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Sequence-specific m6A demethylation in RNA by FTO fused to RCas9

Kristina Rau1, Lukas Rösner1, Andrea Rentmeister1

  • 1Institute of Biochemistry, Department of Chemistry, University of Münster, 48149 Münster, Germany.

RNA (New York, N.Y.)
|July 3, 2019
PubMed

Insights

Scientists developed RCas9-FTO, a novel tool for precise RNA demethylation. This technology allows targeted removal of N6-methyladenosine (m6A) modifications at specific sites, advancing the study of m6A functions.

Area of Science:

  • Molecular Biology
  • Epigenetics
  • RNA Modifications

Background:

  • N6-methyladenosine (m6A) is a prevalent mRNA modification influencing cellular processes.
  • Current methods lack precision in targeting specific m6A sites for modification.
  • Modulating global m6A levels via writer/eraser proteins is insufficient for site-specific studies.

Purpose of the Study:

  • To develop a tool for sequence-specific demethylation of m6A in RNA.
  • To enable precise manipulation of individual m6A sites.
  • To facilitate the study of distinct m6A site functions.

Main Methods:

  • Genetic fusion of the m6A demethylase FTO to the RNA-targeting module RCas9.
  • Utilizing sgRNA and PAMmer for sequence-specific RNA binding.
  • Employing SCARLET analysis to quantify site-specific m6A levels.
  • Assessing demethylation activity and sequence preference of RCas9-FTO.

Main Results:

  • RCas9-FTO demonstrated sequence-specific RNA binding and retained FTO demethylation activity.
  • SCARLET analysis quantified m6A levels and analyzed PAM-to-m6A distance effects.
  • RCas9-FTO exhibited up to 15-fold sequence preference for target RNA.
  • The tool proved adaptable to various RNA sequences.

Conclusions:

  • RCas9-FTO is a novel tool for targeted RNA m6A demethylation.
  • This technology allows for precise manipulation of specific m6A sites.
  • RCas9-FTO opens new avenues for investigating the functional roles of distinct m6A modifications.
Keywords:
Cas9FTON6-methyladenosine (m6A)PAMmerRNA modificationdemethylationsgRNA

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