Dataset Information

How to make a ribosome from mixed-up ribosomal RNA fragments in Chlamydomonas reinhardtii mitochondria

ABSTRACT: Mitochondria are essential components of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few, although essential, mitochondrial messenger RNAs still encoded by mitochondrial genomes. Here, we present the biochemical and structural characterization of the model green alga Chlamydomonas reinhardtii mitoribosome as well as the functional study of some of its Chlamydomonas specific components. We provide a single particle cryo-electron microscopy structure of Chlamydomonas mitoribosome and show how its core is composed by the assembly of 13 rRNA fragments encoded by separate non-contiguous gene pieces. While 5S rRNA was assumed to be absent from Chlamydomonas mitoribosome, a small rRNA representing a highly divergent 5S rRNA occurs in its central protuberance. In contrast, a gene piece that was believed to encode a fragment of rRNA encodes a small RNA that does not occur in the mitoribosome. Eleven novel proteins, mainly helical repeat proteins, including OPR, PPR and mTERF proteins occur in Chlamydomonas mitoribosome, revealing in particular the first structure of an OPR protein in complex with its RNA target. Reverse genetic studies reveal that the functions of some of the novel helical repeat proteins are required for mitoribosome biogenesis. The long studied p32 protein, whose function was related to several mitochondrial diseases in Human uniquely occurs as a constitutive ribosomal protein in the small subunit. Finally, cryo-electron tomography and biochemical studies show that Chlamydomonas mitoribosomes are exclusively attached to the mitochondrial inner membrane via two contact points mediated by Chlamydomonas specific novel proteins. One of them provides a direct interface between the exit of the peptide channel and the insertase Oxa1 for the insertion of nascent proteins in the mitochondrial inner membrane.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Chlamydomonas Reinhardtii

SUBMITTER: Lauriane Kuhn

LAB HEAD: Florent Waltz

PROVIDER: PXD024708 | Pride | 2022-03-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2020_S09_FWaltz_TSalinas_Chlamy_Pic1_500ng.mgf	Mgf
	2020_S09_FWaltz_TSalinas_Chlamy_Pic1_500ng.raw	Raw
	2020_S09_FWaltz_TSalinas_Chlamy_Pic2_500ng.mgf	Mgf
	2020_S09_FWaltz_TSalinas_Chlamy_Pic2_500ng.raw	Raw
	2020_S09_FWaltz_TSalinas_Chlamy_Pic3_500ng.mgf	Mgf

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Publications

How to build a ribosome from RNA fragments in Chlamydomonas mitochondria.

Waltz Florent F Salinas-Giegé Thalia T Englmeier Robert R Meichel Herrade H Soufari Heddy H Kuhn Lauriane L Pfeffer Stefan S Förster Friedrich F Engel Benjamin D BD Giegé Philippe P Drouard Laurence L Hashem Yaser Y

Nature communications 20211209 1

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 mi ...[more]

PMID: 34887394

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Project description:Mitochondrial ribosomes (mitoribosomes) have undergone substantial structural remodelling throughout evolution leading to distinct structural properties compared to their prokaryotic ancestors. Compared to their prokaryotic counterparts, mitoribosomes show a substantial loss of ribosomal RNA (rRNA), whilst acquiring unique mitochondrial proteins located on the periphery of the mitoribosome. This extended protein content suggests mitochondrial-specific functions of these proteins involved in mitochondrial mRNA selective-translation activation, ribosome assembly, translation regulation rather than exclusively structural scaffolding. We investigated the functional properties of the 14 unique (mitochondria-specific or supernumerary) mammalian mitoribosomal proteins (snMRP) in the small mitochondrial subunit (mtSSU) by CRISPR-mediated knockout and describe their specific effect on mitoribosome integrity and function. Unexpectedly, we show that each snMRP knockout leads to a unique pattern of mitoribosome instability with variable effects on mitochondrial protein synthesis as essential structural components and all – with the exception of mS37 (MRPS37, CHCHD1) – to almost complete loss of protein synthesis. Concomitantly, through steady-state proteomic analysis we confirm a modular assembly of the mtSSU. Surprisingly, mS37 was found to have impaired stability in all our tested mtSSU and large mitochondrial subunit (mtLSU) snMRPs knockouts as well as in patient-derived cell lines, suggesting an extended role of the protein in mtSSU pre-translation initiation steps. As mitochondrial intermembrane (IMS) targeted protein – a eukaryotic-confined localisation mechanism – with additional unique MIA40 (CHCHD4)-oxidisable motifs involved in the disulfide relay system, mS37 could have a specific role in mitochondrial translation regulation. Indeed, we find oxidation of the cysteines in the CX9C motif of mS37 to be essential for its stability, confirming mS37 regulating mitochondrial translation regulation.