Mechanism Analysis Of Spliceosome-Associate Protein FgSad1 In Fusarium Graminearum

Pre-mRNA splicing is modulated by the spliceosome,a dynamic molecular machine that is composed of five small nulear ribonucleoprotein(snRNP)particles(U1、U2、U4、U5、U6)and hundreds of non-snRNP proteins.As the largest subunit of spliceosome,the pre-formed tri-snRNP(U4/U6.U5)plays an important role in spliceosome assembly.The interactions between three most important U5-proteins,Brr2,Prp8 and Snu114 are important to keep the integrality of the tri-snRNP before joining the complex A.And the crux is inhibiting the RNA helicase function of Brr2 by regulating the interactions among Brr2,Prp8 and Snu114.Sad1 is involved in maintaining the homeostasis of tri-snRNP and recruiting the tri-snPNP to complex A.However,the mechanism of Sad1’s action is unknown.In this study,we found mutations in FgSad1 partially suppressed Fgprp4,which is impaired in splicing efficiency,in Fusarium graminearum.Via investigating the mutations in FgSAD1,we character the function of FgSad1.Seven mutations that are focused on three differently parts in the 3-D model of FgSad1 were identified after sequencing the FgSAD1 genes in 320 suppressors of Fgprp4 mutant.Although they are recovered in growth rate,all the suppressors still have defects in sexual reproduction and plant infection,indicating that the suppressors did not fully recover the splicing defects of Fgprp4.In sequence alignment,the 7 mutated sites are conserved in filamental fungi.When mapped in its 3D model,all the suppressor mutations did not change the overall structure of FgSad1,but the P258 S,S269P,T324 I and A327 V changed hydrogen bonds in the protein.Interestingly,in comparison with ScSad1,FgSad1 has an extra N-terminal serine and threonine rich region.Four phosphorylation sites were identified in this N-terminal region,but whether they are phosphorylated by FgPrp4 needs further proof.The N-terminal truncation mutant is impaired in growth,sexual reproduction,and plant infection.But the truncation did not affect subcellular localization of the protein.Therefore,the N-terminal region is important for the FgSad1’s function but disposable for its localization.To determine the mechanism of suppressor mutations,we choose 4 mutations according to their position on the 3-D structure of Sad1 and tested their effects on the interactionsbetween FgSad1 and other tri-snRNP proteins with yeast two hybrid and co-IP assays.We are able to show that P258 S and S269 P mutations enhanced the interactions between FgSad1 and FgSnu66,FgBrr2,FgPrp8,or FgSnu114,while D76 N and L512 P decreased the interaction between FgSad1 and FgSnu114.Overall,our results suggest the role of FgSad1 in maintaining tri-snRNP homeostasis.Via mediating the interaction between itself and FgBrr2、FgSnu114、FgSnu66 and FgPrp8,FgSad1 helps to maintain integrality of tri-snRNP.