Modular maturation and novel assembly factors in the biogenesis of human mitochondrial small ribosomal subunit

Assembly of the mitochondrial ribosome (mitoribosome) is a crucial step in mitochondrial gene expression. This process facilitates mitochondrial translation, which produces essential subunits of the oxidative phosphorylation machinery—the cell’s primary energy-producing machinery. Disruptions in mitoribosome assembly can lead to severe human diseases. Given its fundamental importance, detailed structural analysis of mitoribosome assembly pathways is essential for advancing our understanding of mitochondrial function in both health and disease.In this study, we characterize twelve distinct assembly states of mitoribosomal small subunit (mtSSU) isolated from human cells. Our findings reveal the intricate details of the final maturation stages of the mtSSU platform, decoding center, and the 3'-end of 12S rRNA. This process is governed by coordinated actions of assembly factors that ensure precise, stepwise rRNA folding and the integration of mitoribosomal proteins into the developing subunit.Our approach identifies pseudouridine synthase PUS1 and initiation factor mtIF2 as novel assembly factors, expanding their known roles beyond mt-tRNA maturation and translation, respectively. Additionally, we provide structural evidence supporting a mtIF3-independent mechanism for mtSSU maturation and translation initiation. Together, these findings shed light on the modular nature of mtSSU biogenesis in mitochondria and uncover parallel assembly pathways that converge on translation initiation.