A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit

The epitranscriptome plays a key regulatory role in cellular processes in health and disease, including ribosome biogenesis. Here, analysis of the human mitochondrial transcriptome shows that 2’-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, modified by MRM1, MRM2, and MRM3. Ablation of MRM2 leads to a severe impairment of the oxidative phosphorylation system, caused by defective mitochondrial translation and accumulation of mtLSU assembly intermediates. Structures of these particles (2.58Å) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module. Additionally, we present five mtLSU assembly states with different intersubunit interface configurations. Complementation studies demonstrate that the methyltransferase activity of MRM2 is dispensable for mitoribosome biogenesis. The Drosophila melanogaster orthologue, DmMRM2, is an essential gene, with its knock-down leading to developmental arrest. This work identifies a key late-stage quality control step during mtLSU assembly, ultimately contributing to the maintenance of mitochondrial homeostasis. WT HEK-293T cells, and cells knocked-out of either MRM1, MRM2 or MRM3 were harvested. Mitochondria isolation and RNA extraction was conducted as previously described (Sas-Chen, Nir and Schwartz, 2021; doi: 10.1007/978-1-0716-0834-0_9). Strand-specific libraries were generated using low and high concentration of dNTPs. Libraries were sequenced using a NextSeq 500 platform generating short paired-end reads, ranging from 25 to 55 bp from each end.