Cryo-EM Structure of the RNA-Rich Plant Mitochondrial Ribosome

bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Cryo-EM structure of the RNA-rich plant mitochondrial ribosome

Florent Waltz1* & Heddy Soufari1*, Anthony Bochler1, Philippe Giegé2+ & Yaser Hashem1+

1 Institut Européen de Chimie et Biologie, U1212 Inserm, Université de Bordeaux, 2 rue R. Escarpit, F- 33600 Pessac, France 2Institut de biologie de moléculaire des plantes, UPR 2357 du CNRS, Université de Strasbourg, 12 rue du général Zimmer, F-67084 Strasbourg, France

*equally contributing authors +corresponding authors

The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs1. These organelles have a bacterial origin and were acquired during an early endosymbiosis event2. Mitochondria possess specialized gene expression systems composed of various molecular machines including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5, mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species6–10. In contrast to animals and trypanosomatides, plants mitoribosomes have unusually expanded ribosomal RNAs and conserved the short 5S rRNA, which is usually missing in mitoribosomes11. We have previously characterized the composition of the plant mitoribosome6 revealing a dozen plant-specific proteins, in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations, and the plant-specific ribosomal features of unknown structures. Here, we present a cryoelectron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPR) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA- constructive phase of mitoribosome evolution across eukaryotes.

Previously, we determined the full composition as well as the overall architecture of the Arabidopsis thaliana mitoribosome6. However, due to the difficulty to purify large amounts of A. thaliana mitoribosomes, mainly because of the low quantities of plant material usable for mitochondrial extraction, only a low-resolution cryo-EM reconstruction was derived. In order to obtain a high- resolution structure of the plant mitochondrial ribosome, we purified mitoribosome from a closely related specie, Brassica oleracea var. botrytis, or cauliflower (both Arabidopsis and cauliflower belong to the group of Brassicaceae plants), as previously described6(see Methods). We have recorded cryo- EM images for ribosomal complexes purified from two different sucrose gradient peaks (see Methods), corresponding to the small ribosomal subunit (SSU) and the full 78S mitoribosome. After extensive particle sorting (see Methods) we have obtained cryo-EM reconstructions for both types of complexes. The SSU reconstruction displayed an average resolution of 4.36Å (Extended Data Fig. 1). After multibody refinement (3 bodies) and particle polishing in RELION312 (see Methods), reconstructions were derived of the body of the SSU at 3.77Å, the head at 3.9Å and the head extension at 10.5 Å. The combined structure revealed the full plant mitoribosome SSU (Fig. 1a-d). As for the full mitoribosome, multi-body refinement of the LSU, SSU head and SSU body generated reconstructions at 3.50Å, 3.74Å and 3.66Å, respectively (Fig. 1e-g, Extended Data Fig. 1). Density segmentations of our various cryo-EM reconstructions revealed the fine architecture of the plant mitoribosome showing a large rRNA core in interaction with numerous ribosomal proteins. Among those ribosomal proteins, 8 densities located at the surface of both subunits (3 on the LSU and 5 on the SSU) unambiguously display alpha-helical motifs characteristic of pentatricopeptide repeat proteins (Fig. 1a-g). Most of these ribosomal PPRs (rPPRs) are in direct interaction with large rRNA

expansion segments (ESs) such as the head extension of the SSU (Fig. 1a-d). However, some of these SSU rPPRs appear to present a higher level of flexibility in the context of the full 78S, as they appear more scant compared to the SSU-only reconstruction, along with the ESs that they interact with. Consequently, we have focused our structural analysis of these SSU-rPPRs and their associated ESs on the SSU-only reconstruction. Our cryo-EM reconstructions at near-atomic resolutions, along with our previous extensive MS/MS analysis6, allowed us to build a near-complete atomic models of the 78S mitoribosome. In contrast to its mammalian10,13 and trypanosoma8 counterparts, the plant mitoribosome is characterized by its largely expanded rRNAs, completely reshaping the overall structure of this mitoribosome. The 26S, 18S and 5S rRNAs are respectively 3,169, 1,935 and 118 nucleotides (nt) long, thus making the plant mitochondria SSU and LSU rRNAs 20% and 9% larger than their prokaryote counterparts, respectively6 (Fig. 1h-i). Nevertheless, while plant mitoribosomes contain more rRNA in general as compared to bacteria, they have lost a few rRNA helices present in bacteria (Fig. 1j-k). As for the ribosomal proteins (45 in the LSU, 37 in the SSU), most of them are either universally conserved or mitochondria-specific constituting the common protein-core of almost all the known mitoribosomes, e.g. mS23, mS26 and mS29 on the SSU or mL46 and mL59/64 on the central protuberance of the LSU, thus confirming an acquisition of these proteins early during eukaryotes evolution. The structure of the SSU revealed the exact nature of its several specific features, namely its large and elongated head additional domain, the body protuberance and its elongated foot (Fig. 1). The body protuberance is mainly formed by the mitoribosome-specific r-protein mS47, shared with yeast7 and trypanosoma8. Interestingly, mS47 is only absent in mammals, suggesting a loss of this protein during animal evolution. The body protuberance also contains one additional proteins (mS45) and extensions of uS4m (Extended Data Fig. 7), as well as additional protein densities. The foot of the small subunit is mainly reshaped by rRNA ESs and deletions, stabilized by plant-specific r-proteins, namely ribosomal PPR (rPPR) proteins. The SSU-characteristic helix 44 is 47 nt longer, thus slightly extending the SSU and forming part of the foot extension (Figs. 2 and 3e). This extension is stabilized by a rPPR protein itself connected to ES-h6 forming a three-way junction also stabilized by an additional rPPR protein. We could not identify these rPPRs with certainty based on their sequence because of the lack of resolution at these regions, however they can only correspond to rPPR1, 3a or 3b, identified in our previous work6, based on their number of repeats, they are here referred as rPPR*. In contrast, h8, h10 and h17 are reduced compared to their bacterial counterpart (Fig. 1j), leaving spaces for the plant specific additional proteins. Our analysis of the large SSU head extension revealed that it is indeed primarily shaped by a 370nt rRNA novel domain inserted in h39. Due to its high flexibility, it was refined with an average resolution around 10Å. Indeed, due to its movement relative to the head, but also the head movement relative to the body, the overall movement of the head extension is of large amplitude (~30°)(Extended Data Fig. 2), impairing the local resolution of this area. Nevertheless, the composition of the head extension can be determined unambiguously, as our data identifies secondary structure elements for both rRNA and rPPRs (Fig. 3c). It is mainly composed of rRNA, rooting from h39. From there, the extension forms a four-ways junction stabilized by a long PPR protein (rPPR6 or mS80), locking the whole additional domain in a position perpendicular to the intersubunit side. Past the four-ways junction, two of the rRNAs helices organize into two parallel segments, forming the core of the extension - one of the two helices ends in a three-way junction shaping the tip of the head-extension. The two parallel RNA helices are themselves contacted by a rPPR (Fig. 3c). However, local resolution is too low to clearly determine its exact identity (rPPR*). Interestingly, the protein bTHXm, previously identified by mass-spectrometry6 was found buried deep inside the small subunit head. This protein is only found in the plant mitoribosome as well as in chlororibosomes4,5 and ribosomes from the Thermus genus14 . On the back of the SSU, a large cleft extends the exit of the mRNA channel. This cleft is delimited by mS26, uS8m and h26 on one side and by mS47 and the rPPR mS83 on the other side (Fig. 4a). Similarly to all known PPRs, mS83 is predicted to be an RNA binder. In plants, the processes underlying the recruitment and correct positioning of mRNAs during translation initiation is unknown. Similar to other known mitochondrial translation systems, the Shine-Dalgarno (SD) and the anti-Shine-Dalgarno sequences are absent from both plant mRNAs and SSU rRNAs. Moreover, mRNAs have long 5’ untranslated regions (UTRs), similarly to yeast mitochondrial mRNAs7. Interestingly, half of the plant mitochondrial mRNA 5’UTRs harbors an A/purine rich sequence AxAAA located about 19nt upstream of the AUG (Extended Data Fig. 8). This distance correlates with the size of the extended mRNA exit channel and would put the purine-rich sequence in close vicinity to the rPPR protein mS83. We thus hypothesize that this plant-specific cleft may act as a recruitment

