Effects Of Alternative Splicing On The Targeting Sequences Of Proteins And Biological Functions Of ARC5 In Arabidopsis

Alternative splicing(AS)is a process occurring in all eukaryotes by which a single pre-mRNA can generate multiple mRNA variants,often encoding proteins with functional differences.Through AS,the genome of an organism can encode proteins with more varieties,resulting in more complex mechanisms of metabolisms,developmental regulations and stress responses,as well as a better environmental adaptability.Signal peptides,transit peptides and PTS2 at the N-terminus and PTS1 sequences at the C-terminus of proteins are critical for the targeting of a protein to various organelles.Genome-wide analysis of the effects of AS on protein subcellular localization will help us to understand the roles of AS.In this study,the effects of AS on protein subcellular localization in Arabidopsis was investigated at the genome level,mainly based on approaches of protein sequence analysis,Gene Ontology(GO)analysis,RT-PCR,subcellular localization and evolutionary analysis.The results indicated that in the 5,885 alternatively spliced genes,AS affected the N-terminal targeting sequences of 413 genes,and the C-terminal targeting sequence PTS1 of 66 genes.In addition,PTS2 motifs in protein sequence of 5 genes were affected by AS.Proteins with different subcellular localizations caused by AS are involved in various aspects of plant physiological processes.RT-PCR analysis of the 9 candidate genes showed that they could produce two or more different types of mRNAs through AS,and the expression and the AS pattern of 2 genes are tissue-specific.Subcellular localization results showed that AS could affect the existence of chloroplast transit peptides,mitochondrial targeting peptides,signal peptides and PTS2 by affecting the N-terminal sequence of a protein,which further affected their targeting ability to chloroplasts,mitochondria,ER pathways and peroxisomes.AS can also affect the peroxisome targeting ability of proteins by altering their C-terminal PTS1.ARC5 is an important chloroplast division protein.Previous experimental results showed that the PH domain with a potential membrane lipid binding capacity is affected by AS,but the detailed molecular mechanism is not clear.Here,the results show that the PH domain has a special lipid binding property which is critical for the function of ARC5.ARC5-S(short,S)resulted from AS has a stronger chloroplast division ability than ARC5-L(long,L).The lipid binding capacity of PH-L was significantly lower than that of PH-S.In the young green tissue,the total amount of ARC5 and the proportion of ARC5-S are higher for the rapid division of chloroplasts,whereas in the aging or non-green tissues,ARC5 level and the proportion of ARC5-S are relatively lower.In addition,PDV1 and PDV2 proteins play an important role in the dimerization or oligomerization of ARC5-L,whereas they have no effect on the dimerization or oligomerization of ARC5-S.The AS of ARC5 and its effect are conserved in vascular plants,which facilitates the regulation of chloroplast division.