Structural Insights into the Mechanism of Mitoribosomal Large Subunit Biogenesis.

In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.

Mitoribosomal small subunit maturation involves formation of initiation-like complexes.

Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although mitoribosomes from different organisms exhibit considerable structural variations, recent insights into mitoribosome assembly suggest that mitoribosome maturation follows common principles and involves a number of conserved assembly factors. To investigate the steps involved in the assembly of the mitoribosomal small subunit (mt-SSU) we determined the cryoelectron microscopy structures of middle and late assembly intermediates of the Trypanosoma brucei mitochondrial small subunit (mt-SSU) at 3.6- and 3.7-Å resolution, respectively. We identified five additional assembly factors that together with the mitochondrial initiation factor 2 (mt-IF-2) specifically interact with functionally important regions of the rRNA, including the decoding center, thereby preventing premature mRNA or large subunit binding. Structural comparison of assembly intermediates with mature mt-SSU combined with RNAi experiments suggests a noncanonical role of mt-IF-2 and a stepwise assembly process, where modular exchange of ribosomal proteins and assembly factors together with mt-IF-2 ensure proper 9S rRNA folding and protein maturation during the final steps of assembly.

Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery.

Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in Trypanosoma brucei involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo-electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.

Evolutionary shift toward protein-based architecture in trypanosomal mitochondrial ribosomes.

Ribosomal RNA (rRNA) plays key functional and architectural roles in ribosomes. Using electron microscopy, we determined the atomic structure of a highly divergent ribosome found in mitochondria of Trypanosoma brucei, a unicellular parasite that causes sleeping sickness in humans. The trypanosomal mitoribosome features the smallest rRNAs and contains more proteins than all known ribosomes. The structure shows how the proteins have taken over the role of architectural scaffold from the rRNA: They form an autonomous outer shell that surrounds the entire particle and stabilizes and positions the functionally important regions of the rRNA. Our results also reveal the "minimal" set of conserved rRNA and protein components shared by all ribosomes that help us define the most essential functional elements.

Isolation of mitochondria from procyclic Trypanosoma brucei.

The mitochondrion of the parasitic protozoon Trypanosoma brucei shows a number of unique features, many of which represent highly interesting research topics. Studies of these subjects require the purification of mitochondrial fractions. Here, we describe and discuss the two most commonly used methods to isolate mitochondria from insect stage T. brucei. In the first protocol, the cells are lysed under hypotonic conditions, and mitoplast vesicles are isolated on Percoll gradients; in the second method, lysis occurs isotonically by N2 cavitation, and the mitochondrial vesicles are isolated by Nycodenz gradient centrifugation.

Dual targeting of a tRNAAsp requires two different aspartyl-tRNA synthetases in Trypanosoma brucei.

The mitochondrion of the parasitic protozoon Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by partial import of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single copy genes, suggesting the use of the same enzyme in the cytosol and in the mitochondrion. However, the T. brucei genome encodes two distinct genes for eukaryotic aspartyl-tRNA synthetase (AspRS), although the cell has a single tRNAAsp isoacceptor only. Phylogenetic analysis showed that the two T. brucei AspRSs evolved from a duplication early in kinetoplastid evolution and also revealed that eight other major duplications of AspRS occurred in the eukaryotic domain. RNA interference analysis established that both Tb-AspRS1 and Tb-AspRS2 are essential for growth and required for cytosolic and mitochondrial Asp-tRNAAsp formation, respectively. In vitro charging assays demonstrated that the mitochondrial Tb-AspRS2 aminoacylates both cytosolic and mitochondrial tRNAAsp, whereas the cytosolic Tb-AspRS1 selectively recognizes cytosolic but not mitochondrial tRNAAsp. This indicates that cytosolic and mitochondrial tRNAAsp, although derived from the same nuclear gene, are physically different, most likely due to a mitochondria-specific nucleotide modification. Mitochondrial Tb-AspRS2 defines a novel group of eukaryotic AspRSs with an expanded substrate specificity that are restricted to trypanosomatids and therefore may be exploited as a novel drug target.

