Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

Abstract

<jats:title>Abstract</jats:title><jats:p>Kinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis<jats:sup>1</jats:sup>. They possess a single large mitochondrion, essential for the parasite survival<jats:sup>2</jats:sup>. In kinetoplastids mitochondrion, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes<jats:sup>3</jats:sup>. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes<jats:sup>4,5</jats:sup>. Structural studies performed in <jats:italic>Trypanosoma brucei</jats:italic> already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit<jats:sup>3,6</jats:sup>. However, several important aspects mainly related to the large subunit remain elusive, such as the structure and maturation of its ribosomal RNA<jats:sup>3</jats:sup>. Here, we present a cryo-electron microscopy study of the protozoans <jats:italic>Leishmania tarentolae</jats:italic> and <jats:italic>Trypanosoma cruzi</jats:italic> mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the large subunit as well as previously unidentified ribosomal proteins. Most importantly, we introduce the structure of an LSU assembly intermediate in presence of 16 identified maturation factors. These maturation factors act both on the intersubunit and solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.</jats:p>

Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit

“…In addition, the exit tunnel is blocked. In this regard, the present study is in agreement with the recently published work on Leishmania pre-mtLSU (Soufari et al 2020), which suggested that mL67, mL71, mL77, mL78, and mL81 represent assembly factors. The N-terminus of mL71 fills the exit tunnel, and its basic residues form electrostatic interactions with the rRNA that anchor the protein moiety.…”

Structural Insights into the Mechanism of Mitoribosomal Large Subunit Biogenesis

“…During the transition from assembly intermediate state B to the mature LSU, which probably includes multiple steps and intermediates, all of the remaining assembly factors must leave such that the 12S rRNA core, including the PTC, can adopt its mature fold. These observations suggest that even the previously described mitoribosomal LSU (Ramrath et al, 2018) may represent a very late assembly intermediate with still-unstructured PTC and possibly some assembly factors still bound to the exit region of the large ribosomal subunit as described for the Leishmania tarentolae mitoribosome (Soufari et al, 2020). Multiple mitoribosomal proteins must join state B to form the missing parts of the LSU, in particular, the CP and elements of the L1 and the L7/ L12 stalks.…”

Structural Heterogeneities of the Ribosome: New Frontiers and Opportunities for Cryo-EM

“…In addition, mitochondrial ribosomes present important morphological differences with cytoplasmic ribosomes [ 27 ]. As cryo-EM technology allows to computationally sort ribosomes of different classes from the image data, the past few years have seen various new structures of mitochondrial ribosomes from yeast, plants, mammals and other eukaryotic cells [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. In contrast with bacteria, from which mitochondria originate according to the endosymbiotic hypothesis [ 36 ], new or modified ribosomal proteins in mitoribosomes form an extended network around the ribosomal RNA.…”