Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis

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

Kinetoplastids are unicellular eukaryotic parasites, causative agents of several human and livestock pathologies (1). They are potentially lethal, affecting more than 20 million people worldwide (1). Owing in part to their parasitic nature, they strongly diverged from other eukaryotic model species. Kinetoplastids evolved to live in and infect a large variety of eukaryotic organisms in very different molecular environments. Consequently, beyond the general similarities, kinetoplastid species have diverged evolutionarily from one another, and their protein sequence identity can be relatively low. They have a single large mitochondrion, a crucial component of their cellular architecture (2), where gene expression machineries have also largely diverged, notably their mitochondrial ribosomes (mitoribosomes) (3–10). These sophisticated RNA-protein complexes translate the few mRNAs still encoded by mitochondrial genomes. The mitoribosomes’ composition and structure diverged greatly from their bacterial ancestor, with the kinetoplastid mitoribosomes the most extreme case described to date, with highly reduced rRNAs and more than 80 supernumerary ribosomal proteins (r-proteins) compared with bacteria, completely reshaping the overall ribosome structure.Recent structural studies performed in Trypanosoma brucei have highlighted the particularities of this mitoribosome structure and composition, as well as the assembly processes of the small subunit (SSU) (3, 6). However, despite the very comprehensive structural characterization of the full T. brucei SSU and its maturation, several pivotal aspects related to the large subunit (LSU) have remained uncharacterized. For instance, a large portion of the LSU at the intersubunit side, including the whole rRNA peptidyl-transfer center (PTC) along with several r-proteins, were unresolved (3). Moreover, in contrast to the SSU, nearly nothing is known about LSU maturation and assembly. More generally, the maturation of the mitoribosomes in all eukaryotic species remains largely underexplored. Here we present a cryo-electron microscopy (EM) investigation of the full mature mitoribosomes from two different kinetoplastids, Leishmania tarentolae and Trypanosoma cruzi. In addition, we reveal the structure of an assembly intermediate of the LSU displaying unprecedented details on rRNA maturation in these very singular mitoribosomes, some of which likely can be generalized to the maturation of all rRNA.To obtain high-resolution reconstructions of the full kinetoplastid mitoribosomes, we purified mitochondrial vesicles from both L. tarentolae and T. cruzi and directly purified the mitoribosomes from the sucrose gradient (Methods) (SI Appendix, Fig. S1). All of our collected fractions were analyzed by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS) (SI Appendix, Tables S1 and S2) to determine their proteomic composition. We collected micrographs from multiple vitrified samples corresponding to different sucrose gradient density peaks for both species, and following image processing, we obtained cryo-EM reconstructions of the full mitoribosomes, as well as of the dissociated SSU. Moreover, we also derived reconstructions of what appeared to be an assembly intermediate of L. tarentolae LSU. After extensive rounds of two-dimensional (2D) and three-dimensional (3D) classification and refinement we obtained the structure of L. tarentolae and T. cruzi complete and mature mitoribosomes at 3.9 Å and 6 Å, respectively (SI Appendix, Figs. S2–S4). Other notable features include the intersubunit contacts and two distinct rotational states in T. cruzi. Similarly to T. brucei (3), our cryo-EM analysis revealed a reconstruction of an early initiation complex from T. cruzi at 3.1 Å for the body and 3.2 Å for the head of the SSU (SI Appendix, Fig. S5). Further focused refinement on the LSU, SSU head, and SSU body generated reconstructions at 3.6, 3.8, and 4 Å, respectively, for L. tarentolae (SI Appendix, Figs. S2 and S3), and 3.7 and 4.5 Å for T. cruzi LSU and SSU, respectively (SI Appendix, Fig. S4). Combined, these reconstructions, along with the MS data allowed us to build nearly complete atomic models, with only few protein densities remaining unidentified.