Ciliate mitoribosome illuminates evolutionary steps of mitochondrial translation

Abstract

To understand the steps involved in the evolution of translation, we used Tetrahymena thermophila, a ciliate with high coding capacity of the mitochondrial genome, as the model organism and characterized its mitochondrial ribosome (mitoribosome) using cryo-EM. The structure of the mitoribosome reveals an assembly of 94-ribosomal proteins and four-rRNAs with an additional protein mass of ~700 kDa on the small subunit, while the large subunit lacks 5S rRNA. The structure also shows that the small subunit head is constrained, tRNA binding sites are formed by mitochondria-specific protein elements, conserved protein bS1 is excluded, and bacterial RNA polymerase binding site is blocked. We provide evidence for anintrinsic protein targeting system through visualization of mitochondria-specific mL105 by the exit tunnel that would facilitate the recruitment of a nascent polypeptide. Functional protein uS3m is encoded by three complementary genes from the nucleus and mitochondrion, establishing a link between genetic drift and mitochondrial translation. Finally, we reannotated nine open reading frames in the mitochondrial genome that code for mitoribosomal proteins.

Analysis of translating mitoribosome reveals functional characteristics of translation in mitochondria of fungi

“…coordinated movement of the mtSSU rotation and mL108 to the L1 stalk. The involvement of mitochondria-specific elements in coordination of tRNA movement has been recently reported also in other systems [21][22][23] .…”

Structural basis of mitochondrial translation

“…Therefore, at all three sites protein elements unique to the mitoribosome play roles in the tRNA recognition, particularly the elbow region. The multiple recognition events relying on the tRNA elbow and its specific coordination are likely co-evolved with the structural properties of human mt-tRNA, and they are different from fungi ( Naschberger et al, 2020 ) and alveolates ( Tobiasson and Amunts, 2020 ). This suggests that mitochondria-specific proteins promote effective transit through the mitoribosome by stabilizing the tRNA elbow and favoring the unidirectionality of movement.…”

Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit

“…In mitochondria, the mtLSU lacks many of the rRNA components involved in the canonical pathways, and higher complexity of the interactions between the mitoribosomal proteins at the functional sites has evolved (Ott et al, 2016;Greber & Ban, 2016). A functional mtLSU requires a folded rRNA core, a flexible L1 stalk that is involved in tRNA movement, an extended L7/L12 protrusion for binding of translational factors, and a proteinaceous CP formed by mitochondria-specific elements involved in tRNA binding (Aibara et al, 2020;Tobiasson & Amunts, 2020). However, only the final stage of the mtLSU assembly with fully mature functional sites has been visualized (Brown et al, 2017;Itoh et al, 2020), and no preceding steps in the formation have been detected.…”

“…Amunts et al, 2014;Greber et al, 2014;Amunts et al, 2015;Greber et al, 2015;Ramrath et al, 2018;Waltz et al, 2020;Tobiasson & Amunts, 2020).…”