PRP4 encodes the only kinase among the spliceosome components. Although it is an essential gene in the fission yeast and other eukaryotic organisms, the Fgprp4 mutant was viable in the wheat scab fungus Fusarium graminearum . Deletion of FgPRP4 did not block intron splicing but affected intron splicing efficiency in over 60% of the F . graminearum genes. The Fgprp4 mutant had severe growth defects and produced spontaneous suppressors that were recovered in growth rate. 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. The Q86K mutation in FgMSL1 was identified by whole genome sequencing in suppressor mutant S3. Whereas two of the suppressor mutations in FgBrr2 and FgPrp8 were similar to those characterized in their orthologs in yeasts, suppressor mutations in Prp6 and Prp31 orthologs or FgMSL1 have not been reported. Interestingly, four and two suppressor mutations identified in FgPrp6 and FgPrp31, respectively, all are near the conserved Prp4-phosphorylation sites, suggesting that these mutations may have similar effects with phosphorylation by Prp4 kinase. In FgPrp31, the non-sense mutation at R464 resulted in the truncation of the C-terminal 130 aa region that contains all the conserved Prp4-phosphorylation sites. Deletion analysis showed that the N-terminal 310-aa rich in SR residues plays a critical role in the localization and functions of FgPrp4. We also conducted phosphoproteomics analysis with FgPrp4 and identified S289 as the phosphorylation site that is essential for its functions. These results indicated that FgPrp4 is critical for splicing efficiency but not essential for intron splicing, and FgPrp4 may regulate pre-mRNA splicing by phosphorylation of other components of the tri-snRNP although itself may be activated by phosphorylation at S289. Author Summary In eukaryotic organisms, many genes containing introns that need to be spliced by the spliceosome after transcription. Among all the spliceosome components, Prp4 is the only protein kinase. Unlike other organisms, deletion of the FgPRP4 kinase gene was not lethal in the wheat scab fungus Fusarium graminearum . In this study, we found that FgPRP4 is not essential for intron splicing but important for splicing efficiency. The Fgprp4 mutant was not stable and produced spontaneous suppressors recovered in growth rate. Suppressor mutations were identified in the PRP6, PRP31, BRR2, and PRP8 orthologs, key components of the U4/U6-U5 complex in the spliceosome and FgMSL1 by candidate gene or whole genome sequencing. We also showed that the N-terminal 310 amino acid region of FgPrp4 plays a critical role in its localization and functions of FgPrp4 and identified S289 as a critical phosphorylation site. Overall, our result indicated that FgPrp4 is important for splicing efficiency, possibly by phosphorylation of other spliceosome components.
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