Architecture of algae mItochondria translation system and its interplay with mRNA maturation – ARAMIS

To address the ARAMIS project, we combined cutting-edge approaches in molecular biology, genetics, biochemistry, and structural biology. This integrative strategy allowed us to gain diverse insights into the mechanisms of mitochondrial mRNA maturation and translation in the green alga Chlamydomonas. The project was structured around three major axes:

Axis 1. This axis identified two non-canonical poly(A) polymerases (PAPs) involved in the polycytidylation and uridylation of mitochondrial RNAs in Chlamydomonas. Their mitochondrial localization was confirmed using confocal microscopy (via expression of GFP-fused recombinant proteins) and Western blot after mitochondrial purification. Reverse genetics approaches (CLiP mutants, amiRNA knockdown) were employed to assess their function, with phenotyping based on dark growth and respiration. Defects in RNA maturation were analyzed using Northern blot, RNA-seq, 5’-RACE-seq, and proteomics.

Axis 2. This axis aimed to determine the high-resolution structure of the Chlamydomonas mitoribosome and elucidate the role of helical repeat domain proteins (HRPs). Using cryo-electron microscopy (cryo-EM), the team optimized mitoribosome purification (improving yield, purity, and integrity) by employing various detergents to solubilize membrane-associated ribosomes. Particle image sorting enabled the resolution of the mitoribosome structure. Concurrently, functional characterization of HRPs was conducted using laboratory-generated amiRNA mutants, with phenotyping based on dark growth and the stability of ribosomal RNA fragments.

Axis 3. This axis sought to elucidate the mechanisms of translation initiation for leaderless mRNAs in Chlamydomonas and explore potential links to their maturation. A tagged version of the mtIF3 factor was expressed in a Chlamydomonas strain. Following co-immunoprecipitation to purify translation initiation complexes, protein partners were analyzed by mass spectrometry. This work is still under analysis. Additionally, initial cryo-EM trials were launched to resolve their high-resolution structure. Plans include ribosome profiling to compare translation profiles between wild-type and PAP mutant strains by analyzing ribosome-protected RNA fragments.

Under Axis 1, two nucleotidyl-transferases belonging to the family of non-canonical poly(A) polymerases (PAP), specifically PAP4 and PAP6, were characterized. Analysis using 3’ RACE-seq and RNA-seq of mitochondrial transcripts from mutant lines (CLiP library or amiRNA) and the composition of their post-transcriptionally added 3’ tails revealed that PAP4 is likely the enzyme responsible for adding cytidine residues, whereas PAP6 appears to be involved in the uridylation of mRNAs. Recombinant PAP4 and PAP6 proteins, expressed in a bacterial system, were purified and used to develop in vitro activity assays for nucleotide addition on small RNA substrates, confirming the results obtained from mutant strain analyses. Additionally, qPCR analysis of the steady-state levels of Chlamydomonas mitochondrial mRNAs suggests that polyC-rich tails stabilize these mRNAs, while polyU-rich tails lead to their degradation.

As part of axis 2, biochemical and structural characterization of the Chlamydomonas mitoribosome, as well as functional studies of some of its specific components, have been obtained. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene segments. Novel proteins, mainly helical repeat proteins, including OPR, PPR, and mTERF proteins, are found in the Chlamydomonas mitoribosome, revealing the first structure of an OPR protein in complex with its RNA target. Furthermore, the missing 5S rRNA was characterized. Targeted amiRNA silencing indicated that the novel ribosomal proteins are required for mitoribosome integrity. Thanks to a collaboration with the team of B. Engel (Biozentrum, Basel), cryo-electron tomography allowed us to show that Chlamydomonas mitoribosomes are attached to the mitochondrial inner membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study provides the first example of a ribosome composed of numerous rRNA fragments, revealing a strikingly divergent blueprint for building this conserved molecular machine.

As part of Axis 3, the objective was to investigate the mechanisms of translation initiation on leaderless mitochondrial mRNAs in Chlamydomonas, by identifying the complexes formed during the early steps of this process. We first identified the mitochondrial initiation factors IF2 and IF3. A Chlamydomonas strain expressing a FLAG-tagged version of mtIF3 was then generated. Co-immunoprecipitation experiments were performed using purified mitochondria from this strain. Preliminary results revealed the presence of mitoribosomal subunits, thereby validating the relevance of the experimental approach.