platform for incoming mRNAs and / or additional factors. mS83 might recognize the AxAAA motif, thus recapitulating a SD/antiSD-like recognition system, using an RNA-protein interaction instead of an RNA-RNA interaction (Fig. 4a). An example of such possible rPPR-mediated initiation system may be found in mammalian mitochondria where the rPPR mS39 located on the SSU was proposed to accommodate the 5’UTR-less mRNAs from their 3’ end through a U-rich motif15. It is important to note that this A-rich cis-element is not found in all plant mitochondrial mRNAs, thus suggesting that this proposed mechanism for translation initiation would not be universal in plant mitochondria. Likewise, in chloroplasts, a third of the mRNAs do not possess SD-sequence, suggesting that at least two different mechanisms co-exist for translation initiation16. Similar to the SSU, the overall shape of the LSU is also strongly remodeled, even though a large portion of the core components are conserved (Fig. 2). Indeed, core functional components, such as the peptidyl-transferase center (PTC), the L7/L12 stalk and the central protuberance (CP) are similar to those found in bacteria. This conservation is in line with the globally bacterial-like intersubunit bridges (Fig. 2b). However, the LSU is reshaped by the conserved mitochondria-specific proteins e.g. mL41 or mL59/mL64 connecting the body of the LSU to the CP. Unlike all other mitoribosomes described to date, the plant mitoribosome CP includes a 5S rRNA. The core structure of the CP is quite conserved compared to prokaryotes. However, mitochondria-specific ribosomal proteins, namely mL40, mL59/64 and mL46, complement the classical bacterial-like CP and bind on top of the r-proteins bL27m, uL5m and uL18m. Interestingly, the same mitochondria-specific ribosomal proteins forming part of the plant CP are also present in other mitoribosomes even though they have lost their 5S rRNA, indicating that the acquisition of these mitoribosome-specific proteins occurred prior to the loss of the 5S in other mitoribosomes (Extended Data Fig. 5). Interestingly, the universally conserved uL2 is split in two parts in the plant mitoribosome, its N-terminal part is encoded by a mitochondria encoded gene whereas its C-terminal part is encoded by a nucleus encoded gene, which could constitute a way of mito-nuclear crosstalk (Extended Data Fig. 7). In yeast7, mammals10,13 and trypanosoma8 mitoribosomes, the peptide exit channel is highly remodeled by species-specific proteins (e.g mL45 or mL71). In plants however, the major part of the peptide channel and its exit are rather bacterial-like, with only minimal rearrangement of the surrounding rRNAs helices and a small extension of uL29m (Fig. 4b). In contrast to human and yeast17, a significant portion of the mitochondria encoded proteins are soluble proteins in plants (e.g. 7 soluble r-proteins are mitochondria encoded in Arabidopsis), thus it is conceivable that the plant mitoribosome does not systematically requires an association with the inner mitochondrial membrane, as it is the case for human and yeast18,19. However, it is likely that, at least in some cases, a non- ribosomal protein could link the mitoribosome to the mitochondrial insertase Oxa1. This hypothesis is supported by the observation that Oxa1 copurifies with immuno-precipitated plant mitoribosomes6(Fig. 4b). The main plant-specific features of the LSU are the plant-specific proteins and rRNA ESs completely reshaping the back of the LSU, below the L1-stalk region. Indeed, running along the back of the LSU, from uL15m and H31 and contacting the largely extended and remodeled domain I rRNAs (ES-H21-22 and ES-H16-18), the 19-repeats rPPR protein mL102 (rPPR5) stabilizes these additional rRNAs extensions (Fig. 3d). Moreover, the domain III is extensively remodeled and holds several expansion segments, therefore helices of this domain were renamed pH53-59 (Fig. 1h, Extended Data Fig. 4). Indeed, this remodeled domain III has two main helices that largely extend in the solvent and are stabilized by two PPR proteins (rPPR4 and 9 or mL101 and mL104) (Fig 3a-b). These two rPPRs appear rigid and present numerous interactions with the rRNA. Thus, rPPR9 encapsulates the tip of helix H10, and stabilizes the end of pH59, one the newly formed helices of domain III. rPPR9 also directly contacts rPPR4 that wraps around a single stranded rRNA extension (1645-1644nt) and contacts the two major helices of domain III pH55 and pH57 (Fig. 3b). Interestingly, the rPPRs described here, along with those of the SSU, seem to hold a different mode of RNA binding as compared to the RNA recognition process of canonical PPR proteins. Indeed, PPR proteins usually bind ssRNA through a combinatorial recognition mechanism mainly involving two specific residues in each repeat (5 and 35) allowing each repeat to bind a specific nucleotide20, similar to other helical repeat modular proteins21. However, the structure obtained here revealed that several rPPR proteins bind the convex surface of double stranded rRNA, mainly through positively charged residues contacting the phosphate backbone of the rRNAs. This novel mode of RNA binding evidenced for rPPR proteins (Fig. 3) extends our understanding of the diversity of functions and modes of action held by PPR proteins. The position of this remodeled domain III on the full 78S suggests that rPPRs 4 and 9 might be involved in the attachment to the inner mitochondrial membrane, as they appear to strongly stabilize the structure of the whole domain (Fig. 4b).

