No abstract

The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs 1 . These organelles have a bacterial origin and were acquired during an early endosymbiosis event 2 . Mitochondria possess specialized gene expression systems composed of various molecular machines including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes 3 . While chloroplast ribosomes strongly resemble those of bacteria 4,5 , mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species [6][7][8][9][10] . In contrast to animals and trypanosomatides, plants mitoribosomes have unusually expanded ribosomal RNAs and conserved the short 5S rRNA, which is usually missing in mitoribosomes 11 . We have previously characterized the composition of the plant mitoribosome 6 revealing a dozen plant-specific proteins, in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations, and the plant-specific ribosomal features of unknown structures. Here, we present a cryoelectron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPR) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNAconstructive phase of mitoribosome evolution across eukaryotes.Previously, we determined the full composition as well as the overall architecture of the Arabidopsis thaliana mitoribosome 6 . However, due to the difficulty to purify large amounts of A. thaliana mitoribosomes, mainly because of the low quantities of plant material usable for mitochondrial extraction, only a low-resolution cryo-EM reconstruction was derived. In order to obtain a highresolution structure of the plant mitochondrial ribosome, we purified mitoribosome from a closely related specie, Brassica oleracea var. botrytis, or cauliflower (both Arabidopsis and cauliflower belong to the group of Brassicaceae plants), as previously described 6 (see Methods). We have recorded cryo-EM images for ribosomal complexes purified from two different sucrose gradient peaks (see Methods), corresponding to the small ribosomal subunit (SSU) and the full 78S mitoribosome. After extensive particle sorting (see Methods) we have obtained cryo-EM reconstructions for both types of complexes. The SSU reconstruction displayed an average resolution of 4.36Å (Extended Data Fig. 1). After multibody refinement (3 bodies) and particle polishing in RELION3 12 (see Methods), reconstructions were de...

Highlights d Cryo-EM structures of LS48S ICs unravel crucial mRNA sequence-dependent interactions d After start-codon recognition, eIF1A interacts with the G(+4) position in b-globin mRNA d t 6 A(37) modification mediates tRNA i Met binding to the C(À1) mRNA position d The binding of ABCE1 does not affect the conformation of the LS48S IC