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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.

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

  • 实验方案

  • 发表日期：

    2025年

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  • Maxime Wery,
  • Ugo Szachnowski,
  • Sara Andjus,
  • Antonin Morillon

摘要

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.