In conclusion, the plant mitoribosome with its large rRNA ESs illustrates yet another route taken during mitoribosome evolution. It represents an augmented prokaryote-type ribosome. Based on a bacterial scaffold, it has both expanded rRNAs and an expanded set of proteins that were specifically recruited during eukaryote evolution in the plant clade. The structure of the plant mitoribosome could reflect the so-called “constructive phase” of mitoribosome evolution where both rRNAs and protein sets were augmented, in strong contrast with mammals and trypanosoma where rRNAs were considerably reduced3 (Fig. 4c). The structure presented here thus provides further insights into the evolution of mitoribosomes and the elaboration of independent new strategies to perform and regulate translation.

METHODS Methods and any associated references are available in the online version of the paper.

ACKNOWLEDGEMENTS This work has benefitted from the facilities and expertise of the Biophysical and Structural Chemistry platform (BPCS) at IECB, CNRS UMS3033, Inserm US001, University of Bordeaux. We thank A. Bezault for assistance with the Talos Arctica electron microscope. We thank L. Kuhn , J. Chicher and P. Hamman of the Strasbourg Espanade proteomic analysis for the proteomic analysis. We thank M. Sissler for her useful comments during the article redaction. This work was supported by the “Centre National de la Recherche Scientifique”, the University of Strasbourg, by Agence Nationale de la Recherche (ANR) grants [MITRA, ANR-16-CE11-0024-02]] to PG and YH and by the LabEx consortium “MitoCross” in the frame of the French National Program “Investissement d’Avenir” [ANR-11-LABX-0057_MITOCROSS, as well as by a European Research Council Starting Grant (TransTryp ID:759120) to YH.

DATA AVAILABILITY The cryo-EM maps of the mitoribosome have been deposited at the Electron Microscopy Data Bank (EMDB) with accession codes XXXX. Corresponding atomic models have been deposited in the Protein Data Bank (PDB) with PDB codes XXXX.

AUTHOR CONTRIBUTIONS PG, FW, and YH designed and coordinated the experiments. FW purified the mitochondria and mitochondrial ribosomes. HS acquired the cryo-EM data. HS and YH processed the cryo-EM results. HS, AB and FW built the atomic models. FW, HS and YH interpreted the structure. PG, FW, HS and YH wrote and edited the manuscript.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

a head extension b c head extension d

head head head

beak beak

body h44 protuberance

SSU rRNA 90° 90° 90° SSU rRNA SSU rp SSU rp rPPRs rPPRs foot extension h44 h44 beak side intersubunit side platform side solvent side e f g head head CP head L1-stalk CP P-stalk L1-stalk CP

SSU rRNA SSU rRNA SSU rp 90° 90° SSU rp LSU rRNA LSU rRNA LSU rp h44 LSU rp rPPRs h44 rPPRs beak side LSU side platform side h i P-stalk j k P-stalk

CP head H1 CP ES-h39 head h33 ES-h33 beak beak ES-h16 L1-stalk L1-stalk

h17 h44 H63 ES-H21-22 H78 h44 ES-H16 h10 h8 H9 ES-h6 Domain III

intersubunit side ES-h44 LSU side intersubunit side LSU side

Plant-specific rRNA extensions Conserved rRNA Plant-specific rRNA deletions

Fig1. Overall structure of the plant mitochondrial ribosome Composite cryo-EM map of the SSU alone (a-d) and the complete mitoribosome (e-g). rRNAs are colored in light blue (LSU) and yellow (SSU) and ribosomal proteins in blue (LSU) or orange (SSU). rPPR proteins are shown in red. h-k Arabidopsis mitoribosome rRNAs compared to E.coli ribosome. h and j comparison of the SSU, i and k comparison of the LSU, extensions in Arabidopsis rRNAs are shown in green, reductions are shown in blue. CP represents the central protuberance. bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

uS13m mS80 mS80 mS35 uL11m mS29 uS9m rPPR* uS19m uL5m mL40 rPPR* uS14m mL46 uL10m mL59/64 mL46 uL25m uL18m uL2m uL16m bL27m bL9m uL15m bL28m uS7m uS10m mL102 mS37 mS31/mS46 mL60 uS11m 180° mL102 uS3m uL30m bS21m uS2m mS23 mS33 uL4m bS18m uS5m bL21m uL4m bS6m uS4m mL43 mS47 mS47 uL20m uL24m mS83 uS12m uL13m uL22m mL41 uS8m mS45 mL53 mS26 bS16m uL29m uL6m uL23m uS15m mS41 bL35m mL104 bL17m mL87 uS17m rPPR* mL87 mL80 mL101 mL101 mS34 rPPR* bacterial r-prot bacterial r-prot bL19m uL14m mitochondrial r-prot rPPR* mitochondrial r-prot plant-specific r-prot plant-specific r-prot

b CP

head mB8 B2am L1 stalk B2a-bm mB3 B7am

mB3 B2bm B3m

B7bm B3m B2cm B8m body B4m

B8m B5m

mB13 bacterial bridges mitochondrial bridges plant-mitochondrial bridges

Intersubunit side SSU Intersubunit side LSU

Fig2. Atomic model of the plant mitoribosome a The overall model of the plant mitoribosome with individual proteins annotated. rRNAs are colored in blue (LSU) and light brown (SSU) and ribosomal proteins in shades of blue and green (LSU) or shades of yellow and red (SSU), according to their conservation. b Intersubunit interfaces with conserved and novel observed intersubunit bridges highlighted. Bacterial ones are colored in purple, mitoribosomes ones in pink and plant ones in green. bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1635-1646nt a H10 b rPPR6 c rPPR4

rPPR9

ES-h39

1862-1864nt 1913-1921nt

d H31

rPPR5 CP

rPPR*

uL15m platform side

rPPR* SSU & LSU rp H16 SSU rRNA ES-h6 LSU rRNA rPPR* rPPRs

h44 ES-H16 ES-h44

Fig3. The plant mitoribosome PPR proteins Overall view of the mitoribosome with PPR proteins highlighted in red, with zoomed-in view of each rPPR proteins (a-e) and their rRNAtarget and mode of binding. In a and d rPPR4 and rPPR5 stabilize single stranded segments, whereas in b, c and e rPPR proteins mainly interact with the backbone of the rRNAs. bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

