Profiling of circular RNA N⁶‐methyladenosine in moso bamboo (<i>Phyllostachys edulis</i>) using nanopore‐based direct RNA sequencing

Abstract

N⁶ -methyladenosine (m⁶ A) is a prevalent modification in messenger RNAs and circular RNAs that play important roles in regulating various aspects of RNA metabolism. However, the occurrence of the m⁶ A modification in plant circular RNAs has not been reported. A widely used method to identify m⁶ A modifications relies on m⁶ A-specific antibodies followed by next-generation sequencing of precipitated RNAs (MeRIP-Seq). However, one limitation of MeRIP-Seq is that it does not provide the precise location of m⁶ A at single-nucleotide resolution. Although more recent sequencing techniques such as Nanopore-based direct RNA sequencing (DRS) can overcome such limitations, the technology does not allow sequencing of circular RNAs, as these molecules lack a poly(A) tail. Here, we developed a novel method to detect the precise location of m⁶ A modifications in circular RNAs using Nanopore DRS. We first enriched our samples for circular RNAs, which we then fragmented and sequenced on the Nanopore platform with a customized protocol. Using this method, we identified 470 unique circular RNAs from DRS reads based on the back-spliced junction region. Among exonic circular RNAs, about 10% contained m⁶ A sites, which mainly occurred around acceptor and donor splice sites. This study demonstrates the utility of our antibody-independent method in identifying total and methylated circular RNAs using Nanopore DRS. This method has the additional advantage of providing the exact location of m⁶ A sites at single-base resolution in circular RNAs or linear transcripts from non-coding RNA without poly(A) tails.