Multi-Omics Analysis Of RNA-Binding Proteins RZ-1B/1C Regulating Co-Transcriptional Splicing In Arabidopsis

The transcriptional regulation of gene expression is the main mechanism by which plants are used to confer phenotypic plasticity,but compared with other eukaryotes or bacteria,studies on the principles of transcriptional regulation in plants are currently relatively backward.Splicing of pre-mRNA is one of the key steps in eukaryotic gene expression,but little is known about its mechanism and regulation.In this study we analyze CB-RNA-seq(chromatin-bound RNA sequencing)data to map nascent RNA in Arabidopsis wild-type seedlings and plant-specific hnRNP-like RNA binding protein rz-1b rz-1c double mutant.With the analysis of iCLIP(individual-nucleotide resolution UV crosslinking and immunoprecipitation)data,the binding sites of RZ-1C are identified at the genome-wide level.The splicing mechanism and characteristics of plant genes at the level of co-transcription are studied by multi-omics methods.It is found that the genes with high expression in wild-type and rz-1b rz-1c double mutant also have more transcription of nascent RNA,indicating they are positively correlated.The gene with high expression of wild-type nascent RNA have a downward trend from the 5' end to the 3' end in the gene region.In the rz-1b rz-1c double mutant,the gene with high expression of nascent RNA have more obvious decline from the 5' to 3' end of the gene region.The deletion of RZ-1B and RZ-1C affects the transcriptional process of the gene,allowing more nascent RNA to stay at the 5' end.To understand the regulation of RZ-1B and RZ-1C on the splicing process at the level of co-transcription,we analyze the splicing ratio of the intron and exon boundary regions to study the co-transcriptional splicing ratio of wild-type and rz-1b rz-1c double mutant.In wild type,the 5' and 3' splicing sites ratios of intron is 0.237 and 0.243 on average,respectively,indicating that splicing of most of the genes in Arabidopsis occurs at the level of co-transcription.The mean 5' and 3' splicing sites ratios in rz-1b rz-1c double mutants are 0.329 and 0.335,respectively,indicating that the ratio of co-transcriptional splicing in the rz-1b rz-1c double mutant is low,and RZ-1B and 1C affect the splicing at the level of co-transcription.Analyses of the splicing ratios of intron positions,number of introns,and gene length reveal that the splicing ratio in the wild type is affected by the number of introns and the length of the gene,while the relationship with the position of the intron is not significant.As the number of introns increases,the rate of co-transcriptional splicing decreases.The increase in gene length also leads to a decrease in the rate of co-transcriptional splicing of genes.The number and position of introns in the mutant are not significantly different from the ratio of co-transcriptional splicing compared to the wild type,while the ratio of co-transcription is the slowest in the shorter length of the gene,which is opposite to the distribution in the wild type.Different co-transcriptional splicing patterns indicate that RZ-1B and 1C regulate co-transcriptional splicing of genes of different lengths.GO analysis of the splicing ratio difference genes reveals that the differential genes are mainly enriched in various metabolic processes of the cell,chromatin modifications and histone modifications.To explore the relationship between histone modifications and co-transcriptional splicing,we use published histone modifications data to analyze the level of histone modifications in genes with different co-transcriptional splicing ratios in wild-type.The results show that H3K36me3,H3K4me2 and H3K4me3 related to activation or promotion of transcription are negatively correlated with the ratio of co-transcriptional splicing in the intron-exon-intron interval.The H3K36me3 and H3K4me3 levels show a decrease from the 5' to 3' direction,which is consistent with the gene transcription direction,indicating that H3K4me2,H3K36me3 and H3K4me3 are involved in RNA co-transcriptional splicing regulation.The level of H3K27me3 modification is low and is not significantly correlated with the ratio of co-transcriptional splicing.There are few studies on the function of nuclear localization of RNA-binding proteins in plants.We use iCLIP with high-throughput sequencing to analyze the distribution of RZ-1C protein binding sites in Arabidopsis thaliana at the whole genome level.The results show that RZ-1C binding sites are mainly enriched in the exon region.With the expression data of wild-type RNA-seq,it is found that RZ-1C is positively correlated with gene expression.GO analysis of the overlapping genes between the RZ1-C binding genes and the differential splicing genes shows that RZ-1C regulates co-transcriptional splicing related to the synthesis of splicing ribosomal protein complex and various cellular metabolism processes.In this study,CB-RNA-seq,iCLIP-seq and other multi-omics methods are used to study the patterns of nascent RNA in plants,revealing that most of the gene splicing in plants occurs at the level of co-transcription,and RZ-1B/1C is widely involved in gene co-transcription.This study could provide a reference for further study of the characteristics of co-transcriptional splicing in plants.