PTC a b bS18m

h26 uL4m

uL22m mS26 uS8m SSU uL29m mS23 uL23m uL24m

mS83 Channel exit

LSU

Linker mS47 IM Oxa1 OM

c Plants Yeast Mammals Kinetoplastids

LSU LSU LSU r-prot LSU LSU SSU SSU rRNA SSU SSU SSU

Ancestral alpha-proteobacterial ribosome 1:Constructive evolution of rRNAs and proteins 2:Destructive evolution of rRNAs and constructive of proteins

Fig4. Specific features of the plant mitoribosome a View of the SSU back channel and its proteins constituents. The possible mRNA path is highlighted by the dashed blue line and the blue star indicated the position of the AxAAA motif present in the 5’UTR of half of plant mitochondrial mRNAs. b Highlight of the plant mitoribosome peptide channel and possible mode of membrane attachment of the plant mitoribosome. Similar to yeast with Mba122 plant mitoribosome would require an accessory protein factor (green linker) to tether the mitoribosome to the Oxa1 insertase. The green line represents the possible surface of interaction with the inner membrane (IM) of the mitochondria revealing an extensive surface mainly formed by the remodeled Domain III of the LSU. c Comparison of the different mitoribosomes described to date. Schemes of the ancestral alpha-proteobacterial is represented compared to the actual mitoribosomes. Light blue represent core bacterial rRNAs and yellow core bacterial r-proteins, blue and orange represent mitochondria specific rRNAs and r-proteins acquisition. Two main different evolutive paths have been taken by mitoribosomes, a mainly constructive one (1) represented by the yeast and plant mitoribosomes, where few proteins were lost and novel r-proteins were acquired as long as extended rRNAs, whereas in human and trypanosoma (2) rRNAs were highly reduced and more novel r-prot were acquired. bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

ONLINE METHODS

Mitochondrial ribosome purification: Cauliflower (Brassica oleracea var. botrytis) was used here as it is best suited for large scale biochemical, structural analyses as compared to Arabidopsis. Cauliflower belongs to the same family of plants as Arabidopsis, Brassicaceae, thus making it an optimal model for this study, allowing the preparation of large quantities of highly pure mitochondria. However, the genome of Cauliflower is not sequenced, and the closest fully sequenced member of the family (Brassica oleracea subsp. oleracea) is poorly annotated. Still, protein sequence identities between members of the Brassicaceae family are higher than 90%, thus facilitating proteomics identification of cauliflower proteins. Combining the proteomics results from Waltz 2019 and those obtained in this study it is evident that no difference in terms of protein composition could be observed between Cauliflower and Arabidopsis. Hence, to facilitate comprehension and analysis we positioned Arabidopsis proteins in the cauliflower map.

For the mitochondria purification, fresh cauliflower inflorescence tissue was blended in extraction buffer containing 0.3 M mannitol, 30 mM sodium pyrophosphate (10.H2O), 0.5 % BSA, 0.8 % (w/v) polyvinylpyrrolidone-25, 2mM beta-mercaptoethanol, 1 mM EDTA, 20 mM ascorbate and 5 mM cysteine, pH 7.5. Lysate was filtered and clarified by centrifugation at 1.500 g, 10 min at 4°C. Supernatant was kept and centrifuged at 18.000 g, 15 min at 4°C. Organelle pellet was re-suspended in wash buffer (0.3 M mannitol and 10 mM phosphate buffer, 1 mM EDTA, pH 7.5) and the precedent centrifugations were repeated once. The resulting organelle pellet was re-suspended in wash buffer and loaded on a single-step 30 % Percoll gradient (in wash buffer without EDTA) and run for 1h30 at 40.000 g. Mitochondria are were retrieved, washed two times before being flash frozen in liquid nitrogen.

For mitoribosome purification, mitochondria were re-suspended in Lysis buffer (20 mM HEPES-KOH, pH 7.6, 100 mM KCl, 30 mM MgCl2, 1 mM DTT, 1.6 % Triton X-100, 100 µg/mL chloramphenicol, supplemented with proteases inhibitors (Complete EDTA-free)) to a concentration of 1 mg/mL and incubated for 15 min in 4°C. Lysate was clarified by centrifugation at 30.000 g, 20 min at 4°C. The supernatant was loaded on a 40% sucrose cushion in Monosome buffer (same as lysis buffer without Triton X-100 and 50 µg/mL chloramphenicol) and centrifuged at 235.000 g, 3h, 4°C. The crude ribosomes pellet was re-suspended in Monosome buffer and loaded on a 10-30 % sucrose gradient in the same buffer and run for 16 h at 65,000 g. Fractions corresponding to mitoribosomes were collected, pelleted and re-suspended in Monosome buffer.

Grid preparation 4 µL of the samples at a concentration of 2 µg/µl was applied onto Quantifoil R2/2 300-mesh holey carbon grid, which had been coated with thin home-made continuous carbon film and glow-discharged. The sample was incubated on the grid for 30 sec and then blotted with filter paper for 2.5 sec in a temperature and humidity controlled Vitrobot Mark IV (T = 4°C, humidity 100%, blot force 5) followed by vitrification in liquid ethane pre-cooled by liquid nitrogen.

Single particle cryo-electron microscopy data collection For the two data-sets (Full and dissociated complexes), data collection was performed on a Talos Artica instrument (FEI Company) at 200 kV using the EPU software (FEI Company) for automated data acquisition. Data were collected at a nominal underfocus of -0.5 to -2.7 µm at a magnification of 120,000 X yielding a pixel size of 1.21 Å. Micrographs were recorded as movie stack on a Falcon II direct electron detector (FEI Compagny), each movie stack were fractionated into 20 frames for a total exposure of 1 sec corresponding to an electron dose of 60 ē/Å2.

Electron microscopy image processing Drift and gain correction and dose weighting were performed using MotionCor225. A dose weighted average image of the whole stack was used to determine the contrast transfer function with the software Gctf26. The following process has been achieved using RELION 3.027. Particles were picked using a Laplacian of gaussian function (min diameter 260 Å, max diameter 460 Å). For the full mitoribosome, after 2D classification, 153,608 particles were extracted with a box size of 400 pixels and binned four fold for 3D classification into 10 classes. Four classes depicting high-resolution features have been selected for refinement. The complex has been focused refined with a mask on the LSU, the body and the head of the SSU, yielding respectively 3.50, 3.66 and 3.74 Å resolution. Determination of the local resolution of the final density map was performed using ResMap28.

For the dissociated subunits, following a 2D classification, 132,130 particles for the LSU and 120,350 particles for the SSU were extracted with a box size of 400 pixels and binned four fold for 3D classification into 8 classes for each subunits. Five subclasses depicting high-resolution features have been selected for the SSU refinement with 73,670 particles. After Bayesian polishing a multi-body refinement has been performed using mask on the body, the head and the RNA expansion on the head, yielding respectively 3.77, 3.9 and 10.5 Å resolution. Three classes have been selected for the LSU refinement yielding a resolution of 3.96 Å. Determination of the local resolution of the final density map was performed using ResMap28.

