Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation.

RNA fragments deriving from tRNAs (tRFs) exist in all branches of life and the repertoire of their biological functions regularly increases. Paradoxically, their biogenesis remains unclear. The human RNase A, Angiogenin, and the yeast RNase T2, Rny1p, generate long tRFs after cleavage in the anticodon region. The production of short tRFs after cleavage in the D or T regions is still enigmatic. Here, we show that the Arabidopsis Dicer-like proteins, DCL1-4, do not play a major role in the production of tRFs. Rather, we demonstrate that the Arabidopsis RNases T2, called RNS, are key players of both long and short tRFs biogenesis. Arabidopsis RNS show specific expression profiles. In particular, RNS1 and RNS3 are mainly found in the outer tissues of senescing seeds where they are the main endoribonucleases responsible of tRNA cleavage activity for tRFs production. In plants grown under phosphate starvation conditions, the induction of RNS1 is correlated with the accumulation of specific tRFs. Beyond plants, we also provide evidence that short tRFs can be produced by the yeast Rny1p and that, in vitro, human RNase T2 is also able to generate long and short tRFs. Our data suggest an evolutionary conserved feature of these enzymes in eukaryotes.

Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.

Transfer RNA-derived fragments (tRFs) exist in all branches of life. They are involved in RNA degradation, regulation of gene expression, ribosome biogenesis. In archaebacteria, kinetoplastid, yeast and human cells, they were also shown to regulate translation. In Arabidopsis, the tRFs population fluctuates under developmental or environmental conditions but their functions are yet poorly understood. Here, we show that populations of long (30-35 nt) or short (19-25 nt) tRFs produced from Arabidopsis tRNAs can inhibit in vitro translation of a reporter gene. Analyzing a series of oligoribonucleotides mimicking natural tRFs, we demonstrate that only a limited set of tRFs possess the ability to affect protein synthesis. Out of a dozen of tRFs, only two deriving from tRNA Ala (AGC) and tRNA Asn (GUU) strongly attenuate translation in vitro. Contrary to human tRF(Ala), the 4 Gs present at the 5' extremity of Arabidopsis tRF(Ala) are not implicated in this inhibition while the G18 and G19 residues are essential. Protein synthesis inhibition by tRFs does not require complementarity with the translated mRNA but, having the capability to be associated with polyribosomes, tRFs likely act as general modulation factors of the translation process in plants.

The unicellular photosynthetic organism, Chlamydomonas reinhardtii, represents a powerful model to study mitochondrial gene expression. Here, we show that the 5′- and 3′-extremities of the eight Chlamydomonas mitochondrial mRNAs present two unusual characteristics. First, all mRNAs start primarily at the AUG initiation codon of the coding sequence which is often marked by a cluster of small RNAs. Second, unusual tails are added post-transcriptionally at the 3′-extremity of all mRNAs. The nucleotide composition of the tails is distinct from that described in any other systems and can be partitioned between A/U-rich tails, predominantly composed of Adenosine and Uridine, and C-rich tails composed mostly of Cytidine. Based on 3′ RACE experiments, 22% of mRNAs present C-rich tails, some of them composed of up to 20 consecutive Cs. Polycytidylation is specific to mitochondria and occurs primarily on mRNAs. This unprecedented post-transcriptional modification seems to be a specific feature of the Chlorophyceae class of green algae and points out the existence of novel strategies in mitochondrial gene expression.

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.

No abstract

These authors contributed equally to the work. SUMMARYThe maturation of tRNA precursors involves the 5 0 cleavage of leader sequences by an essential endonuclease called RNase P. Beyond the ancestral ribonucleoprotein (RNP) RNase P, a second type of RNase P called PRORP (protein-only RNase P) evolved in eukaryotes. The current view on the distribution of RNase P in cells is that multiple RNPs, multiple PRORPs or a combination of both, perform specialised RNase P activities in the different compartments where gene expression occurs. Here, we identify a single gene encoding PRORP in the green alga Chlamydomonas reinhardtii while no RNP is found. We show that its product, CrPRORP, is triple-localised to mitochondria, the chloroplast and the nucleus. Its downregulation results in impaired tRNA biogenesis in both organelles and the nucleus. CrPRORP, as a single-subunit RNase P for an entire organism, makes up the most compact and versatile RNase P machinery described in either prokaryotes or eukaryotes.