Towards the unterstanding of the pre-mRNA splicing reaction: structure and dynamic studies of the 2'-5' AG lariat forming ribozyme and of its complex with catalysis inhibitors
Final Report Abstract
Conformational switches are the basis of most molecular processes that control the cell life cycle. The cellular splicing machinery, the spliceosome, is a dynamic object where specific docking of the conformations of proteins and nucleic acids is required at various steps duringthe assembly and disassembly of complexes in the catalytic cycle. However, it has been proposed that the catalytic activity of the spliceosome resides mostly in the RNA component, while proteins support the machinery in stabilizing the structure, enhancing regulation and optimizing the catalytic efficiency. About 10 years ago, a 2’-5’ AG branch forming ribozyme has been identified, that undergoes a transterification reaction which shows striking similarity to the first step of both pre-mRNA and group II introns splicing. The ribozyme contains a conserved ACAGAGA box that is essential for catalytic activity; moreover the branching formation has the same sequence specificity as in pre-mRNA, including the attack of the 2’ OH group of an internal adenosine to the a phosphate of a 5’ terminal guanosine with formation of a 2’-5’branched lariat and release of a diphosphate. The 2’-5’ AG branch forming ribozyme is small enough (59mer) to be investigated structurally in solution. To date there is no structural information on the catalytic core of introns II or of the spliceosome. The structural investigation of this 2’-5’ AG branch forming ribozyme would allow for the first time to visualize and understand the structural basis of RNA catalyzed lariat formation. Our original grant proposal included the characterization of the structure of this ribozyme at various stages along the reaction pathway. Furthermore, we had planned to understand how small positively charged molecules can influence this conformational landscape and consequentely inhibit the catalytic activity. Due to the conspicuous cut in the granted funding, we concentrated on the study of the structure of the linear form of the 2’-5’AG branch forming ribozyme, namely of the ground-state structure before addition ofmagnesium ions to start the catalytic reaction. Our structure investigation reveals an important role for the GAGA portion of the conserved ACAGAGA box. This portion guides the adenosine to interact closely with the 5’-terminal guanosine, thus generating a folding that supports catalysis. Surprisingly, the role of the ACAGAGA is only structural in this ribozyme, while the nucleotides necessary for the specific recognition of the adenosine and for the catalysis are located in another region of the ribozyme. These results are in agreement with previous studying showing that the phosphate of the ACAGAGA box do not coordinate any magnesium ion necessary for catalysis. The structure determination of the linear form of the 2’-5’ AG branch forming ribozyme is being completed. We expect that the knowledge won in this project will be of extreme importance to understand the principles of RNA-catalysed lariat formation. However, it is still uncertain how much of this information could be used to understand spliceosomal catalysis, as some of the characteristics of this ribozyme seem to differ from the spliceosome. Notwithstanding this, we will be able to provide insights in the structural basis of RNA catalytic activity and give an example of the enourmous plasticity of RNA molecules. In addition, this structural study by NMR is extremely challenging due to the extensive structural overlap and the high dynamic character of the molecule. Solving the structure of this molecule in solution will represent a landmark for structural determination of nucleic acids by NMR.