Structure building and model refinement The atomic model of the plant mitoribosome was built into the high-resolution maps using Coot, Phoenix and Chimera. Atomic models from E.coli ribosome (5kcr)29, yeast mitoribosome (5mrc)7, human mitoribosome (6gaw)15 and trypanosoma mitoribosome (6hiv)8 were used as starting points for protein identification and modelisation. The online SWISS-MODEL service was used to generate initial models for bacterial and mitochondria conserved r-proteins. Models were then rigid body fitted to the density in Chimera30 and all subsequent modeling was done in Coot31. For the LSU and SSU ribosomal RNA, the 16S and 23S from E.coli were docked into the maps and used as templates from positioning and reconstruction. A multiple sequence alignment of several plant mitochondrial ribosomes and E.coli ribosome was performed in order to determine the additional or depleted domains of A.thaliana mitochondrial ribosome. Ponctual differences were done in Chimera using the “swapna” command line. In order to build the additional rRNA domains of A.thaliana, the co-variation algorithm LocARNA webservice (http://rna.informatik.uni-freiburg.de) was used to determine secondary structure of these domains. At that point, the secondary structure prediction was used to build the 3D model in Chimera using the “build structure” tools followed by manual adjustments. Proteins with clear homologs in either mammalian or yeast mitoribosomes as long as E.coli ribosome were build using Phyre2 and SWISS-Model. The global atomic model was refined with VMD using the Molecular Dynamic Flexible Fitting (MDFF) then with PHENIX using a combination of real and reciprocal space refinement for proteins and ERRASER for RNA.

Proteomic and statistical analyses of mitochondrial ribosome composition Mass spectrometry analyses of the ribosome fractions were performed at the Strasbourg-Esplanade proteomic platform and performed as previously6. In brief, proteins were trypsin digested, mass spectrometry analyses and quantitative proteomics were carried out by nano LC-ESI-MS/MS analysis on AB Sciex TripleTOF mass spectrometers and quantitative label-free analysis was performed through in-house bioinformatics pipelines.

Figure preparation Figures featuring cryo-EM densities as well as atomic models were visualized with UCSF ChimeraX32.

References

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bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made DATA-SET 1 available under aCC-BY-NC-ND 4.0 International licenseDATA-SET. 2 Full Dissociated 2D Classification 2D Classification

107,710 132,130 120,350 particles particles particles b 3D classification 3D classification 3D classification 65,280 particles 61,900 particles 73,670 particles

3D Refinement 3D Refinement LSU 3D Refinement Full SSU 3.96Å

3.86Å 4.36Å

3 4 5 6 7 8 Å 3 4 5 6 7 8 Å

Focused Multi-body refinement refinement

3.50Å 10.57Å

3.9Å

3.77Å

3.74Å 3.66Å

c 1.0 1.0

Full mitoribosome (3.86Å) SSU before multibody (4.36Å) 0.8 0.8 Focused SSU body (3.66Å) SSU body (3.77Å)

0.6 Focused SSU head (3.74Å) 0.6 SSU head (3.9Å) Focused LSU (3.50Å) SSU head extension (10.57Å) Correlation Correlation 0.4 0.4

0.2 0.2

0.0 0.0 Fourier Shell Fourier Shell

-0.2 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 -0.20.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 resolution (1/A) resolution (1/A)

Extended Data Fig. 1 Data processing workflow Graphical summary of the processing workflow described in Methods, with 2D classes presented in a for both datasets and 3D processing, presented in b, with ResMap of the full mitoribosome and SSU only before further processing. c FSC curves of the full mitoribosome and SSU before and after focused classification and multibody refinement. bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a head extension b 30 °

head

body

90°

body

Extended Data Fig. 2 Multibody refinement, additional SSU head domain movement amplitude a Views of the two extreme states of the head and head extension, relative to the body of the SSU, calculated using the multibody refinement implemented in RELION312, showing the movement in two different planes. b All ten states reveals a movement amplitude of 30°. bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

