Influence of cisplatin on RNA structure in solution Escherichia coli tRNA(Ala) and human Wnt-5a 3' UTR model system studies

Sammanfattning: RNA molecules in the cellular environment have several important functions. In many cases, proper RNA function is intimately linked to proper three-dimensional structure of the molecule. Thus, it seems reasonable to assume that structural changes caused by external influence can strongly inhibit important cellular functions maintained by RNAs. Studies in this thesis have had a focus on the influence of the anticancer drug cisplatin and its effect on RNA structure in solution. Consequences of the structural distortions caused by platination, were studied in two model systems i) Escherichia coli tRNAAla, and ii) the human Wnt-5a 3? UTR. Chemical and enzymatic probing techniques were used to document the preferred and specific location for interactions of cisplatin with the RNA, and also the structural changes resulting from these interactions. The tRNA models used for structural probing were based on the synthetically prepared tRNAAla together with truncated models based on the acceptor stem and anticodon seuquences, respectively. Functional studies were carried out by use of the aminoacylation reaction maintained by Escherichia coli alanyl-tRNA synthetase (AlaRS). The enzyme was cloned in Escherichia coli M15(pREP4) strain and purified with chelating chromatography in one step. The second target used in this work represents a model of an AU rich region corresponding to the initial 260 bases of the Wnt-5a 3? UTR. To detect functional perturbations after platination, the effect of cisplatin on protein expression was investigated with a reporter vector containing the Wnt-5a 3? UTR coupled to the luciferase reporter gene (pcDNA3-Luc/W-UTR(1-259)). The studies show that cisplatin-RNA adduct formation takes place with a rate comparable to that of DNA-binding, but with an adduct formation profile that seems to be influenced by the structure of the targeted RNA. If accessible, GG-sequences seem to be preferentially platinated. However, subtle variations of the flanking sequences have a profound influence on the nature of the final adduct. Further, platination was shown to have a significant influence on the resulting both structure and function, in the latter case for example illustrated by inhibition of the aminoacylation reaction. In conclusion, the data obtained supports the assumption of RNA as a biologically relevant target for cisplatin and related metal-based drugs, which may operate in parallel with already established ones on the DNA-level.