Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing

Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing. Overall design: To determine the capping status of the RNAs that accumulate upon CHX treatment and/or NMD inactivation, we performed strand-specific total RNA-seq in control (DMSO) and CHX-treated WT and upf1 mutant cells, including a treatment of the RNA extracts with the Terminator 5´-Phosphate-Dependent Exonuclease, which degrades RNAs with 5'-monophosphate ends but not those with an intact m7G cap (2 biological replicates per condition).