ESR 05 Project - Structures of the human mitochondrial ribosome
Objectives: Against the background of its medical importance, little is known about pathways of mitoribosome assembly and roles of auxiliary factors. Mitoribosomal biogenesis is an intricate multistep process having an additional level of complexity over the cytoplasmic system, as it requires cooperative effort involving precise regulation of two genomes: nuclear and mitochondrial. All human mitochondrial ribosomal proteins are encoded by nuclear DNA and actively imported into mitochondria by translocases in a spatially and temporally synchronized manner. Inside the mitochondrial matrix, they are coordinatively assembled onto mitochondrially transcribed and matured rRNA, being gradually processed to form pre-mitoribosomal subunits. At this functionally inactive state, both subunits are still complexed with ribosome-biogenesis trans-acting factors preventing association in a highly regulated way. The release of trans-acting factors constitutes the final step of mitoribosomal biogenesis, producing translationcompetent intact particles capable of mt-mRNA translation into proteins. In this project, we will use the recent advances in cryo-EM supplemented by genetic modifications to investigate mitoribosome assembly intermediates. Our strategy will be to label auxiliary factors that will allow direct pulling down of mitoribosome assemblies. This approach will allow us for the first time to directly determine atomic structures of ribosomes from their native environment in mitochondrial matrix. We will combine these efforts with the characterization of knockout strains in mitoribosomal auxiliary factors, to complement the functional importance of different states.
Expected results: These projects will unravel structural basis for mitoribosome assembly. Atomic structures of intermediates will allow understanding the sequence of events leading to functional mitochondrial translation. On the technical level, this will be the first time that atomic structure determination of any native ribosomal assembly will be approached. The results will therefore not only increase our knowledge on the processes leading to maturation of mitochondrial translation, but also will provide a methodological advance.