Abstract
Posttranscriptional regulation of RNA has emerged as an important regulator of genetic information flow in eukaryotic systems. In particular, chemical modifications of RNA have recently been established as key regulatory marks that affect the lifetime, location, trafficking, and function of messenger RNA (mRNA). In mammalian systems, N6-methyladenosine (m6A) is the most prevalent mRNA modification, and the writer, eraser, and reader proteins that install, remove, or recognize m6A have been rapidly uncovered and studied at the whole cell level. Understanding the effects of specific m6A modifications and their regulation at the single transcript level is the key next step to understanding the mechanism and consequences of epitranscriptomic regulation. We recently developed programmable m6A reader proteins to study the effects of epitranscriptomic regulatory factors at individual RNA transcripts. In this chapter, we discuss the application of targeted m6A readers to study RNA regulation at single endogenous sites. We briefly introduce what is currently known about the N6-methyltranscriptome and the Cas13 RNA-targeting family of proteins before detailing our protocol to study RNA modifications with targeted reader proteins.
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