The Study Of M~6A Function For Nascent RNA In Arabidopsis Thaliana Based On MeRIP-seq Method

N6-methyladenylate(m~6A)is the most common and abundant chemical modification known on m RNA,mainly distributed in the stop codons and the 3’UTRs of m RNA,and mainly located on the RRACH motif.With the widespread application of high-throughput sequencing technology,m~6A modification has been shown to play an important role in the regulation of eukaryotic transcription and m RNA degradation.Previous studies on m~6A modification mainly focused on m RNA.Through the study on nascent RNA,we can further understand the distribution change of m~6A modification between nascent RNA and m RNA and the function of m~6A in transcript processing and maturation.With the development of research,the regulatory effect of m~6A modification on nascent RNA on biological processes has been gradually discovered.In order to explore the characteristic changes of m~6A modification on nacent RNA and m RNA and its effect on biological functions,chromatin-binding RNA(cb RNA)was used to represent nacent RNA in this study.By analyzing cb RNA and m RNA Me RIP-seq data,the m~6A modification map was obtained.The m~6A modification map showed that m~6A modification was mainly distributed in CDS in cb RNA,and m~6A modification was mainly distributed in CDS and 3’UTR in m RNA,and the proportion of m~6A in 3’UTR region of m RNA was significantly higher than that of cb RNA.In addition,most transcripts contained only one m~6A peak,and the proportion of transcripts containing multiple m~6A peaks in cb RNA was higher than that in m RNA.The significant m~6A modification regions identified by MACS2 were divided into three types according to the modification changes between cb RNA and m RNA:5260 m~6A modification regions specific to cb RNA,10964 m~6A modification regions specific to m RNA,and 2829 m~6A modification regions common to both.GO enrichment analysis showed that cb RNA-specific m~6A modifiers were mainly concentrated in response to stress,response to salt stress,and response to stimulus.The enrichment results of m RNA-specific m~6A modifiers no longer contained stress related items,which were mostly related to anabolism and transport.The results shows that during the conversion of cb RNA to m RNA,the m~6A modifications on many stress-related genes are removed.In order to further verify the relationship between m~6A modification and stress response,Arabidopsis thaliana was treated with salt to further explore the response of cb RNA m~6A modification to salt stress.The results showed that the distribution trend of m~6A in cb RNA was basically unchanged after salt treatment,but the total number of modifications was decreased,and the number of transcripts containing two or more m~6A modifications was significantly reduced.The distribution changes of nascent RNA and m RNA m~6A modifications before and after salt treatment were statistically analyzed.It was found that under salt stress,the amount of m~6A modifications on cb RNA accounted for 32%of that under normal condition.Only 25%of the m~6A modification sites were removed during the conversion of cb RNA to m RNA,much lower than the 46%in the normal state.Most of the modifications were retained during the conversion of cb RNA to m RNA.The GO analysis of differentially modified genes showed that the genes only modified on cb RNA were enriched in biological processes such as response to oxygen-containing compounds and response to stress,while the genes newly modified with m~6A on the m RNA after salt treatment were also enriched in stress-response related items.These results indicate that salt stress can change the distribution of m~6A modification in Arabidopsis thaliana,and m~6A modification can involved in the plant response to stress.MTA is a key gene of m~6A methyltransferase,and mutation of MTA will lead to a significant decrease in the synthesis of m~6A modification.RNA-Seq analysis was performed on cb RNA data of wild-type and mta mutants under normal conditions in this study to further explore the relationship between m~6A modification and gene expression in cb RNA.The results of this study showed that after mta mutation,the number of m~6A modifications on cb RNA was significantly reduced from 5260 to 17.A total of 815 differentially expressed genes were found between wild-type and mta mutants by RNA-seq analysis,among which739 genes were up-regulated and 76 genes were down-regulated.GO enrichment analysis was performed on the genes with reduced m~6A modification and decreased expression after mutation and the genes with reduced m~6A modification and increased expression,respectively.The results showed that there was no significant enrichment in the genes with down-regulated m~6A modification and decreased gene expression.However,genes with decreased m~6A modification and increased gene expression were enriched in response to stimulus and response to stress.The results further proved that m~6A modification could be involved in the process of cell response to stress,and that the decrease of m~6A modification was accompanied by the decrease of the expression levels of some stress-related genes.Histone methylation is also a dynamic epigenetic modification in.Previous studies have shown that histone methylation can also be involved in the transcriptional regulation and growth and development of eukaryotes.In order to study the relationship between m~6A modification and histone modification,this study continued to explore the modification data of H3K4me3,H3K27me3 and H3K36me3.The results showed that the H3K4me3 and H3K36me3 modifications were strongly correlated with the m~6A modifications on the cb RNA,but the correlation basically disappeared during the conversion of cb RNA to m RNA,while the H3K27me3 modifications had no correlation with the m~6A modifications.In this study,based on MeRIP-seq data of nacent RNA and m RNA,combined with histone modification data,the distribution characteristics and function of m~6A modification during the transformation of nascent RNA into m RNA were further studied.In this study,we build the m~6A modification map of cb RNA and m RNA on the whole genome level.We explored the biological processes involved in m~6A modification and their relationship with gene expression.At the same time,the relationship between m~6A modification and histone methylation was explored based on histone methylation modification data.It is helpful to further explore the biological function of m~6A modification in plants and provide new ideas and methods for the study of plant post-transcriptional regulation.