Proteins in a cell are universally synthesized by ribosomes. Mitochondria contain their own ribosomes, which specialize on the synthesis of a handful of proteins (8 in yeast, 13 in human cells) required for oxidative phosphorylation (OXPHOS). The pathway of mitoribosomal biogenesis and the factors involved are poorly characterized. A case in point are the DEAD-Box proteins, widely known to participate in the biogenesis of bacterial and cytoplasmic eukaryotic ribosomes as either RNA helicases or RNA chaperones, whose mitochondrial counterparts remain completely unknown. We propose to uncover the molecular function/s of the yeast DEAD-box protein Mrh4 and its best BLAST match in humans, the DEAD-box protein DDX28, in mitochondrial ribosome assembly and protein synthesis. Preliminary studies indicate that yeast Mrh4 is essential for large mitoribosome subunit (LSU) biogenesis. Mrh4 interacts with the 21S rRNA, mitoribosome subassemblies and fully assembled mitoribosomes. In the absence of Mrh4, the 21S rRNA is matured and forms part of a large on-pathway assembly intermediate missing proteins Mrpl16 and Mrpl39. Human DDX28 localizes to the mitochondrial matrix as part of RNA granules and interacts with the LSU. RNAi-mediated silencing of DDX28 in HEK293T cells leads to reduced levels of 16S rRNA and LSU ribosomal proteins, impaired LSU assembly without apparent accumulation of large intermediates, deeply attenuated mitochondrial protein synthesis and consequent failure to assemble OXPHOS complexes. Studies outlined in this proposal will involve yeast genetics, gene disruption in human cells and mechanistic biochemistry in yeast, human cell lines, isolated mitochondria and purified native and recombinant proteins to gain insight into the role/s of Mrh4 and DDX28 in mitoribosome assembly and protein synthesis. These studies will be complemented with structural analysis of pre-ribosomal particles by high-resolution cryo-electron microscopy. We will test the hypothesis that Mrh4 and DDX28 play related but not equal functions in ribosome assembly by promoting remodeling of the rRNA-protein interactions during LSU assembly. We will establish the hierarchy of these helicases in the LSU assembly pathway in relation to the GTPase Mtg1, another LSU assembly factor identified by our group. Finally, we will investigate the determinants of DDX28-mediated ribosome assembly within or in the vicinity of RNA granules in mammalian mitochondria.
The goal of this RO1 grant application is to uncover the molecular function/s of two DEAD-box proteins, putative RNA helicases, in the assembly of yeast and human mitochondrial ribosomes and the process of mitochondrial translation. Disorders arising from impaired mitochondrial protein synthesis result in a variety of pathologies including encephalomyopathies and cardiomyopathies. Identifying and characterizing mitoribosome biogenesis and translation factors is a prerequisite towards understanding the molecular basis of these disorders.
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