Dual targeting of a single tRNA(Trp) requires two different tryptophanyl-tRNA synthetases in Trypanosoma brucei.

The mitochondrion of Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by the import of a small fraction of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single-copy genes, suggesting the use of the same enzyme in the cytosol and mitochondrion. However, the T. brucei genome contains two distinct genes for eukaryotic tryptophanyl-tRNA synthetase (TrpRS). RNA interference analysis established that both TrpRS1 and TrpRS2 are essential for growth and required for cytosolic and mitochondrial tryptophanyl-tRNA formation, respectively. Decoding the mitochondrial tryptophan codon UGA requires mitochondria-specific C-->U RNA editing in the anticodon of the imported tRNA(Trp). In vitro charging assays with recombinant TrpRS enzymes demonstrated that the edited anticodon and the mitochondria-specific thiolation of U33 in the imported tRNA(Trp) act as antideterminants for the cytosolic TrpRS1. The existence of two TrpRS enzymes, therefore, can be explained by the need for a mitochondrial synthetase with extended substrate specificity to achieve aminoacylation of the imported thiolated and edited tRNA(Trp). Thus, the notion that, in an organism, all nuclear-encoded tRNAs assigned to a given amino acid are charged by a single aminoacyl-tRNA synthetase, is not universally valid.

Eukaryotic-type elongator tRNAMet of Trypanosoma brucei becomes formylated after import into mitochondria.

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of cytosolic, nucleus-encoded tRNAs. Thus, most trypanosomal tRNAs function in both the cytosol and the mitochondrion, and all are of the eukaryotic type. This is also the case for the elongator tRNA(Met), whereas the only other trypanosomal tRNA(Met), the eukaryotic initiator, is found exclusively in the cytosol. Unlike their cytosolic counterparts, organellar initiator tRNAs(Met) carry a formylated methionine. This raises the question of how initiation of translation works in trypanosomal mitochondria, where only elongator tRNA(Met) is found. Using in organello charging and formylation assays, we show that unexpectedly a fraction of elongator tRNA(Met) becomes formylated after import into mitochondria. Furthermore, in vitro experiments with mitochondrial extracts demonstrate that only the trypanosomal elongator and not the initiator tRNA(Met) is recognized by the formylation activity. Finally, RNA interference assays identify the gene encoding the trypanosomal formylase activity. Whereas the predicted protein is homologous to prokaryotic and mitochondrial methionyl-tRNA(Met) formyltransferases, it has about twice the mass of any of these proteins.

Non-canonical eukaryotic glutaminyl- and glutamyl-tRNA synthetases form mitochondrial aminoacyl-tRNA in Trypanosoma brucei.

Glutaminyl-tRNA synthetase is thought to be absent from organelles. Instead, Gln-tRNA is formed via the transamidation pathway, the other route to this essential compound in protein biosynthesis. However, it was previously shown that glutaminyl-tRNA synthetase activity is present in Leishmania mitochondria. This work identifies genes encoding glutaminyl- and glutamyl-tRNA synthetase in the closely related organism Trypanosoma brucei. Down-regulation of their respective gene products by RNA interference showed that (i) they are essential for the growth of insect stage T. brucei and (ii) they are responsible for essentially all of the glutaminyl- and glutamyl-tRNA synthetase activity detected in both the cytosol and the mitochondria. In vitro aminoacylation experiments with the recombinant T. brucei enzymes and total tRNA confirmed the identity of the two aminoacyl-tRNA synthetases. Interestingly, T. brucei uses the same eukaryotic-type glutaminyl-tRNA synthetase to form mitochondrial and cytosolic Gln-tRNA. The formation of Glu-tRNA in mitochondria and the cytoplasm is catalyzed by a single eukaryotic-type discriminating glutamyl-tRNA synthetase. T. brucei, similar to Leishmania, imports all of its mitochondrial tRNAs from the cytosol. The use of these two eukaryotic-type enzymes in mitochondria may therefore reflect an adaptation to the situation in which the cytosol and mitochondria use the same set of tRNAs.