Our work allowed us to identify the proteins involved in the addition of polyC- and polyU-rich tails to mitochondrial mRNAs in Chlamydomonas. We initially assumed that polyA-rich tails were added by the same enzyme responsible for polyU-rich tails; however, our recent analyses suggest that a distinct enzyme is involved. This enzyme remains to be identified, as does the specific role of these polyA-rich tails. In addition, although the enzyme responsible for adding polyC-rich tails has been identified, the mechanism by which these tails contribute to transcript stabilization remains to be elucidated.

Structural resolution of the mitoribosome has revealed proteins specific to Chlamydomonas: some interact with ribosomal RNA to ensure its stabilization, whereas others mediate its anchoring to the inner mitochondrial membrane. However, several identified proteins remain of unknown function. Moreover, the current resolution (3 Å for the large subunit and 5 Å for the small subunit) does not yet enable a comprehensive identification of all proteins that compose the mitoribosome. Additional densities observed by cryo-electron tomography also remain to be characterized. Finally, the mechanism by which the 13 ribosomal RNA fragments, dispersed throughout the mitochondrial genome, assemble into a functional mitoribosome remains an open question.

A major challenge lies in understanding the mechanism by which mitochondrial mRNAs are loaded into the small mitoribosomal subunit to ensure correct positioning of the start codon. In contrast to the situation in humans, where a specific protein recognizes a U-rich sequence downstream of the start codon, this mechanism does not appear to be conserved in Chlamydomonas. Tools have been developed to investigate this process, and preliminary results are promising. Further work will be required to address this fundamental question.

Around two billion years ago, the acquisition of mitochondrion, a double membrane-bound endosymbiotic organelle, has dramatically influenced eukaryotic cells evolution. Indeed, mitochondria are central to eukaryotic bioenergetics, with their primary role in ATP generation by oxidative phosphorylation and their important roles in fundamental processes like apoptosis, aging, or development.

Since the establishment of the integrated endosymbiotic bacterium within an ancestral proto-eukaryotic cell, mitochondria have considerably evolved. These organelles now combine bacterial traits inherited from their prokaryote ancestor with distinctive features that evolved during eukaryote history. Their mt gene expression depends on the coordinated expression between the mitochondrial and the nuclear genomes, and consequently, on the interaction between factors of dual prokaryotic and eukaryotic origins.

Several recent results have underlined the extraordinary diversification of mitochondria. Among them, high-resolution structures of mitoribosomes have been obtained for various eukaryotes, revealing tremendous structural differences with bacterial ribosomes and between them. Their architectures show significant variations highlighting an unexpected diversity of mitochondrial translation systems. In particular, the composition and structure of land plant mitoribosomes that has been recently determined is particularly remarkable. As a follow-up to this recent research, deciphering the evolutionary drift of mitochondria in different eukaryotic lineages represents a fascinating challenge. Further studies are thus essential to fully understand the diversity of mt genome expression processes in many unexplored eukaryote lineages.

Here, the ARAMIS project aims at providing detailed mechanistic insights on mt mRNA maturation and translation systems in a prime model organism, the unicellular green alga, Chlamydomonas reinhardtii. Its mt genome possesses remarkable features. All mt mRNAs directly start at the AUG initiation codon and end with post-transcriptionally added poly-cytidine tails, a feature that we recently discovered. Ribosomal RNAs are fragmented and our ongoing characterisation of Chlamydomonas mitoribosome, using single particle cryoEM combined with biochemical and functional studies reveals several peculiarities. We identified a novel small RNA resembling the 5S rRNA and the occurrence of about ten specific novel proteins possessing helical repeat motifs, in particular octotricopeptide repeat proteins.

Based on robust preliminary results and a consortium of four partners with a long-lasting history of fruitful collaborations, characterizing the model alga Chlamydomonas mitochondrial translation apparatus and understanding how its activity is linked with mRNA maturation is the central concern of the ARAMIS project. An integrative analysis performed at the biochemical, genetic, and structural levels will reveal:
- The protein factors associated with mitochondrial mRNA maturation
- The composition, structure, and function of the mitoribosome and the role of novel octotricopeptide repeat proteins
- The molecular mechanisms associated with mitochondrial translation initiation and its interplay with mRNA maturation
As a whole, ARAMIS will be instrumental to understand the biology of mt genome expression in algae. It will thus participate to understand the diversity and evolution of mitochondrial gene expression systems across eukaryotes.