ABSTRACT
Prp31 is one of the key tri-snRNP components essential for pre-mRNA splicing although its exact molecular function is not well studied. In a previous study, suppressor mutations were identified in the PRP31 ortholog in two spontaneous suppressors of Fgprp4 mutant deleted of the only kinase of the spliceosome in Fusarium graminearum. To further characterize the function of FgPrp31 and its relationship with FgPrp4 kinase, in this study we identified additional suppressor mutations in FgPrp31 and determined the suppressive effects of selected mutations. In total, 28 of the 35 suppressors had missense or nonsense mutations in the C terminus 465-594 aa (CT130) region of FgPrp31. The other 7 had missense or deletion mutations in the 7-64 aa region. The nonsense mutation at R464 in FgPRP31 resulted in the truncation of CT130 that contains all the putative Prp4 kinase-phosphorylation sites reported in humans, and partially rescued intron splicing defects of Fgprp4. The CT130 of FgPrp31 displayed self-inhibitory interaction with the N-terminal 1-463 (N463) region, which was reduced or abolished by the L532P, D534G, or G529D mutation in yeast two-hybrid assays. The N463 region, but not full-length FgPrp31, interacted with the N-terminal region of FgBrr2, one main U5 snRNP protein. The L532P mutation in FgPrp31 increased its interaction with FgBrr2. In contrast, suppressor mutations in FgPrp31 reduced its interaction with FgPrp6, another key component of tri-snRNP. Furthermore, we showed that FgPrp31 was phosphorylated by FgPrp4 in vivo. Site-directed mutagenesis analysis showed that phosphorylation at multiple sites in FgPrp31 is necessary to suppress Fgprp4, and S520 and S521 are important FgPrp4-phosphorylation sites. Overall, these results indicated that phosphorylation by FgPrp4 at multiple sites may release the self-inhibitory binding of FgPrp31 and affect its interaction with other components of tri-snRNP during spliceosome activation.
Subject(s)
Amino Acid Sequence , Fungal Proteins/metabolism , Fusarium/metabolism , Mutation, Missense , Protein Serine-Threonine Kinases/metabolism , Sequence Deletion , Amino Acid Substitution , Fungal Proteins/genetics , Fusarium/genetics , Phosphorylation/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/geneticsABSTRACT
BACKGROUND: This study compared the one-time success rate of blind and endoscopy-guided postpyloric feeding tube placement after implementing a comprehensive nursing scheme of intestinal blind placement for patients with ischemic stroke. METHODS: This retrospective cohort study included 179 patients with blind bedside placement and 118 with endoscopy-guided placement. The primary outcome was the one-time success rate of radiologically confirmed postpyloric placement. The secondary endpoints included the position of the tube tip, length of insertion, time of placement, and expenses. The safety endpoints were the incidence of complications caused by placement. RESULTS: The results showed that the method of tube placement did not significantly affect the outcome of the first tube placement (odds ratio [OR] = 0.41; 95% CI = 0.137-1.207; P = 0.105). Compared with endoscopy-guided placement, blind placement was half the cost. We also found that a history of abdominal surgery (OR = 0.003; 95% CI = 0.000-0.059; P < 0.001) and longer intensive care unit (ICU) days (OR = 0.94; 95% CI = 0.903-0.981; P = 0.004) were inversely associated with the one-time success rate. CONCLUSION: Our study suggested that blind intestinal feeding tube placement has an equivalent one-time success rate compared with endoscopy-guided placement in hospitalized patients with ischemic stroke if operators can be trained well. However, the expenses of endoscopy-guided placement were twice those of blind bedside methods. We also found that patients with abdominal surgery history and longer ICU stay were more likely to fail at the first placement. Further research is needed to replicate our single-center observations in a larger population of patients.