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.