Spontaneous mutations in FgSAD1 suppress the growth defect of the Fgprp4 mutant by affecting tri-snRNP stability and its docking in Fusarium graminearum

Environ Microbiol. 2019 Dec;21(12):4488-4503. doi: 10.1111/1462-2920.14736. Epub 2019 Jul 29.

Abstract

FgPrp4, the only kinase in the spliceosome, is not essential for viability, but is important for splicing efficiency in Fusarium graminearum. The Fgprp4 deletion mutant had severe growth defects but often produced spontaneous suppressors with faster growth rate. To better understand the suppression mechanism, we identified and characterized spontaneous mutations in the tri-snRNP-specific protein, FgSad1, which suppressed the growth defects of Fgprp4. The L512P mutation was verified for its suppressive effects on Fgprp4, suggesting that mutations in FgSad1 may have effects involving FgPrp4 phosphorylation on FgSad1. Phosphoproteomics analysis showed that FgSad1 may not be the direct substrate of FgPrp4 kinase. Furthermore, truncation analysis showed that the N-terminal, extra RS-rich region of FgSad1 is critical for its function and is post-translationally modified. The P258S or S269P mutations in FgSad1 increased its interactions with the U5 protein FgPrp8 and the U4/U6 protein FgPrp31, which may result in tri-snRNP stabilization. Additionally, the D76N mutation increased the association of FgSad1 with the U2 snRNP. These data indicate that suppressor mutations in FgSad1 increase the stability of the tri-snRNP and/or the affinity of FgSad1 with U2 snRNP and therefore potentially facilitate the docking of tri-snRNP into the spliceosome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Fusarium / genetics*
  • Fusarium / growth & development
  • Fusarium / metabolism
  • Humans
  • Phosphorylation
  • Protein Stability
  • Protein-Serine-Threonine Kinases / genetics*
  • Protein-Serine-Threonine Kinases / metabolism
  • RNA Splicing
  • RNA, Fungal
  • Ribonucleoprotein, U4-U6 Small Nuclear / metabolism*
  • Sequence Deletion
  • Spliceosomes / enzymology
  • Spliceosomes / metabolism

Substances

  • RNA, Fungal
  • Ribonucleoprotein, U4-U6 Small Nuclear
  • Protein-Serine-Threonine Kinases