Identifizierung von neuen Faktoren des Gbp2-assoziierten mRNA-Exportes und den Untersuchungen an mRNA-bindenden Proteinen im Modellorganismus Saccharomyces cerevisiae.

Die räumliche und zeitliche Trennung von RNA- und Proteinsynthese in eukaryotischen Zellen bietet eine Menge Möglichkeiten für Kontrolle und Regulation dieser Vorgänge. Gleichzeitig erfordert dies eine funktionierende Maschinerie, die den korrekten Ablauf beider Prozesse und den Transport zwischen d...

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Bibliographic Details
Main Author: Janning, Melanie Christiane
Contributors: Krebber, Heike (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2017
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The separation of RNA- and proteinsynthesis by space and time provides the opportunity for an individual regulation of these processes, but it also demands a functioning machinery which ensures that the processes are performed correctly. Defects in mRNA processing are for example known to be involved in the pathophysiology of neurodegenerative disease and cancer. Basic components are often conserved between Saccharomyces cerevisiae and mammalian cells. This work focuses on mRNA-binding proteins in S. cerevisiae, that shuttle between the nucleus and the cytoplasm. A cytoplasmic variant of the SR-like protein Gbp2 (gbp2(S15A)-GFP) was used in an attempt to identify specific factors and functions that are associated with the mRNA-export of Gbp2. In an EMS mutagenesis screen, 33 temperature-sensitive mutants were analyzed for gbp2(S15A)-GFP-export defects. Mtr4, with is associated with the TRAMP-complex and involved in nuclear surveillance of faulty mRNA and the splicing protein Prp8 were identified as factors that influence export of gbp2 associated mRNAs. GBP2 is not essential, but overexpression is toxic and leads to a nuclear accumulation of poly(A)+-RNA. This work shows, that both the cytoplasmic variant (gbp2(S13/15/17A)) and Gbp2, which is mainly localized in the nucleus at steady state are toxic for the cells. Hence, Gbp2 possibly has a function in both compartments. And indeed later work could show that Gbp2 is an import factor in the surveillance of spliced mRNAs and it marks faulty spliced mRNA for degradation at the nuclear exosome via TRAMP and Mtr4. Nab2 is an essential, shuttling, mRNA-binding protein that interacts with the export receptor Mex67. A Nab2 variant that lacks the binding domain for the import receptor Kap104 was generated (nab2Δ200-249). This mutant fails to replace the temperature-sensitive NAB2 strain (nab2-21). Presumably because of the impaired import of this mutant the location was cytoplasmic at steady state compared to the mainly nuclear localization of wild type Nab2. Localization studies of nab2Δ200-249 in different mutant strains revealed that export of nab2Δ200-249 is dependent on the functional mRNA export since the protein remains in the nucleus in strains with defective MEX67 (mex67-5) and in rat7-1, which lead to accumulation of mRNA in the nucleus. Also in contrast to Nab2 the export of nab2Δ200-249 is independent of ubiquitination by Tom1.