Insights into early biogenesis steps of the human small mitochondrial ribosomal subunit

Abstract

The rise of high-resolution cryo-EM structures of the human mitochondrial ribosome increased the understanding of the function and assembly. As the mitochondrial ribosome differs in structure and composition compared to its bacterial counterpart, it is tempting to believe that the assembly pathway of the mitochondrial ribosome also reveals differences to the bacterial system. Although the assembly of the bacterial ribosome has been excessively studied, the human mitochondrial ribosome biogenesis has yet to be solved. Within the last years, an increasing number of human diseases was reported, which were caused by mutations in genes encoding for proteins required by the mitochondrial ribosome. Therefore, it is crucial to understand the complex mechanism by which the mitochondrial ribosome assembly is facilitated. In this study, the early assembly pathway of the 28S mtSSU was analyzed in more detail by dissecting the role of the early-binding MRPs uS7m, mS27 and mS40. Ablation of uS7m, mS27 or mS40 did not affect their sub-module formation, albeit late-binding MRPs were strongly decreased in the absence of uS7m, mS27 or mS40. Furthermore, loss of either uS7m, mS27 or mS40 influenced the mtLSU assembly and indicates to a potential cross-dependence between the biogenesis of the mtSSU and mtLSU. In a second part, analysis of uS7m p.Met184Val revealed that the patient mutation leads to a destabilization of uS7m. Although the patient mutation of uS7m retains its ability to be incorporated during mtSSU biogenesis, the destabilizing nature of the mutation led to a strong decrease of the mtSSU assembly efficiency and results into the decline of mitochondrial translation. During this work, analysis of the mS40 interactome enabled the identification of NT5DC2 as a mitochondrial matrix protein, which is potentially involved in mitochondrial translation. NT5DC2 could be shown to be strongly decreased when mitochondrial ribosome function was disturbed. Furthermore, ablation of NT5DC2 revealed a decrease in mitochondrial translation, underlining a potential involvement of NT5DC2 in mitochondrial gene expression. The findings of this study allowed the outline of a potential assembly pathway of the early-binding proteins uS7m, mS27 and mS40 and unraveled a potential interdependency during mtSSU and mtLSU biogenesis.