37 A A C G U U G C 1080 G C U U 1090 23a U A 1100 700 710 39 720 U U A 1110 1120 1130 1140 690 C A 23 C C A A A A 38 C U U G A A G U G A A G A A G A G C U U AG AG A G G A U C U A C U A C G A A G C G C G A G A U A C G A G C C C U C G U C U U G U G G C U C A C A U G C G C C UA GA G A A A 1150 A A G C A A A G A 36 C U G U G G G G A G G U G U G C U U U A A G G A C G G G C U G U G C U C G G G G G U G G A G C A C C G C C G U G C G U G G C U U G A G U A A 1470 C C A A 1160 730 1070 A 1530 GA C G G 680 G C 35 1060 U 1500 1490 A G U 670 A U 790 C A G A G CU A A G G A C C CC G G C A A UG C G 1480 A A A G G A A U U U 1540 G G C 1510 A C U A C G A C A G G G C G C A C A U C 22 A C U U A A A A G C G U G C 34 G C G 1460 G1420 660 A G U G 40 C G A G G C A U C G C A G G U 24 U A C G U C C G A G G G A C G 780 C G U A U A U A G 1050 G U C 1550 A U U G A C 1520 C G A G C 740 A U G C G C G C G A C G A C C 1020 A U A C G U G G U G C G 800 33 1030 G C A U U A A C G U A A 1170 G G U C G G G A UG G U A C U C A C 1450 C G G C G A U U G C G 650 A C 770 G C U C G A A G C 620 630 640 G C A U G C U A U G G U U C C U A U C C A A AC C U GA G G C G U A A U U A A A A A G C C G A U G G C G G A A G C U U U C G A C C A A U U C A C A A U A C A U U U C 750 1010 1000 G A G C 1560 U A A G C G U C A U U A C G C U G G G C A U U A A A C C G C U G U G A A A G U U G G C U A A G U G A C A C U A G G A U A G A G G C G 760 G G G G G A G 810 U A C G U G GU 610 A A 600 590 G U A U C C 990 C U A A A G G A U 840 U C G A C A 26 G A AG C G 3'M 1440 A U 1180 21 20 U C G G A A 830 G A G C 32 1390 G G 580 C C U U 980 G C G U A U A U G G C C G C G 520 A C A C A U U C G 1570 C G G 820 25 G C A C G G C U C G C C A G C G A 19 G A G U G U UC G G A G U A G A G 970 G U A G G C 850 A 1610 G C G A C U A U C A G A C 570 U G C A G 1370 C C A C G C C U C A C A A G A A A G 1380 G 1190 480 A 26a C C C 1620 G C 1193 C A G A 530 G C U G C A U U U A G C 870 C G U UU A A G G C G A C G U C G A G U U U A G 17 A G A G G U G C A U 41 G A G C G G C U C A C U A G C G G A 18 C 880 890 G C A 1600 G C G C U G C A G C U C A A A U C C G C G 560 A U G C C A A G 950 G C A U G G C C G G G A 510 C G G G G G G A U A G G U C G A A U A A C A C C U G U C C U C U A 1220 1210 1202 470 U U A 860 A G C 1580 C G G C 490 A A C G A A 31 U G A G G G U G G C U C A C C A G A 960 30 U A A A A A A U U A 1630 G C 550 C 30 A A A C G G G C G U A A A 900 C G 1590 A G U C G 540 U A 27 940 G C A G C G C U G C G U G C A C G A C G A U U G U 450 U A U A G U C G G 1640 460 U C G U G G G A A C G G C G A A U 2 G C A C C UAA U A U G A G U U A 29 U 16 C G C UUG A G C U C A AG A 930 U C G C A U A 920 28 GG A G 40 G U G A C A G U U A U C 20 G C G A G U G A C G G A U G A G U G C G U A U U G A C G G G G C C U G A C A A U U C A 3 A C G 1650 440 A G 420 A 1 G G G A A C G A C 1690 A G A U C A A C U G U C C C G G G C U G U A 430 A U A C A U 410 AU C C C A U A G A A U 1740 A U 42 U 50 G A G 1680 U G A G C C G A U C C A G C A A 5 1730 U U A A U U C 1900 A A C U A G 4 A 1700 A U 1660 G C G G G U A G G UC 1750 C A A G G A C C U 1910 U A 400 A C U U U G C G U G U A 390 A A C A G C C A G 6 U A G C C G U A G G G G G G U 15 GG A 80 G C A G C 43 G G G C 60 C U A 1720 C G U C G G C C 380 U A U C G U C G G U G U C C A G A A G C G U G C G C U UC A A5 U A G U U A 45 C A U G U G 1920 G C 1670 14 C G 70 A C A C G U A 370 G A C A A U U A G G AG C A C A A G 1930 C C G G C A U A 160 A A G G G UG A C G A G A U A 1710 C C G C C G 1760 C G U U C C G 170 C G 360 A C G C 1935 5' G A G U G 350 C C A G U C G G C C C G C A U G C 340 G G A G G A 1880 13 G C G A G A A A U G C A 330 6a A C A U G U C G U U G C A G C A 180 320 G A U 1770 U U C G G G C U G G U C G U G U 37 U C U 3'm U A U G 12 U 310 U A 23 23a 38 U 260 A 44 U G 300 C G U G 1870 36 G A G A C G U U G U G A U G C G U 35 39 A C G C U A C G 40 C A U G U 1780 C G G G U G 190 C C 34 A G G A 24 290 A U C G A 22 C U A C G A C C C U G G A G C A G A G G 270 250 A U C A 33 U U G C A G C U U U U 1860 G A G 7 U G A C A A 11 G C C 26 3'M A G G C 21 20 A U U G 200 1790 G G 32 280 U A A C 25 U A A G 19 G C C G 31 240 U A 210 A U 17 41 C G U A U 18 A G C G U A C G G G C C U A U 1850 A A 26a 10 A 8 G C U 30 A U C G A A G U C C AA A A U A U 27 A G U G 2 29 230 220 1800 C G 28 9 A U 16 3 C G 1 C G 42 C U 4 A U 1840 43 G U 6 C 15 A U 5 C C 14 45 1810 U G U G C A 5' 13 U A G C A U A 1830 6a G C U C 44 3'm A G 12 A U 1820 C G A A 7 C U 11

