Hyper-Stabilizing Transient Protein-Protein Interactions

The spliceosome is a large and highly dynamic RNA-protein molecular machine, responsible for cleavage of introns out of the eucaryotic pre-mRNA, resulting in a mature RNA. As in all complex cellular processes splicing is also known to be a potential source of error during gene expression as for each splicing event, a spliceosome is assembled de novo on the substrate pre-mRNA, extensively remodeled and, after catalysis, disassembled in an ordered fashion. In humans, about 200 proteins, five small nuclear RNAs and the pre-mRNA intimately participate in these processes. During spliceosome assembly, catalysis and disassembly, many protein-protein, protein-RNA and RNA-RNA interactions are formed and broken in a controlled manner. Presently, the exact sequence of remodeling steps and the functional roles of individual components of the spliceosome during assembly and catalysis are incompletely understood. In order to gain further insights into the process of spliceosomal assembly, I propose to interfere with specific steps of splicing using small molecule inhibitors. Specifically, I suggest developing low molecular weight substances, which hyper-stabilize crucial protein-protein interactions that normally form only transiently during splicing. I plan to resort to available compound libraries (from the ChemBioNet resource; http://www.chembionet.info/) and employ structure-based in silico screening, biochemical and biophysical pre-selection and finally crystallographic ligand screening to identify substances that bridge a transient protein complex in the spliceosome. Initial hits may subsequently be improved by rational synthetic optimization guided by crystal structure analysis and will be tested in splicing assays in vitro and in vivo. By selectively inhibiting certain steps of the reaction by hyper-stabilizing transient interactions, one has extremely powerful magic bullets in the hands to further investigate the spliceosomal assembly, resulting in potential therapeutic benefit. This project will give me the possibility to work with two leading institutes on that particular fields: in the group of Prof. Markus Wahl at the Freie Universität Berlin, leading expert in the field of spliceosomal research; and in the group of Dr. Uwe Müller, head of the macromolecular crystallography beamlines at the Helmholtz-Zentrum Berlin, comprising one of the state-of-the-art MX-beamlines in Europe. I will be able to expand my knowledge in both directions, establish a network of collaborations with the leading scientists on that field, which will giving me the ideal starting point for my future career.

The spliceosome is a large and highly dynamic RNA-protein molecular machine, responsible for cleavage of introns out of the eucaryotic precursor-messenger-RNA (pre-mRNA), resulting in a mature RNA. As in all complex cellular processes splicing is also known to be a potential source of error. In humans, about 200 proteins, five small nuclear RNAs and the pre-mRNA intimately participate in these processes, in which many protein-protein, protein- RNA and RNA-RNA interactions are formed and broken in a controlled manner. Presently, the exact sequence of remodeling steps and the functional roles of individual components of the spliceosome during assembly and catalysis are incompletely understood. In order to gain further insights into the process of spliceosomal assembly, it was proposed to interfere with specific steps of splicing using small molecule inhibitors. Specifically, it was suggested to develop low molecular weight substances, which hyper-stabilize crucial protein- protein interactions that normally form only transiently during splicing. This project offered a great possibility to work with two leading institutes on that particular fields: in the group of Prof. Markus Wahl at the Freie Universität Berlin, leading expert in the field of spliceosomal research; and in the group of Dr. Uwe Müller, head of the macromolecular crystallography beamlines at the Helmholtz-Zentrum Berlin, comprising one of the state-of-the-art MX-beamlines in Europe.