解的新型技术。