BOLA3 and NFU1 link mitoribosome iron-sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome

The human mitochondrial ribosome contains three [2Fe-2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe-S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe-2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe-4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently, fibroblasts from subjects suffering from “multiple mitochondrial dysfunction” syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, one of the mitoribosomal [2Fe-2S] clusters and the [4Fe-4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly

Post-transcriptional events in the biosynthesis of mitochondrial ATP synthase.

Available from STL, Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

Mitochondrial RNA Granules Are Centers for Posttranscriptional RNA Processing and Ribosome Biogenesis

Cytoplasmic RNA granules play a central role in mRNA metabolism, but the importance of mitochondrial RNA granules remains relatively unexplored. We characterized their proteome and found that they contain a large toolbox of proteins dedicated to RNA metabolism. Investigation of four uncharacterized putative RNA-binding proteins—two RNA helicases, DHX30 and DDX28, and two proteins of the Fas-activated serine-threonine kinase (FASTKD) family, FASTKD2 and FASTKD5—demonstrated that both helicases and FASTKD2 are required for mitochondrial ribosome biogenesis. RNA-sequencing (RNA-seq) analysis showed that DDX28 and FASTKD2 bound the 16S rRNA. FASTKD5 is required for maturing precursor mRNAs that are not flanked by tRNAs and that therefore cannot be processed by the canonical mRNA maturation pathway. Silencing FASTKD5 rendered mature COX I mRNA almost undetectable, which severely reduced the synthesis of COX I, resulting in a complex IV assembly defect. These data demonstrate that mitochondrial RNA granules are centers for posttranscriptional RNA processing and the biogenesis of mitochondrial ribosomes

Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation

AbstractBiogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome

Stomatin-like protein 2 deficiency results in impaired mitochondrial translation

Mitochondria translate the RNAs for 13 core polypeptides of respiratory chain and ATPsynthase complexes that are essential for the assembly and function of these complexes.This process occurs in close proximity to the mitochondrial inner membrane. However, themechanisms and molecular machinery involved in mitochondrial translation are not fullyunderstood, and defects in this process can result in severe diseases. Stomatin-like protein(SLP)-2 is a mainly mitochondrial protein that forms cardiolipin- and prohibitin-enrichedmicrodomains in the mitochondrial inner membrane that are important for the formation ofrespiratory supercomplexes and their function. Given this regulatory role of SLP-2 in processesclosely associated with the mitochondrial inner membrane, we hypothesized that thefunction of SLP-2 would have an impact on mitochondrial translation. 35S-Methionine/cysteinepulse labeling of resting or activated T cells from T cell-specific Slp-2 knockout miceshowed a significant impairment in the production of several mitochondrial DNA-encodedpolypeptides following T cell activation, including Cytb, COXI, COXII, COXIII, and ATP6.Measurement of mitochondrial DNA stability and mitochondrial transcription revealed thatthis impairment was at the post-transcriptional level. [...

BOLA3 and NFU1 link mitoribosome iron–sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome

Abstract The human mitochondrial ribosome contains three [2Fe–2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe–S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe–2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe–4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently, fibroblasts from subjects suffering from ‘multiple mitochondrial dysfunction’ syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, one of the mitoribosomal [2Fe–2S] clusters and the [4Fe–4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly

BOLA3 and NFU1 Link Mitoribosome Iron–Sulfur Cluster Assembly to Multiple Mitochondrial Dysfunctions Syndrome

The human mitochondrial ribosome contains three [2Fe–2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the e xpression of Fe–S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe–2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe–4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently , fibroblasts from subjects suffering from ‘multiple mitochondrial dysfunction’ syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, w e report that, in addition to their structural role, one of the mitoribosomal [2Fe–2S] clusters and the [4Fe–4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly

Stomatin-like protein 2 deficiency results in impaired mitochondrial translation.

Mitochondria translate the RNAs for 13 core polypeptides of respiratory chain and ATP synthase complexes that are essential for the assembly and function of these complexes. This process occurs in close proximity to the mitochondrial inner membrane. However, the mechanisms and molecular machinery involved in mitochondrial translation are not fully understood, and defects in this process can result in severe diseases. Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein that forms cardiolipin- and prohibitin-enriched microdomains in the mitochondrial inner membrane that are important for the formation of respiratory supercomplexes and their function. Given this regulatory role of SLP-2 in processes closely associated with the mitochondrial inner membrane, we hypothesized that the function of SLP-2 would have an impact on mitochondrial translation. 35S-Methionine/cysteine pulse labeling of resting or activated T cells from T cell-specific Slp-2 knockout mice showed a significant impairment in the production of several mitochondrial DNA-encoded polypeptides following T cell activation, including Cytb, COXI, COXII, COXIII, and ATP6. Measurement of mitochondrial DNA stability and mitochondrial transcription revealed that this impairment was at the post-transcriptional level. Examination of mitochondrial ribosome assembly showed that SLP-2 migrated in sucrose-density gradients similarly to the large ribosomal subunit but that its deletion at the genetic level did not affect mitochondrial ribosome assembly. Functionally, the impairment in mitochondrial translation correlated with decreased interleukin-2 production in activated T cells. Altogether, these data show that SLP-2 acts as a general regulator of mitochondrial translation