The Molecular Mechanism Of FgSnu114 In Regulation Of Spliceosome Dynamics In Fusarium Graminearum

Pre-mRNA splicing is an important cellular process of Eukaryotes.Splicing is catalyzed by the spliceosome,a dynamic molecular machine that is comprised with five canonical subunits,namely U1,U2,U4,U5 and U6 sn RNPs,and numerous non-sn RNP factors.The U4/U6.U5 tri-sn RNP,the most important component of spliceosome,consists of U5 sn RNP),extensively base-paired U4/U6 di-sn RNP,and more than 30 proteins,including the key components Prp8,Brr2 and Snu114.The GTPase amd the largest U5 sn RNP protein Prp8 regulates the unwinding of U4/U6 Sn RNAs by modulating the helicase activity Brr2.Prp4,the only kinase component of the spliceosome,is essential in fission yeast.In a previous study,we obtained the Fgprp4 deletion mutant in Fusarium graminearum,which is extremely instabible and easy to be suppressed.In this study,we first identified suppressor mutations of Fgprp4 in FgSNU114.After sequencing the 320 suppressor strains,we identified 4 suppressor mutations of FgSNU114 in10 supressors.These four mutations were located in the D1,D2,D3 and D4 domains of FgSNU114,and they did not affect the overall structure of the protein,but have an effect on the side chain and hydrogen bonding patterns inside the protein.Although the examined supressors are normal in growth rate,they still have defects in sexual reproduction and/or plant infection.RNA-Seq analysis of supressor S172 showed that it still have intron splicing defect in comparison with the wild type PH-1.MS analysis and phosphorylation minic experiments showed that FgSnu114 is not the substrate of Fg Prp4.Co-IP and yeast two hybrid analysis demonstrated that suppressor mutation D222 N decreased the interaction between FgSnu114 and Fg Prp8,andΔK695reduced the interaction between FgSnu114 and Fg Brr2.In total,our results demonstrate that the suppressor mutations on FgSNU114 inhibit the phenotype of the Fgprp4.And suppressor mutations in FgSnu114 changed its interaction with Fg Brr2 or Fg Prp8,and further rescue the splicing defects caused by deletion of Fg PRP4.