RNABIC · Structure, assembly and metal ion binding properties of the catalytic core of a group II intron ribozyme

Structure, assembly and metal ion binding properties of the catalytic core of a group II intron ribozyme

Project context Since the discovery of catalytically active RNAs in the eighties, research on RNA has gained rapidly increasing scientific interest. Different RNAs are involved in many crucial biological processes, and their importance calls for an in-depth investigation into their role, activity and structure. For many years, Prof. Roland Sigel's group has been committed to obtaining structural and functional information on different families of RNAs by combining different techniques, like sm-FRET, Nuclear Magnetic Resonance (NMR) spectroscopy, CD and UV-VIS spectroscopy, X-ray, etc. Here we concentrate on group II intron ribozymes, which are naturally occurring catalytically active RNAs, mainly found in organellar genes of plants, fungi, bacteria, and lower eukaryotes. Introns are non-coding sequences that must be removed from the primary sequence to attain functional RNA. Group II introns catalyse their own excision, followed by ligation of the exonic parts, but they have also been observed to reinsert into RNA and DNA, making them appealing instruments for biomedical applications. Moreover, not only are they believed to be putative ancestors of the eukaryotic spliceosome, with which they share the splicing mechanism, but they are also closely related to large parts of the eukaryotic genome. A crucial role in these large ribozymes is played by metal ions, which are essential for both folding and activity: they screen the negative charge associated with the phosphate sugar backbone and are directly involved in catalysis. Despite their manifold importance, little structural information is available on this class of RNAs. Project objectives The aim of this Marie Curie IEF project is to attain structural information and elucidate the metal ion binding properties of the catalytic core of a group II intron ribozyme by NMR. A small region within domain 1 of the yeast group II intron Sc.ai5gamma containing the kappa and zeta elements represents, together with domain 5, the minimal structure of the catalytic core. One RNA construct containing the kappa-zeta region was optimised for NMR having 49 nucleotides in total and comprising highly dynamic structural features, like a three-way junction and several loops. Our NMR studies showed that the three-way junction is intrinsically unstable but is stabilised by addition of Mg2+, the natural RNA cofactor. The structure of this molecule was solved in the presence of Mg2+. In addition, M2+ binding properties were studied in detail by substituting Mg2+ with Cd2+ and [Co(NH3)6]3+, able to mimic inner-sphere and outer-sphere coordination properties of Mg2+, respectively. Project results This study, which is now coming to an end, proved to be very challenging because of the intrinsic RNA dynamics and the indispensable role played by Mg2+, which was not obvious at the beginning. Our findings represent a solid counterpart to already available biochemical studies and provide further insights into group II intron architecture in solution. This work at the interface between biological inorganic chemistry and RNA biochemistry added a brick to the knowledge of the structure and behaviour of these fascinating RNA molecules, which are considered ancestors of large parts of the eukaryotic genome. It is clear that any new structural information on biological systems is extremely important, as it allows a better comprehension of the relationship between structure and function. The latter is crucial to unravelling complex biological processes, and represents the starting point for future studies aimed at biomedical applications, like the design of RNA-targeting drugs.

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