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Schrader, Rainer (2004): Funktionelle Analyse des Hypusin enthaltenden Proteins in Saccharomyces cerevisiae. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Hypusine is an unusual and unique posttranslational modification, conserved in evolution from halobacteria to man. However, despite being essential for life the function of the modification and the protein carrying it (Hypp) remains unclear so far. In yeast temperature sensitive mutants were generated and phenotypicly characterized. The investigation included proteomic and transcriptomic techniques. Furthermore binding assays using several Hypp fusion proteins were performed in order to find cellular protein and RNA interaction partners. Describing the phenotype of a highly temperature sensitive point mutated allele of Hypp a limited viability at restrictive temperature was shown leading to apoptotic cell death suggesting an antiapototic property of the protein. On the transcriptomic level studies using high density oligonucleotide micro arrays covering the whole yeast genome were performed. As also found on the protein level by 2D-gel analysis the impairment of mitochondrial functions was confirmed. Respectively 70% of the enriched transcripts of the mutant were also accumulated in strains in which components of the NMD pathway were disrupted indicating a function of the hypusine containing protein in this special 5’-3’-mRNA degradation pathway to which NMD belongs. A respiratory deficiency also was the first observed phenotype of strains bearing a NMD dysfunction. The mutant showed an attenuation of the telomer positioning effect leading to elevated transcriptional activity of genes near the telomers. It could be demonstrated that the Hypp mutant showed shortening of telomers similarly observed in NMD deficient strains. No evidence for the protein to be a general nuclear export factor of a special RNA subset could be given. By affinity chromatographic purification of N-terminal GST-Hypp fusion proteins an interaction of Hypp to the major coat protein of the yeast specific virus L-A was demonstrated, a protein that itself posesses an enzymatic activity for the decapping of mature RNA molecules. Before it was shown that N-terminally GST-tagged Hypp was able to take over the function of the wild-type protein unrestrictedly. Our results give evidence that the protein posesses cellular cytosolic functions in the posttranscriptional control of gene activity of viral and normal genes in Saccharomyces cerevisiae. A connection to a special RNA degradation pathway is evident and should be investigated further on.