Mitochondrial RNA processing defect caused by a SUPV3L1 mutation in two siblings with a novel neurodegenerative syndrome.

SUPV3L1 encodes a helicase that is mainly localized in the mitochondria. It has been shown in vitro to possess both double-stranded RNA and DNA unwinding activity that is ATP-dependent. Here we report the first two patients for this gene who presented with a homozygous preliminary stop codon resulting in a C-terminal truncation of the SUPV3L1 protein. They presented with a characteristic phenotype of neurodegenerative nature with progressive spastic paraparesis, growth restriction, hypopigmentation, and predisposition to autoimmune disease. Ophthalmological examination showed severe photophobia with corneal erosions, optic atrophy, and pigmentary retinopathy, while neuroimaging showed atrophy of the optic chiasm and the pons with calcification of putamina, with intermittent and mild elevation of lactate. We show that the amino acids that are eliminated by the preliminary stop codon are highly conserved and are predicted to form an amphipathic helix. To investigate if the mutation causes mitochondrial dysfunction, we examined fibroblasts of the proband. We observed very low expression of the truncated protein, a reduction in the mature ND6 mRNA species as well as the accumulation of double-stranded RNA. Lentiviral complementation with the full-length SUPV3L1 cDNA partly restored the observed RNA phenotypes, supporting that the SUPV3L1 mutation in these patients is pathogenic and the cause of the disease.

Mitochondrial RNA granules are critically dependent on mtDNA replication factors Twinkle and mtSSB.

Newly synthesized mitochondrial RNA is concentrated in structures juxtaposed to nucleoids, called RNA granules, that have been implicated in mitochondrial RNA processing and ribosome biogenesis. Here we show that two classical mtDNA replication factors, the mtDNA helicase Twinkle and single-stranded DNA-binding protein mtSSB, contribute to RNA metabolism in mitochondria and to RNA granule biology. Twinkle colocalizes with both mitochondrial RNA granules and nucleoids, and it can serve as bait to greatly enrich established RNA granule proteins, such as G-rich sequence factor 1, GRSF1. Likewise, mtSSB also is not restricted to the nucleoids, and repression of either mtSSB or Twinkle alters mtRNA metabolism. Short-term Twinkle depletion greatly diminishes RNA granules but does not inhibit RNA synthesis or processing. Either mtSSB or GRSF1 depletion results in RNA processing defects, accumulation of mtRNA breakdown products as well as increased levels of dsRNA and RNA:DNA hybrids. In particular, the processing and degradation defects become more pronounced with both proteins depleted. These findings suggest that Twinkle is essential for RNA organization in granules, and that mtSSB is involved in the recently proposed GRSF1-mtRNA degradosome pathway, a route suggested to be particularly aimed at degradation of G-quadruplex prone long non-coding mtRNAs.

RNA Crosslinking to Analyze the Mitochondrial RNA-Binding Proteome.

Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly established. Shotgun proteomics has been used successfully to define whole-cell polyadenylated RNA (poly(A)-RNA) interacting proteomes using the nucleotide analogue 4-thiouridine (4SU) combined with UV crosslinking, poly(A)-RNA isolation and mass spectrometry to identify all poly(A)-RNA bound proteins. Although in this case also a considerable number of mitochondrial proteins were identified, the method was not specifically directed at the mitochondrial poly(A)-RNA bound proteome. Here we describe a method for enrichment of the mitochondrial poly(A)-RNA bound proteome based on 4SU labeling and UV crosslinking. The method can be applied either for isolated mitochondria prior to UV crosslinking or for whole-cell crosslinking followed by mitochondrial isolation.

A Prioritized and Validated Resource of Mitochondrial Proteins in Plasmodium Identifies Unique Biology.

Plasmodium species have a single mitochondrion that is essential for their survival and has been successfully targeted by antimalarial drugs. Most mitochondrial proteins are imported into this organelle, and our picture of the Plasmodium mitochondrial proteome remains incomplete. Many data sources contain information about mitochondrial localization, including proteome and gene expression profiles, orthology to mitochondrial proteins from other species, coevolutionary relationships, and amino acid sequences, each with different coverage and reliability. To obtain a comprehensive, prioritized list of Plasmodium falciparum mitochondrial proteins, we rigorously analyzed and integrated eight data sets using Bayesian statistics into a predictive score per protein for mitochondrial localization. At a corrected false discovery rate of 25%, we identified 445 proteins with a sensitivity of 87% and a specificity of 97%. They include proteins that have not been identified as mitochondrial in other eukaryotes but have characterized homologs in bacteria that are involved in metabolism or translation. Mitochondrial localization of seven Plasmodium berghei orthologs was confirmed by epitope labeling and colocalization with a mitochondrial marker protein. One of these belongs to a newly identified apicomplexan mitochondrial protein family that in P. falciparum has four members. With the experimentally validated mitochondrial proteins and the complete ranked P. falciparum proteome, which we have named PlasmoMitoCarta, we present a resource to study unique proteins of Plasmodium mitochondria. IMPORTANCE The unique biology and medical relevance of the mitochondrion of the malaria parasite Plasmodium falciparum have made it the subject of many studies. However, we actually do not have a comprehensive assessment of which proteins reside in this organelle. Many omics data are available that are predictive of mitochondrial localization, such as proteomics data and expression data. Individual data sets are, however, rarely complete and can provide conflicting evidence. We integrated a wide variety of available omics data in a manner that exploits the relative strengths of the data sets. Our analysis gave a predictive score for the mitochondrial localization to each nuclear encoded P. falciparum protein and identified 445 likely mitochondrial proteins. We experimentally validated the mitochondrial localization of seven of the new mitochondrial proteins, confirming the quality of the complete list. These include proteins that have not been observed mitochondria before, adding unique mitochondrial functions to P. falciparum.

A Combined Mass Spectrometry and Data Integration Approach to Predict the Mitochondrial Poly(A) RNA Interacting Proteome.

In order to synthesize the 13 oxidative phosphorylation proteins encoded by mammalian mtDNA, a large assortment of nuclear encoded proteins is required. These include mitoribosomal proteins and various RNA processing, modification and degradation enzymes. RNA crosslinking has been successfully applied to identify whole-cell poly(A) RNA-binding proteomes, but this method has not been adapted to identify mitochondrial poly(A) RNA-binding proteomes. Here we developed and compared two related methods that specifically enrich for mitochondrial poly(A) RNA-binding proteins and analyzed bound proteins using mass spectrometry. To obtain a catalog of the mitochondrial poly(A) RNA interacting proteome, we used Bayesian data integration to combine these two mitochondrial-enriched datasets as well as published whole-cell datasets of RNA-binding proteins with various online resources, such as mitochondrial localization from MitoCarta 2.0 and co-expression analyses. Our integrated analyses ranked the complete human proteome for the likelihood of mtRNA interaction. We show that at a specific, inclusive cut-off of the corrected false discovery rate (cFDR) of 69%, we improve the number of predicted proteins from 185 to 211 with our mass spectrometry data as input for the prediction instead of the published whole-cell datasets. The chosen cut-off determines the cFDR: the less proteins included, the lower the cFDR will be. For the top 100 proteins, inclusion of our data instead of the published whole-cell datasets improve the cFDR from 54% to 31%. We show that the mass spectrometry method most specific for mitochondrial RNA-binding proteins involves ex vivo 4-thiouridine labeling followed by mitochondrial isolation with subsequent in organello UV-crosslinking.