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Isolierung und Charakterisierung von Hupf1-enthaltenden Protein Komplexen und ihre Rolle in nonsense-codon vermitteltem mRNA Abbau
mRNAs containing premature translation termination codons (PTCs) are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway. The mechanism of NMD in mammals is largely unknown. The cytoplasmic protein Hupf1 was the only trans-acting factor of NMD in mammals that was known at the beginning of this study. In order to identify and characterize proteins that are required for NMD in mammals, Hupf1-containing protein complexes were affinity purified from HeLa cells that stably express recombinant, double affinity tagged Hupf1-2tag at physiological levels. The analysis of these purified complexes by mass spectrometry revealed the presence of the proteins Hupf2 and Poly(A)-binding protein 1 (Pabp1). Using size exclusion chromatography it was shown that the majority of Hupf1 occurs as a monomer, but two complexes of distinct composition could be isolated. Further analysis of affinity purified Hupf1-containing complexes by Western Blot revealed that the presence of intact RNA stabilizes the interaction of Hupf1 with Hupf2 as well as the interaction of Hupf1 with the recently identified NMD factors Hupf3a and Hupf3b. The interaction between Pabp1 and Hupf1 was demonstrated to be strictly RNA-dependent. Even though the components of the exon-exon-junction complex (EJC) or the translational release factor eRF3 have been implicated in NMD in mammals, these proteins could not be found in the purified Hupf1-containing complexes, indicating that these proteins interact with Hupf1 transiently or not at all.
The results described in this thesis indicate that Hupf1 occurs in different stable protein complexes and that Hupf2 and Pabp1 are novel interaction partners of Hupf1 and might be involved in NMD in mammals. RNA was found to play an important role for the stability of the interactions of Hupf1 with other proteins.
The dynamic interactions of Hupf1 are likely to play an important role for the recognition of PTC-containing mRNAs and their subsequent destabilization.
|SWD-Schlagwörter:||Molekularbiologie , Genexpression , RNS-Stoffwechsel , Genmutation|
|Freie Schlagwörter (deutsch):||nonsense-Mutationen , Protein-Komplexe , TAP|
|Freie Schlagwörter (englisch):||TAP , Hupf1/Rent1 , mRNA-decay|
|Institut:||Institut für Biologie 2|
|Fakultät:||Fakultät für Biologie (bis Sept. 2002)|
|Erstgutachter:||Neuhaus, Gunther (Prof. Dr.)|
|Tag der mündlichen Prüfung:||28.06.2002|