10 8

Extended Data Fig. 3 Secondary structure diagram of the plant 18S rRNA 2D representation of the 18S rRNA colored by domain. The rRNA expansions specific to the plant mitoribosome are highlighted in cyan. Extensions that could not be modelled are indicated by dashed lines. A simplified secondary structure diagram of the E. coli 16S rRNA is also shown in the black frame, helices not present in the plant mitoribosome are shown in gray. Secondary structure templates were obtained from the RiboVision suite (http://apollo.chemistry.gatech.edu/RiboVision) bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made UA 1850 78 C AUA A G A G 2450 availableU U under aCC-BY-NC-ND 4.0 International license. G A U C A U U G G G 77 G G C p58 CU A U 2430 G C C G A 2440 1840 U A G U AG CA G C G A G A U A GGUGG U 2460 A C U C G U 1860 G C G G 1835 C U U UC AC C U A G 1610 III G G G C AG G U U A U A A U 2420 U U A U C C A U 2470 A GG GA A A G U A U A U 1600 G C 1640 C C G C C C U 1680 C A A C G 2480 G G 1620 1650 C U G U A U U U AGG A C U A A U 76 A C U GG C U U C C 1870 IV U A A G U U G C C C 1660 U A G A U A p56 C A C A C U U G 1590 U G C A G A 2180 G C 2190 U A CU C U C 1670 A G A U 2410 C G 2610 C G 1630 U U U U G A p57 A U C G A U UCC U C C G A G G U G AG A U A 68 G U 2490 C 84 UA C U U G A G A U 79 A U C GCC A C G G A C G 2502 A G C A C U C 1580 A C U G A G CUAA C G C CG A U A U C C G C C G U U GUA A UA A G U A G GC A 85 AC G 1880 G A 2170 A G A A U p55 A G C A A 2200 G GGC G G C GG 2130 C A G U U 2630 U A U A U G A U A 2521 2600 A 1570 C G C G C 2400 C GCAC C G U A 1890 2120 A G AA C G C GAG G CAA A A A A C G 2530 A U G C G U U G C 75 U A A A A U U A G G A C G G C A 81 A 83 U C U U G U G 80 U C U G U G UU A A p59 A 2210 69 C G C G 2590 G C 2640 C G U U A C C A G U 2160 C G G U GGG C G G C A GC U A G UA A G G C U 2650 1560 G A G G ACU G U A 2580 AA G C A A C G U AA A G U C G G A A A 1900 1910 G U U A 2140 G C U C U G C G C G UGUGAGACCG C A U U 66 C G G G U A U U A GUGC C A p54 G U G CC U U C A AA U A U G U U U G U 1920 A U C G C C C G U A 82 C G C G C C G 2110 G C C G G U U C G GAC AAGC UGGU A G U C 63 2100 G U 2150 G C G G A U 2560 C AA U A U C U A A G U 2230 A G A G G U C U C CG C U C G 2660 CA G 2050 A U AAAAC A AC AC C C A 2730 C A U A 2670 1550 U U A A C U A C U C GU G G C 86 C G U 67 U U A U C U G AGU A G A A C G U UGUGG C G A A C G AA p53 A C G AUUUGU U A G G U A 2690 1220 U 1930 U A C A A A C GCA 1230 G 1240 C AG CA G A U C G 43 U A A G U C A 2290 A UG A G U 2770 C U A A A G U A 2070 65 C GA G A C C 2700 87 G AGC C A UC C U G GCGU G 1940 2030 2040 C G 2280 C G 2240 2380 C G G C A A 1540 G GA G C G C 2740 G C GCU A U A GA A G C GC A U A C U A G AA A G A 44 A C A C C GG GGGGU G A U G C G G C 2710 88 2760 C G C G C GGG UGGA C U C GAU G C U C C C 2080 64 70 C G A U C U UU G A G C U A UA U C G A GGUG UC UAU G C C C G A UC C C C C A C G C U A 2250 74 C G AC C 1210 1250 U C U G CU A G G C A G C A C G U G UG G A U 1530 G C 49b A A U U GG C 2270 C UU C G A A U A GG C A C G C A A C C A GC UGA G C 1500 A A A C AG GC 2780 1200 C G C A AU 51 A C CG 62 A A G G G UC A U A UC 52 U G A C G C U U 2750 AGG U A U 1260 C G 1510 G C 49a G C U C G C C U 89 C A G A A A C A G G C C 2790 A G U GGAC A 60 2010 A U C GC G CA G G 2000 G U G A 71 U G U A C G U 1960 A U G AAU 2260 A C UAGC CC A C A G U A 1970 A UG G A C G AA G U A C G 2800 A A 1270 1280 GC A U A GG C A A A G U G UG 1520 C U C G G A U C C 2370 A A 1190 C U G 1490 G U G U U CC A C A AAAUG C C 1470 A A 1990 G G 61 U V G A U A U U A U C G AU U G 49 C G A C U G G C C C 1460 48 1980 A A C A 42 AA A C UG C G G C U 2310 U GG G AU G G A GC G G G C UU G C G U C G A G G U C 1180 A A C C G G 2810 A G CUG 1480 U A G A A 0 C C UG G AC 90 C U A C U G C G U G U A U 1290 U G U C 72 73 A C G A U U GU C 2820 U C 2340 G U 2880 UG 91 A C 50 U A 47 A A C 2320 U A U A A AU A U C 1390 G A A G AAGA UA U A GAG G G C A U G A A 46 G C C U U G U 2360 C G C C A U AC C C UGGGGUU A G 41 A C A G 26a C U 2900 A A G CAUG A G U 1370 G U G AC C G UG U G G C G U G G 2830 U G A A U 1430 A G C G G UG GG AC C C UGG A G U C AU 1300 G G CA C G C A A U G A 2870 A A U G A AC U UGC A A A U A A C 93 G C AU UC GA U G AAG A A C G 2350 A C A G A U 2840 A C 45 C U G 1440 U U 2330 U A C G GU C G 2970 C A U 1170 A G U G A G U C G C U C G UU C C G A A U G U A A G U C G G C G C U 1400 GU A AUU U A G 2920 2860 C G 1160 A C G A U U 1410 1420 A G 95 A U C G A U A 1320 A U A C C A U G U 92 C G A G C A A G A G A U 2950 C G U C G G C G GA G U A G G C G C U 2850 C A C 1310 U A U U A A U U G A U G UUG AU G U A C U C C C G A G G G 1150 C A C G 1360 U G A G C G U 2930 94 2940 A C 2990 3000 GC C C GU U G U C 710 G C C U G C CC C G GA A U C 730 A U C G G A U C G AUG 1340 G G U A A U AAAGGGAG A A C UGCG A A AUGGUG UA UUGUUAUG C G C 26 U U U C 40 A U A G A U A A 2980 C 96 AG G G 1350 C C G U G A GC 3080 AC AU A AUC GA GGG GAC GAUAA C C A 720 C G AG U A C C C 1130 A CC 1140 U A U A A 670 A C G A G CG A A AA 25a C G C U U U 3030 3010 AG CGG G 700 C A U U 3020 GG G C A U C U 3070 C G C C G UU U A A A 16 GGUUU AG U G U A GCC G C G A C U U C A C U A U U A 98 U A 50 3 G G U A C C A A CAAG AC U U A C A 36 U G U A 740 U C U 1120 A 950 G C A U A G A G U U 3100 CC GG 40 C C A U G G C C A C G C A G A 60 C A C U A U G C G 20 A U A U 39 GU G U G 25 G C A 3110 A A 3040 G U 1110 AA A A C C G A A U 3060 C G A CA 970 U A 3161 U A A G VI 35 C U G A GAUC AG 37 UC U C G C A A C G A UUC U A G U A 27 2 A U G C G G A A U A GU A 980 A A U A A G C 97 C C 2 80 4 U GG U G U U A C G G U A U G G A G A G C G A U A 32 660 C G U U G U CGU 1100 U U G U A G C C G 30 A G U A GA A G C GAC G A 800 U G 750 C G AA U C 70 G CU U A C C C A G C 28 C G G C G UU U G 1090 A G U C C UA U A U G A U C A G A CUU U U A G UU G 760 3150 A C C G C G AU GG G A A G A 820 C A A UC G U G 90 A CC 90 U U A U U GC UU GC C AU A U A UC U G 20 GU C G U G C C A U AC G A U GUA A GA 1080 U A C 790 G G 3050 G C G G G U A G A GC GG GUGGA C G 3120 A UG G U 100 C G A A G C A U A G 30 G UG G U C C U U 930 C U G U A G A G A C C C U U G A A G U U C 5 II U G G G C 31 G CCU 640 G AA 80 G C 7 C A A GG GA U GAAA A 650 U U 10 A A GU G 830 CA UA U G G C 100 110 C U 990 A U U A 780 G GAG 3140 U A 5S C G C G AAG C G U AACC G G C A U G C 1070 G G 920 G C G U A 101 G C G C AC A G C C G G C G C U A G GGG G C C G A C 35a A G U 3 G C 3130 70 G A C G C C G U 630 U A A A G C A G 6 C G 1 A G U G AUC C C A A G A A C A A A G A A U U A 1000 910 U A 33 A C G C G U A G A G C G 840 620 G G G C 1 A A 108 38 A G A AGG 610 G CUUU U G U G C 24 G G 40 AGA C C 8 C A U A 23 U G G C 110 GA 120 140 C G A G GC C C C G A C G G A 10 G G A G C A G A G A 1060 U A 900 G U 34 U A UU 4 A U C C 130 C U C G G C U G 600 A GU G A C UGG C 1010 G C A G C A G U A A G 5 G CUCCG A A C C A A A 850 AA A A G G AG G A UA C G G C CG C A 50 U CA UC U A C AA C A 880 G A AUG 580 UC A AUC GCG A U A G A GA 590 U C A AU GGU G C U GUU CU G C C U U C U AG G A G G CC UC A C 150 U A GU 35 870 G 22 C 160 A U G U C A 570 U U 890 A A C G C G ACA G C A U 1050 C G G C 560 240 13 U C G 860 540 550 G U 14 A AG C C 1020 G C U AGCG C G A U G G UA C 250 G G I C CGA A G C A CG A A A GAAA U G A C G A G U CG A C G U G G U G U A A U U AG AG 230 U G U A GUU A U U 530 C U CC C 170 1040 C G G U C 220 200 U G C CA G U U U G G AG 21 GA U A A AA C G U A A G U 12 A U A UC A A C C A G A C A AAU CA 260 A C A A A G G G C 11 520 G G U G A A U C GC GA U G 180 G C A 277 GA A U C 510 C 440 A U G A AG GA AUC A GU C 270 GA G A UAC 500 U A C U G GA AC U A U A AA C 295 19 G U 58 A GA UU G C C UC A U 16 UU 420 A G 450 78 U 300 430 AA G C U G U G C C GC C AG UUU AC GUAAUUGU CA 77 U A A GC UGG C UU UUAG GA U A U C GC A U U A 57 59 68 490 480 GC A 470 C G IV C G 460 18 C U 20 76 V G G 56 79 84 AUA III 66 63 85 75 80 81 69 83 55 82 53 54 67 86 43 62 65 87 44 64 70 88 52 49b 74 51 89 60 71 50 42 49 48 61 0 90 46 47 72 73 91 41 26a 93 45 95 92 94 26 40 25a 96 3 98 39 36 25 99 100 37 2 97 VI 2 32 27 28 1 7 4 101 5 5S II 31 29 3 1 35a 33 6 8 38 24 23 5 9 35 4 10 34 13 I 22 14 21 11 12 16 19

