Untersuchungen zur Interaktion von Prp4p-Kinase und dem Spleißfaktor Prp1p

Die Prp4p-Kinase wurde bei Versuchen mit prä-mRNA-Spleißmutanten der Spalthefe S. pombe identifiziert. Sie zeigt genetische Interaktionen mit den Spleißfaktoren Spp41p, Spp42p, Cdc5p, Prp5p, Prp10p, Rnps1p und Prp1p. Prp1p ist ein Protein mit 19 TPR- Motiven, das als Bindeglied zwischen U5- und U4/U6-snRNP diskutiert wird. In dieser Arbeit wurde die Beziehung zwischen Prp1p und Prp4p näher untersucht. Stämme mit den temperatursensitiven Allelen prp4-73ts bzw. prp1-4ts arretieren bei der restriktiven Temperatur in der G1- und in der G2-Phase des Zellzyklus. Die Aktivität von Prp4p und Prp1p scheint für den Übergang von der G1- in die S-Phase sowie von der G2- Phase in die Mitose erforderlich zu sein. Bakteriell produziertes Prp1p wird in vitro von Prp4p phosphoryliert. Die in vivo-Markierung eines Stammes mit dem prp4-73ts-Allel zeigte, daß Prp1p bei der restriktiven Temperatur nicht länger phosphoryliert wird. Dies ist ein Hinweis darauf, daß Prp1p ein physiologisches Substrat von Prp4p ist. Mit den SR-Proteinen Srp1p und Srp2p konnten zwei weitere in vitro- Substrate von Prp4p identifiziert werden. Prp1p wird in vitro hauptsächlich an Threoninen phosphoryliert. Es gibt mindestens drei Phosphorylierungsstellen von Prp4p in Prp1p. Es wurde versucht, diese mit Hilfe der Massenspektrometrie zu identifizieren. In einem anderen Ansatz sollte der Nachweis der Phosphorylierungsstellen über die Inkubation von Prp1-Peptiden mit bakteriell exprimierter Prp4p-Kinase erfolgen. Die dabei detektierten Phosphorylierungssignale erwiesen sich als unspezifisch. Die Spaltung von in vitro phosphoryliertem Prp1p mit Ameisensäure zeigte, daß die Phosphorylierungsstellen am N-Terminus des Proteins liegen. Der die TPR-Motive umfassende C-terminale Bereich von Prp1p wird nicht phosphoryliert.The Prp4p kinase was identified in experiments with pre-mRNA splicing mutants of the fission yeast S. pombe. Genetic interactions with the splicing factors Spp41p, Spp42p, Cdc5p, Prp5p, Prp10p, Rnps1p and Prp1p could be shown. Prp1p, a protein containing 19 TPR motifs, is discussed as a bridge between the U5- and the U4/U6-snRNPs. In this work the interactions between Prp1p and Prp4p have been further characterized. At the restrictive temperature strains carrying the temperature sensitive allels prp4-73ts respectively prp1-4ts arrest in the G1- and the G2-phase of the cell cycle. Prp4p and Prp1p activity seems to be important for transition from G1-phase into S-phase and from G2-phase into mitosis. Bacterially produced Prp1p is phosphorylated by Prp4p in vitro. In vivo labelling of a strain containing the prp4-73ts allel showed that at the restrictive temperature Prp1p is not phosphorylated any longer, indicating that Prp1p is a physiological substrate of Prp4p. Furthermore the SR-proteins Srp1p and Srp2p could be identified as in vitro substrates of Prp4p. In vitro Prp1p is mainly phosphorylated on Threonines. There are at least three phosphorylation sites of Prp4p in Prp1p. It was tried to identify them by mass spectrometry. It was tried as well to find the phosphorylation sites by incubation of Prp1p peptides with bacterially produced Prp4p kinase. The detected phosphorylation signals could be shown to be unspecific. Cleavage of in vitro phosphorylated Prp1p with formic acid showed that the N-Terminus of the protein contains the phosphorylation sites. The C-terminal part of Prp1p with the TPR- motifs is not phosphorylated

The N-terminus of Prp1 (Prp6/U5-102 K) is essential for spliceosome activation in vivo

The spliceosomal protein Prp1 (Prp6/U5-102 K) is necessary for the integrity of pre-catalytic spliceosomal complexes. We have identified a novel regulatory function for Prp1. Expression of mutations in the N-terminus of Prp1 leads to the accumulation of pre-catalytic spliceosomal complexes containing the five snRNAs U1, U2, U5 and U4/U6 and pre-mRNAs. The mutations in the N-terminus, which prevent splicing to occur, include in vitro and in vivo identified phosphorylation sites of Prp4 kinase. These sites are highly conserved in the human ortholog U5-102 K. The results presented here demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes

Proteomic analysis of the U1 snRNP of Schizosaccharomyces pombe reveals three essential organism-specific proteins

Characterization of spliceosomal complexes in the fission yeast Schizosaccharomyces pombe revealed particles sedimenting in the range of 30–60S, exclusively containing U1 snRNA. Here, we report the tandem affinity purification (TAP) of U1-specific protein complexes. The components of the complexes were identified using (LC-MS/MS) mass spectrometry. The fission yeast U1 snRNP contains 16 proteins, including the 7 Sm snRNP core proteins. In both fission and budding yeast, the U1 snRNP contains 9 and 10 U1 specific proteins, respectively, whereas the U1 particle found in mammalian cells contains only 3. Among the U1-specific proteins in S. pombe, three are homolog to the mammalian and six to the budding yeast Saccharomyces cerevisiae U1-specific proteins, whereas three, called U1H, U1J and U1L, are proteins specific to S. pombe. Furthermore, we demonstrate that the homolog of U1-70K and the three proteins specific to S. pombe are essential for growth. We will discuss the differences between the U1 snRNPs with respect to the organism-specific proteins found in the two yeasts and the resulting effect it has on pre-mRNA splicing

Functional Analysis of the Kinome of the Wheat Scab Fungus Fusarium graminearum

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study

Fission yeast Prp4p kinase regulates pre-mRNA splicing by phosphorylating a non-SR-splicing factor

We provide evidence that Prp4p kinase activity is required for pre-mRNA splicing in vivo and show that loss of activity impairs G(1)–S and G(2)–M progression in the cell cycle. Prp4p interacts genetically with the non-SR (serine/arginine) splicing factors Prp1p and Prp5p. Bacterially produced Prp1p is phosphorylated by Prp4p in vitro. Prp4p and Prp1p also interact in the yeast two-hybrid system. In vivo labelling studies using a strain with a mutant allele of the prp4 gene in the genetic background indicate a change in phosphorylation of the Prp1p protein. These results are consistent with the notion that Prp4p kinase is involved in the control of the formation of active spliceosomes, targeting non-SR splicing factors