Secondary Mutation-Induced Alternative Splicing Suppresses RNA Splicing Defect of the jhs1 Mutant

jing he sheng1 (jhs1) is a mutant of the DNA2 homolog in Arabidopsis (Arabidopsis thaliana), which was previously identified as being involved in DNA damage repair, cell cycle regulation, and meristem maintenance. A mutation at the 3' intron splice site of the 11th intron causes alternative splicing of this intron at two other sites, which results in frame shifts and premature stop codons. Here, we screened suppressors of jhs1 to further study the function and regulatory networks of JHS1 Three suppressors with wild-type-like phenotypes were obtained. Sequencing analysis results showed that each of the suppressors has a second mutation in jhs1 that causes further alternative splicing of the intron and corrects the shifted reading frame with small insertions. Precursor mRNA sequence analysis and intron splice site evaluation results suggested that intron splicing was disturbed in the suppressors, and this switched the splice site, resulting in small insertions in the coding regions of JHS1. Structural analysis of JHS1 suggested that the insertions are in a disordered loop region of the DNA2 domain and do not seem to have much deleterious effect on the function of the protein. This work not only has implications for the evolution of protein sequences at exon junctions but also provides a strategy to study the mechanism of precursor mRNA splicing.