18 20

Extended Data Fig. 4 Secondary structure diagram of the plant 26S and 5S rRNA 2D representation of the 26S rRNA colored by domain. 5S rRNA is shown in dark green. The rRNA expansions specific to the plant mitoribosome are highlighted in cyan. Extensions that could not be modelled are indicated by dashed lines. Simplified secondary structure diagram of the E. coli 23S rRNA is also shown in the black frame, helices not present in the plant mitoribosome are shown in gray. Secondary structure templates were obtained from the RiboVision suite (http://apollo.chemistry.gatech.edu/RiboVision) bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

mL60

mL59/64

bL33m

uL29m

uL25m uL27m mL46 uL31m mL40 uL5m

Extended Data Fig. 5 The central protuberance of the plant mitoribosome contains a 5S rRNA Atomic model of the central protuberance of the plant mitoribosome. Protein components are each indicated in different colors and the 5S rRNA is shown in blue. The plant mitoribosome, contrary to yeast, mammalian and trypanosoma mitoribosome has a 5S rRNA in its CP, like in bacteria. However, the mitochondria specific proteins mL40, mL46, mL59/64 and mL60 are present, augmenting the overall volume of the CP. Moreover, this indicates that the loss of the 5S in yeast, mammals and trypanosoma occurred after the acquisition of these proteins, which seem to constitute core components of the ancestral mitoribosome. bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a b

rPPR* (9-10 mS80 - rPPR6 (12 repeats) repeats) ES-h39 c d

rPPR* rPPR* ES-h44 ES-h6 (8 repeats) (10 repeats)

e f

Tyr72 Tyr397 Tyr398 Ile380 Leu99 Phe400

Leu75 Tyr368

His96 Tyr112

mL101 - rPPR4 (12 repeats) mL104 - rPPR9 (12 repeats) g h

mL102 - rPPR5 (19 repeats)

mS83 - rPPR10 (6 repeats)

Extended Data Fig. 6 rPPR proteins in their respective densities a-h All rPPR present in the model in their respective filtered densities. Assignment of rPPRs was made mainly thanks to their numbers of repeats that constitute reliable “finger prints”. rPPR 4, 6 and 9 share the same number of repeats, in which case their assignment was possible thanks to the analysis of their bulky side-chains. a, c and d are designated rPPR* as the local resolution could not allow to distinguished them, even based on the number of repeats as rPPR1 , 3a and 3b all are predicted to have 10 repeats. e-f Detailed with of selected side-chain in their densities that allowed unambiguous assignment. g mS83, even though resolved at low resolution, it was identified based on its unique number of repeats (6), which correlates with the predicted number of repeats from the TPRpred software23. bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a c

additional domain Bacterial uL2

Plant uS4m Bacterial uS4

Plant additional domain uL2m

C-ter nucleus encoded

density of the N-ter mitochondria non-modeled encoded 250aa

Plant uS3m Bacterial uS3

Extended Data Fig. 7 Specificities of plant mitoribosome proteins a Compared view of uS4m and bacterial uS4. The N-terminal part is shown in yellow and the C-terminal part in blue. The additional domain of uS4m is shown in red. This additional domain makes a part of the SSU body protuberance. b Similar to uS4m, uS3m also present a large additional domain. Due to low resolution in this area only part of the insertion was modelized, however there is no doubt that the 250 amino-acids missing would constitute the large density observed on the head of the SSU, shown here in brown. N-terminal parts of the proteins are shown in yellow and C-terminal parts in blue. The additional domain of uS3m is shown in red. c In plant mitochondria, the uL2m protein was already speculated to be composed of two parts24. The structure confirmed this hypothesis. The N-terminal part of the protein (red) is encoded by a mitochondrial gene and the C- terminal part of the protein (yellow) is encoded by a nuclear gene. bioRxiv preprint doi: https://doi.org/10.1101/777342; this version posted September 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. a b 5' UTR -30 -23 -19 0 10 consensus atp4 atp6 atp9 ccmC ccmFC ccmFN1 ccmFN2 cox1 cox2 cox3 nad3 nad9 mttB rpl2 rpl5 rps3 rps12

Extended Data Fig. 8 5’UTR of the mitochondrial mRNAs a Alignment of sequences surrounding the initiation codons of the 17, out the 33, protein coding genes encoded in the Arabidopsis mitochondrial genome. A characteristic AxAAA consensus is observed and illustrated by the WebLogo (https://weblogo.berkeley.edu/logo.cgi) representation in b.