Molecular Biology

Chemical modifications on RNA play important roles in regulating its fate and various biological activities. However, the impact of RNA modifications varies depending on their locations on different transcripts and cells/tissues contexts; available tools to dissect context-specific RNA modifications are still limited. Herein, we report the detailed protocol for using a chemically inducible and reversible platform to achieve site-specific editing of the chosen RNA modification in a temporally controlled manner by integrating the clustered regularly interspaced short palindromic repeats (CRISPR) technology and the abscisic acid (ABA)-based chemically induced proximity (CIP) system. The procedures were demonstrated using the example of inducible and reversible N⁶-methyladenosine (m⁶A) editing and the evaluation of its impact on RNA properties with ABA addition and reversal with the control of ABA or light.

Generation of the epitranscriptome through chemical modifications of protein-coding messenger RNAs (mRNAs) has emerged as a new mechanism of post-transcriptional gene regulation. While most mRNA modifications are methylation events, a single acetylated ribonucleoside has been described in eukaryotes, occurring at the N4-position of cytidine (N4-acetylcytidine or ac4C). Using a combination of antibody-based enrichment of acetylated regions and deep sequencing, we recently reported ac4C as a novel mRNA modification that is catalyzed by the N-acetyltransferase enzyme NAT10. In this protocol, we describe in detail the procedures to identify acetylated mRNA regions transcriptome-wide using acetylated RNA immunoprecipitation and sequencing (acRIP-seq).