Human mitoribosome assembly and mitochondrial translation
Abstract
Human mitochondrial ribosomes play a central role in cellular metabolism by synthesizing the thirteen hydrophobic polypeptides of the oxidative phosphorylation machinery. They consist of a large subunit (LSU) with 16S rRNA, tRNA-Val and n proteins and a small subunit (SSU) with 12S rRNA and m proteins. The assembly of these mitoribosomes is a highly regulated process that includes several auxiliary factors. The architecture and composition of human mitoribosomes differ from their bacterial counterparts, and their assembly involves a coordinated and step-wise incorporation of components from both the mitochondrial and nuclear genomes. Recent research has identified specific factors involved in the late stages of large subunit and small subunit maturation, revealing their roles through the elucidation of structures in natively purified complexes.
The process of ribosome assembly requires concerted action by a large array of molecular players including GTP-binding proteins (GTBPs), RNA helicases, RNA modifying enzymes such as methyl transferases and pseudouridine synthetases at different stages. Recently, there have been several structural studies elucidating the pathway of LSU assembly (10.1016/j.tcb.2021.09.004). They together explain the structural basis of action of various auxilliary factors including methyl transferases such as MRM2 and MRM3 that methylate RNA residues around the peptidyl transferase center; GTPases such as GTPBP5, 6, 7 and 10 that together with NSUN4 and MTERF4 heterodimer perform scaffolding functions for the rRNA as it matures. For the SSU, there are only two structural studies reported that elucidate relatively late stages of assembly (10.1038/s41586-022-04795-x; 10.1038/s41586-022-05621-0). The structures reveal the role of GTPBPs, EraL1 and NoA1, methyl transferases: METTL15, METTL17 and TFB1M and RNA chaperones, MCAT and RbfA that assist in maturation of SSU faciliating incorporation.
Given the limited work done on SSU maturation pathway, especially the early stages of SSU assembly, there is a significant gap in our knowledge. We do not fully understand the mechanism of key rRNA modifications near the decoding center that are important for correct rRNA folding. Also, the mechanism of action of GTPases on SSU assembly is poorly understood as a bound guanosine nucleotide could not be observed in the reported structures. Literature suggests the role of other auxiliary factors such as guanosine exchange factors (GEFs) like RCC1L that interact with EraL1 and NoA1 to facilitate ribosome assembly. The structural basis of this is not known. To this end, we have prepared HEK cell lines over-expressing FLAG-tagged ribosome assembly GTPases, NoA1, EraL1 and the putative GEF, RCC1L and plan to carry out single particle cryo-EM analysis.
