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

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.

Quantitative profiling of N6-methyladenosine at single-base resolution in stem-differentiating xylem of Populus trichocarpa using Nanopore direct RNA sequencing

“…The products were then eluted in 21-μl Elution Buffer (1-μl elution was used to quantify the concentration using QubitTM dsDNA HS Assay Kit), mixed with nuclease-free water and RNA Running Buffer (RRB) to a total of 75-μl library prior to loading onto the flow cell, and ran on GridION (repeat1) and MinION (repeat2) sequencer, respectively. The RNA strand was ligated to the 1D sequencing linker bearing the RNA motor protein, which ensured efficient translocation of RNA rather than cDNA through the Nanopore [27,28]. Two independent biological repeats were sequenced using a GridION X5 sequencer with flow cell R9.4.1 and a MinION flow cell (FLO-MIN106) to generate real-time single-molecule sequencing.…”

N6-Methyladenosine Modification Opens a New Chapter in Circular RNA Biology

“…Through depletion of rRNA, treatment with RNase R to digest linear RNAs and m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq), m6A modification of circRNAs has been found to exist widely. In plant, Wang et al (2020) found that about 10% of the EcRNAs contained m6A sites in moso bamboo. In human embryonic stem cells (hESCs) and HeLa cells, Zhou et al (2017) identified 1,404 m6A circRNAs and 987 m6A circRNAs, respectively, and they found that m6A circRNAs were expressed in cell-type-specific methylation patterns.…”

Research progress concerning m6A methylation and cancer (Review)

“…With the progress of high-throughput sequencing and antibody-specific enrichment technology, a new detection method, methylated RNA immunoprecipitation sequencing (MeRIP-seq), was developed with the advantage of identifying almost all m 6 A modifications in different types of RNA, such as mRNA (77), lncRNA (78) and circular RNA (79). In MeRIP-seq, specific antibody of m 6 A is used to extract co-immunoprecipitated RNA fragments, which are further identified using high-throughput sequencing (80).…”