m6A2Circ: A comprehensive database for decoding the regulatory relationship between m6A modification and circular RNA

Abstract

Circular RNA (circRNA) is a class of noncoding RNAs derived from back-splicing of pre-mRNAs. Recent studies have increasingly highlighted the pivotal roles of N6-methyladenosine (m6A) in regulating various aspects of circRNA metabolism, including biogenesis, localization, stability, and translation. Despite the importance of m6A in circRNA metabolism, there remains a substantial gap in comprehensive resources dedicated to exploring m6A modification in circRNA. To bridge this significant gap, we present m6A2Circ (http://m6a2circ.canceromics.org/), a pioneering database designed to systematically explore the regulatory interactions between m6A modification and circRNA. The m6A2Circ database encompasses 198,804 m6A-circRNA associations derived from diverse human and mouse tissues. These associations are meticulously categorized into four levels of evidence supported either by experimental data or by high-throughput sequencing data. Moreover, the database offers extensive annotations, facilitating research into circRNA function and its potential disease implications. Overall, m6A2Circ aims to benefit the research community and bolster novel discoveries in terms of crosstalk between m6A and circRNA.

Keywords: Circular RNA; Database; N6-methyladenosine.

Fig. 1

Overall design and construction of m6A2Circ. The m6A-circRNA associations cataloged in m6A2Circ are categorized into four evidence types: validated—derived from manual curation; NGS.Lv1-m6A—inferred from MeRIP-seq data analysis; NGS.Lv2-Perturb—inferred from m6A WER perturbation followed by RNA-seq and Ribo-seq data analysis; and NGS.Lv2-Bind—inferred from m6A WER RIP-seq data analysis. Additionally, m6A2Circ offers many annotations, including mature sequences, predicted m6A sites, disease associations, miRNA binding sites, RBP binding sites, internal ribosome entry sites (IRESs), and open reading frames (ORFs).

Fig. 2

Overview of m6A-circRNA associations in m6A2Circ. (A) A table displaying the number of m6A-circRNA associations and non-m6A-circRNAs within m6A2Circ, categorized by various tissue types. The bars at the top of the table indicate the number of high-throughput sequencing samples from different tissues. (B) A stacked column chart illustrating m6A-circRNAs in m6A2Circ across different tissue types, classified into four confidence levels. If an m6A-circRNA is associated with multiple confidence levels, it is represented multiple times in the chart, with each confidence level assigned a distinct color. (C) Overlap between records with different types of evidence for the same m6A-circRNA. (D) Validation of 10 candidate m6A-circRNAs via qRT—PCR and RNase R treatment in KYSE30 cells. (E) Validation of the m6A modification of 10 m6A-circRNAs using MeRIP-qPCR after RNase R treatment in KYSE30 cells. The data in E and F are the means ± SDs (n = 3). * , P < 0.05; * *, P < 0.01; * ** , and P < 0.001 for Student’s t test compared with each control.

Fig. 3

Characterization of m6A-circRNAs in humans. (A) Violin plot of the lengths of m6A-circRNAs and non-m6A-circRNAs in human circRNAs. (B) The percentage of circRNAs (y-axis) was calculated on the basis of the number of exons that each circRNA spans (x-axis) for m6A-circRNAs and non-m6A-circRNAs in humans. (C) Exon lengths of m6A-circRNAs and non-m6A-circRNAs spanning different numbers of exons in humans. The distributions of exon length (y-axis) for input m6A-circRNAs and non-m6A-circRNAs are plotted on the basis of the number of exons spanned by each circRNA (x-axis). (D) Boxplot of the differential expression of m6A-circRNAs and non-m6A-circRNAs in different tissue types in humans. (E) Differences in the annotation shares of human m6A-circRNAs and non-m6A-circRNAs in m6A2Circ (IRES: internal ribosome entry site). (F) Cumulative fraction of coding probability scores predicted by CPAT for m6A- and non-m6A-circRNAs in humans. (G) Cumulative fraction coding probability scores predicted by the CPAT for m6A-circRNAs of specific WERs in humans. Statistical tests: Wilcoxon test for A, C, D; chi-square test for E. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Fig. 4

Introduction of the m6A2Circ web interface. (A) The browsing interface of m6A2Circ. (B) The main modules of the search interface in m6A2Circ. (C) Detailed information about m6A-circRNA associations can be accessed by clicking on the m6A2Circ ID. Clicking on the analysis ID on the m6A2Circ ID page links to more details about the specific analysis.