The Role Of Prp4 Kinase In Pre-mRNA Splicing In Fusarium Graminearum

Pre-mRNA splicing, an important gene expression regulation mechanism in eukaryotic cells, is mediated by the spliceosome that is formed by ordered interaction of the U1, U2, U4/U6, U5 snRNPs, and non-snRNP proteins. Reversible protein phosphorylation affects most cellular activities in eukaryotes, including pre-mRNA splicing. The phosphorylation of spliceosomal proteins are important during the splicing process. Among all the spliceosome components, Prp4 is the only protein kinase.Although it is an essential gene in the fission yeast and other eukaryotic organisms, the budding yeast has limited number of small introns and lacks a distinct Prp4 ortholog and deletion of PRP4 was not lethal in the wheat scab fungus Fusarium graminearum. Prp4 contains a C-terminal kinase domain and an extended N terminus subdomain rich in arginine-serine dipeptides. Deletion analysis showed that the N-terminal 310-aa rich in SR residues plays a critical role in the localization and functions of Prp4. Phosphoproteomics analysis with Prp4 identified S289 as the phosphorylation site that is essential for its functions.Based on RNA-seq data, Prp4 is not essential for intron splicing but important for splicing efficiency, and introns affected by PRP4 deletion had longer distance between 5’ss and BP. The PRP4 mutant had severe growth defects and produced spontaneous suppressors that were recovered in growth rate. More than 300 spontaneous fast-growing suppressors were isolated. Among the 49 suppressor strains assayed for defects in conidiation, sexual reproduction, and plant infection, none of them were fully rescued the defects of PRP4 mutant. RNA-seq analysis of S2 and S47 showed that the suppressor strains were only partially recovered in intron splicing efficiency. Therefore, the spontaneous suppressor strains may be not fully recovered in splicing efficiency. Suppressor mutations were identified in the PRP6, PRP31, BRR2, and PRP8 orthologs in nine suppressor strains by sequencing analysis with candidate tri-snRNP component genes. Whereas two of the suppressor mutations in Brr2 and Prp8 were similar to those characterized in their orthologs in yeasts, suppressor mutations in Prp6 and Prp31 orthologs have not been reported.Prp31 was selected for further characterization not only because that Prp31 is one of the key protein components of the tri-snRNP that is essential for pre-mRNA splicing, but the non-sense mutation at R464 resulted in truncation of the C-terminal 130 aa region that contains all the conserved putative Prp4-phosphorylation sites. In our study, 28 suppressor mutations were identified in the C-terminal tail of Prp31 that displayed self-inhibitory interaction with its N-terminal region in yeast two-hybrid and Co-IP assays, after sequencing 260 suppressor strains. Suppressive truncation or point mutations in Prp31 decreased its interaction with Prp6 but increased its interaction with Brr2. G308 E suppressor mutation of Brr2 also increased its interaction with Prp31. We further confirmed that Prp31 is phosphorylated by Prp4. Moreover, site-directly mutagenesis analysis with the conserved Prp4-phosphorylation sites showed that mutations at multiple phosphorylation sites of PRP31 were necessary to suppress PRP4 deletion, and phosphorylation at S520, S521 and T525 may be more important for Prp31 function. Phosphorylation of Prp31 by Prp4 may release its self-inhibitory binding and affects its interaction with Brr2 and other components of tri-snRNP for B-complex activation.Overall our data indicated that Prp4 is critical for splicing efficiency but not essential for intron splicing, and Prp4 may regulate pre-mRNA splicing and the interactions between components of the U4/U6-U5 tri-snRNP by phosphorylation of Prp31.