Analysis Of Micro-Exon Genes And Their Related Gene Families In Rice

Micro-exons are a type of exons with a length not more than 51 nucleotides.In some early studies,they are generally ignored due to their short length,and would be annotated incorrectly.However,recent studies indicate their unusual splicing properties and important functions.Despite the discoveries and descriptions of micro-exons in the studies of plant genes,there is still little systematic researches based on whole genomes.To fill this gap,this study identified and analyzed micro-exons and their related gene families from two indica rice varieties,Zhenshan 97 and Minghui 63.The genome annotations of Zhenshan 97 and Minghui 63 showed that micro-exons accounted for only 6%of total exons,but they existed in 23%of the total genes.This study used RNA-seq data to identify 7,645 and 8,137 internal micro-exons,of which33%and 35%had not been annotated before.Furthermore,these novel micro-exons were located in the annotated genes,which might contribute to the improvement of the genome annotation.Besides,micro-exon genes were ancient genes,while micro-exons might emerge in a later period based on phylostratum analysis.And it was found that micro-exons were related to the region of domains.Among the domains,AP2 domain and K-box domain were significantly correlated with micro-exons.In addition,micro-exons were related to the coiled coil regions and tyrosine phosphorylation sites,indicating that micro-exons have the function of changing protein-protein interactions and inducing post-translational modification sites.Among variant samples,60%of the micro-exons were constitutively spliced in all samples,while the other micro-exons were alternatively spliced under different tissues and conditions.The micro-exons had a certain tissue specificity in splicing level but had no obvious specificity under different temperatures or day light conditions.And the functional enrichment studies indicated that the tissues-specific micro-exon genes were enriched in specific metabolism processes and biosynthesis processes.Moreover,micro-exon genes panicle-specific in were enriched in the molecular functions,such as transcription factor activity and protein dimerization activity.Besides,the prediction of protein structure showed that a local structural change might occur in the process of alternative splicing of the micro-exon(a multiple of three nucleotides).On the other hand,micro-exons were found related to the core structure of the domain and post-translational modification sites,which might still affect the function of the protein.Further study was performed in two micro-exon related gene families of plants transcription factors.The AP2/ERF family include AP2 domains,and micro-exons were concentrated in the AP2 domain regions of AP2 subfamily.Genes of the AP2 subfamily were rarely found in algae,while a large number of them were in terrestrial plants.In addition,the two AP2 domains R1 and R2 of AP2 subfamily of different species were separately clustered in the phylogenetic tree except Chlamydomonas reinhardtii whose R1 was aggregated with R2 of other species.And the region of these domains contained no micro-exons.The micro-exons existed in different groups of species,suggesting that they might emerge in divergent periods.Besides,sequences of the micro-exons and AP2 domains were conserved in various plants,and the micro-exons in Oryza tended to use rare condons.The MADS-box family(MIKC subfamily)is another important transcription factor in plants,which contain both a MADS-box domain and a K-box domain.Similar to the study of the AP2/ERF family,most MIKC genes were also only found in terrestrial plants,and the micro-exons were concentrated in regions of the K-box domain.The phylogenetic analysis showed that K-box domains from Chlamydomonas reinhardtii or Selaginella moellendorffii were clustered in a single group,while that in other species were distributed in multiple clades,indicating the differentiation of protein sequences and functions.The regions of K-box domains generally contained two micro-exons,which were in tandem at the C-terminus of the domain and constituted a motif.And previous research showed that the motifs were related to protein polymerization activity.Besides,it was found that the micro-exons had significantly higher K_a/K_s values than that of the K-box domain.