Functional Regulation Of Splicing-Related Proteins By H₂S-Mediated S-Sulfhydration In Arabidopsis Thaliana

Hydrogen sulfide（H₂S）,a gaseous signal molecule,plays a key role in plant growth and development and response to abiotic stress.One of its mechanisms is that H₂S participates in the regulation of protein function through persulfidation modification.Alternative splicing（AS）is a major contributor to proteome complexity and plays an important role in variety of cellular functions,including signal transduction,immunity and development,which are mainly regulated by alternative spliced proteins.At present,there are few reports on the relationship between hydrogen sulfide and alternative splicing in plants.Previous transcriptome data in our laboratory showed that H₂S affected the alternative splicing patterns of many genes,and it has been reported that 5%of the proteins in Arabidopsis thaliana proteome may undergo persulfidation modification.These proteins are involved in a wide range of biological functions,regulating carbon metabolism,RNA translation,plant response to abiotic and biological stress and other important processes,but there is no further confirmation.Therefore,we speculated that H₂S may affect the alternative splicing modes of genes by modifying some pre-m RNA splicing related proteins by persulfidation modification,thus regulating the ability of plants to cope with abiotic stress.Through a large number of bioinformatics analysis and combined with the literature reports,we selected seven proteins CC1,CC1-LIKE,ATO,RSZ22A,MOS4,CBP80 and SWAP to study.The experimental results are as follows:1.Cloning of CC1（AT2G16940,1263 bp）,CC1-LIKE（AT5G09880,1584 bp）,ATO（AT5G06160,1515 bp）,RSZ22A（AT2G24590,591 bp）,MOS4（AT3G18165,762 bp）,CBP80（AT2G13540,2547 bp）and SWAP（AT1G14650,2358 bp）gene.Nine prokaryotic expression vectors were constructed,namely p Cold-ATO,p Cold-CBP80,p Cold-SWAP,p Cold-MOS4,p Cold-RSZ22A,p Cold-CC1,p Cold-CC1-LIKE,XF245-CC1-LIKE and XF245-CC1.Seven proteins,p Cold-ATO,p Cold-CBP80,p Cold-SWAP,p Cold-MOS4,p Cold-RSZ22A,XF245-CC1-LIKE and XF245-CC1,were expressed in Escherichia coli（E.coli）in vitro and purified by His tag.2.Four of the seven splicing proteins can be persulfidated by H₂S using the improved biological switching method,they are RSZ22A,ATO,CC1,CC1-LIKE,respectively.MOS4can not be persulfidated by H₂S.SWAP and CBP80 failed to be persulfidated.3.Wild type Arabidopsis thaliana as genetic background,homozygous CRISPR-RSZ22A gene editing lines and OE-RSZ22A,OE-CC1 homozygous plants were constructed,and the cc1 were identified as homozygous T-DNA insertion mutants.4.We performed RNA sequencing in 2-week-old WT and rsz22a seedlings with or without treatment by H₂S.GO enrichment analysis showed that the deletion of RSZ22A splicing factor was related to abnormal alternative splicing of histone modification-related genes in epigenetic regulation,and H₂S was involved in this regulation mechanism.Therefore,we analyzed the alternative splicing event pattern of histone modification related genes.Genes with intron retention were ATX2,JMJ27;genes with exon skipping were SUVH9,B160,HAG1,OTLD1,JMJ24,HOS15,SRT2;the gene with mutually exclusive exons was HDA9;genes with alternative 3’splice site were HUB2,HUB2,ULCS1,SGF29b,B160,ASG3,AT2G44970,ELP2,AT3G52030,SGF29a;genes with alternative 5’splice site were RH30,HUB2,HAC1,EAF6.To sum up,H₂S can persulfidate RSZ22A,ATO,CC1,CC1-LIKE splicing factors,H₂S can not persulfidate MOS4 splicing factor.H₂S probably regulates pre-mRNA splicing of histone modification genes by persulfidating RSZ22A.