The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine.

Center for Genetic and Neurological Diseases Institut de recherches cliniques de Montréal Montréal Quebec Canada

Department of Biochemistry and Molecular Biology and Institute of Comparative Genomics Dalhousie University Halifax Nova Scotia Canada

Department of Biochemistry and Robert Cedergren Centre for Bioinformatics and Genomics Université de Montréal Montréal Quebec Canada

Division of Experimental Medicine McGill University Montréal Quebec Canada

Faculty of Sciences University of South Bohemia České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

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