Identification,analysis Of Salt-responsive Alternative Splicing In Grapevine And Construction Of Grape Bioinformatics Website

Alternative splicing(AS)is a post-transcriptional regulatory mechanism widely present in eukaryotes,which greatly enhances the plasticity of transcriptome and proteome by generating multiple RNA transcripts from a single gene through different splicing patterns,and plays a critical role in various physiological processes of organisms.Grapevine is an economically important fruit tree in China and exhibits relatively strong salt tolerance,but the role of AS in grapevine salt tolerance remains unclear.In this study,we optimized the method for identifying AS events and comprehensively identified salt-responsive AS events in grapevine roots.We analyzed the related mechanisms and regulatory pathways to enrich the molecular mechanism of grapevine salt tolerance.Additionally,we established a grape bioinformatics website that can be used for querying AS-related information.The main results of this study are as follows:(1)The bioinformatics-based method for identifying AS events relies on genome and annotation files,and thus we optimized these two aspects.We constructed a pan-genome for grape family to optimize the genome file,collected 765 publicly available grape genome data,including 405 Eurasian species,98 sylvestris species,81 North American species,57 East Asian species,and 12 Muscadinia species,etc.Pan-genome construction based on Map-to-pan method,resulting in 1.33 Gb of novel sequences that did not belong to the reference genome,including 9,479 new genes and 1538 new alternative splicing events.Genome annotation file optimization: Firstly,a genome annotation file was obtained through full-length transcriptome sequencing.Then,it was merged with multiple publicly available annotated files.Finally,the information on splicing isoforms was enriched using long reads and RNA-seq short reads.The optimized annotation file identified more AS events than the publicly available annotation files.(2)A total of 1,466 salt-responsive AS events were identified in grapevine roots,of which retained introns(RI)is the most dominant type,followed by alternative 3′ splice sites(A3)and alternative 5′ splice sites(A5).Differential splicing events mainly produced exclusion isoforms rather than inclusion isoforms.AS affected the integrity of protein products: Some genes related to ion transport and stress response,such as HKT1,SKOR,and one NB-ARC domaincontaining protein,are upregulated in expression while the level of intron retention is decreased,thereby maintaining the level of truncated proteins at a low level.AS may affect the function of transcription factors through peptide interference(PEPi)mechanism: for example,EFM is a MYB domain-containing protein with an RI event,under salt stress conditions,the level of intron retention decreases,weakening the inhibitory effect of truncated products(incomplete MYB domain)on the normal protein.AS may affect mRNA stability through nonsensemediated decay(NMD): 73.5% of salt-responsive RI events contained premature termination codons and were regulated by NMD mechanisms to avoid excessive non-functional protein production.AS affected miRNA-mediated regulation: 410 salt-responsive AS events in grapevine roots affect the targeting of 76 miRNAs(such as vvi-miR3637-5p)to mRNA.Although 42 AS events occurred in lncRNAs,it does not seem to participate in any known lncRNA mechanisms.(3)The AS regulatory mechanism mediated by multiple factors in grapevine was analyzed.The impact of spliceosome-associated proteins on AS in grapevine: Several spliceosomeassociated genes(such as LSM2,SRSF1,PRP18,and RS31,etc.)were induced by salt stress;Meanwhile,some spliceosomal protein genes(such as RS31,etc.)were also regulated by splicing.The impact of sequence features on AS in grapevine: By analyzing 4,387 publicly available RNA-seq datasets,introns in grapevine genes were classified into four categories:spliced,retained,housekeeping,and regulated.A total of 136 RNA sequence features were extracted,and binary logistic analysis showed that there are many similarities in RNA sequence features associated with intron retention(retained vs.spliced)and intron retention level regulation(regulated vs.housekeeping).The most significant feature is the high G/C content of the intron,and low splice site(donor and acceptor)strength,location in UTR,lack of PTC,relatively high intron rank(closer to the 3′ end),and high downstream exon G/C content are all positively correlated with high intron retention levels and high regulatory potential.The impact of transcriptional regulation on AS in grapevine: Genes with up-regulated expression were more likely to produce excluded splicing isomers under short-term salt stress,and similar results were obtained by analyzing other published RNA-seq data.In order to study the effect of posttranscriptional processing on AS in grapevine,the ASAPA pipeline was developed based on Iso-Seq data.After analysis,it was found that there may be a correlation between two AS events,and the most common scenario involves two adjacent RI events,where two introns are typically spliced or retained together.Alternative transcription start sites may affect the splicing of the first intron(usually in RI or A5 events),and the proximal transcription start site is usually accompanied by the proximal splice donor site.Alternative polyadenylation may affect the splicing of the last intron,and the proximal poly A site is usually accompanied by the proximal splice acceptor site.There may also be a potential interaction mechanism between alternative transcription start sites and alternative polyadenylation.(4)A bioinformatics website for grapevine(http://www.winberige.cc/)has been established,which can be used for retrieval and analysis of AS.For example,Search and GeneID Finder can be used to query splicing isoforms of genes across multiple genomes;Protein Annotator can perform functional analysis on multiple splicing isoforms,simultaneously;Genome Browser can display the location of AS on chromosomes;Ref Seq Structer can show the position of splice junctions in multiple sequence alignment;A total of259,376 gene structure maps(Snapgene Pictures)were provided for download,and each labeled with the location information of UTR,CDS and splice junctions;RNA-seq Processor was developed based on 4,387 RNA-seq data,which enables pairwise correspondence between environment,gene and AS.This means that it is possible to query which genes are differentially expressed and differentially spliced in which environmental treatments,as well as which environmental treatments affect the expression and splicing of which genes.