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J Integr Plant Biol
Profiling of circular RNA N6-methyladenosine in moso bamboo (Phyllostachys edulis) using nanopore-based direct RNA sequencing
Yongsheng Wang1† , Huihui Wang2† , Feihu Xi1 , Huiyuan Wang2 , Ximei Han3 , Wentao Wei2 , Hangxiao Zhang2 , Qianyue Zhang3 , Yushan Zheng3 , Qiang Zhu2 , Markus V. Kohnen2 , Anireddy S. N. Reddy4 and Lianfeng Gu2 *

1Basic Forestry and Proteomics Research Center, College of life science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2Basic Forestry and Proteomics Research Center, College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, China
4Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA

† These authors contributed equally to this work.
* Correspondence: Lianfeng Gu (lfgu@fafu.edu.cn)
doi: 10.1111/jipb.13002
Abstract

N6‐methyladenosine (m6A) 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 m6A modification in plant circular RNAs has not been reported. A widely used method to identify m6A modifications relies on m6A‐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 m6A 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 m6A 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 m6A 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 m6A sites at single‐base resolution in circular RNAs or linear transcripts from non‐coding RNA without poly(A) tails.

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Received: 13 June 2020      Accepted:    Online on:31 July 2020
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