Identifying RNA Modifications by Direct RNA Sequencing Reveals Complexity of Epitranscriptomic Dynamics in Rice

Transcriptome analysis based on high-throughput sequencing of a cDNA library has been widely applied to functional genomic studies. However, the cDNA dependence of most RNA sequencing techniques constrains their ability to detect base modifications on RNA, which is an important element for the post-transcriptional regulation of gene expression. To comprehensively profile the N-6-methyladenosine (m(6)A) and N-5-methylcytosine (m(5)C) modifications on RNA, direct RNA sequencing (DRS) using the latest Oxford Nanopore Technology was applied to analyze the transcriptome of six tissues in rice. Approximately 94 million reads were generated, with an average length ranging from 619 nt to 1013 nt, and a total of 45,707 transcripts across 34,763 genes were detected. Expression profiles of transcripts at the isoform level were quantified among tissues. Transcriptome-wide mapping of m(6)A and m(5)C demonstrated that both modifications exhibited tissue-specific characteristics. The transcripts with m(6)A modifications tended to be modified by m(5)C, and the transcripts with modifications presented higher expression levels along with shorter poly(A) tails than transcripts without modifications, suggesting the complexity of gene expression regulation. Gene Ontology analysis demonstrated that m(6)A- and m(5)C-modified transcripts were involved in central metabolic pathways related to the life cycle, with modifications on the target genes selected in a tissue-specific manner. Furthermore, most modified sites were located within quantitative trait loci that control important agronomic traits, highlighting the value of cloning functional loci. The results provide new insights into the expression regulation complexity and data resource of the transcriptome and epitranscriptome, improving our understanding of the rice genome.