Mitochondrial RNA Helicase Research Articles

A New Case of Mitochondrial RNA Helicase SUPV3L1-Associated Neurodegenerative Disease: Ataxia, Spasticity, Optic Atrophy, and Skin Hypopigmentation (ASOASH).

The SUPV3L1 gene encodes ATP-dependent RNA helicase SUPV3L1, which is a part of the mitochondrial degradosome complex or SUV3. SUPV3L1 unwinds secondary structures of mitochondrial RNA (mtRNA) and facilitates the degradation of mtRNA molecules. A nonsense homozygous variant in the SUPV3L1 gene was recently associated with mitochondrial disease. Our study presents the second documented case of SUPV3L1 pathology in humans. Whole-genome sequencing was performed on the NovaSeq 6000 platform using pair-end reading. Data analysis was performed with an in-house developed pipeline. The 17-year-old female patient exhibited a diverse array of symptoms, including ataxia, spastic paraparesis, cognitive deficit, optic atrophy, and horizontal gaze-evoked nystagmus. Early onset of symptoms, such as ataxic gait and nystagmus, was noted, with subsequent progression of neurological manifestations. At the time of the observation, the proband had extensive regions of hypopigmented skin patches on the body and extremities, which have progressed over time. Whole-genome sequencing revealed compound heterozygous variants in the SUPV3L1 gene: c.272-2A>G and c.1924A>C; p.(Ser642Arg). RNA analysis demonstrated splicing changes attributable to the c.272-2A>G variant. ELISA assay showed increased Complex I content in the patient's fibroblasts. This case underscores the phenotypic diversity associated with SUPV3L1 mutations, emphasizing the importance of considering mitochondrial RNA helicase dysfunction in the differential diagnosis of neurodegenerative disorders. Further elucidation of the molecular mechanisms underlying SUPV3L1-associated pathology may provide valuable insights into targeted therapeutic interventions.

Mitochondrial double-stranded RNA triggers antiviral signalling in humans

Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures1,2. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.

Conditional control of gene function by an invertible gene trap in zebrafish

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.

Uncoupling the Roles of the SUV3 Helicase in Maintenance of Mitochondrial Genome Stability and RNA Degradation

Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ΔCC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ΔCC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.

Widespread expression of the Supv3L1 mitochondrial RNA helicase in the mouse

Supv3L1 is an evolutionarily conserved helicase that plays a critical role in the mitochondrial RNA surveillance and degradation machinery. Conditional ablation of Supv3L1 in adult mice leads to premature aging phenotypes including loss of muscle mass and adipose tissue and severe skin abnormalities. To get insights into the spatial and temporal expression of Supv3L1 in the mouse, we generated knock-in and transgenic strains in which an EGFP reporter was placed under control of the Supv3L1 native promoter. During development, expression of Supv3L1 begins at the blastocyst stage, becomes widespread and strong in all fetal tissues and cell types, and continues during postnatal growth. In mature animals reporter expression is only slightly diminished in most tissues and continues to be highly expressed in the brain, peripheral sensory organs, and testis. Together, these data confirm that Supv3L1 is an important developmentally regulated gene, which continues to be expressed in all mature tissues, particularly the rapidly proliferating cells of testes, but also in the brain and sensory organs. The transgenic mice and cell lines derived from them constitute a valuable tool for the examination of the spatial and temporal aspects of Supv3L1 promoter activity, and should facilitate future screens for small molecules that regulate Supv3L1 expression.

Two DEAD-Box Proteins May Be Part of RNA-Dependent High-Molecular-Mass Protein Complexes in Arabidopsis Mitochondria

Posttranscriptional processes are important for regulation of gene expression in plant mitochondria. DEAD-box proteins, which form a huge protein family with members from all kingdoms, are fundamental components in virtually all types of processes in RNA metabolism. Two members of this protein family, designated PMH1 and PMH2 (for PUTATIVE MITOCHONDRIAL RNA HELICASE), were analyzed and characterized in mitochondria of Arabidopsis (Arabidopsis thaliana). Green fluorescent protein tagging with N-terminal PMH1 and PMH2 sequences supports the mitochondrial localization of these proteins. Northern experiments, as well as histochemical beta-glucuronidase staining of transgenic plants carrying respective promoter:beta-glucuronidase fusion constructs, revealed differing transcription patterns for the two genes. In response to cold, however, transcript levels of both genes increased. Immunodetection analyses of mitochondrial protein complexes after two-dimensional blue native/urea SDS-PAGE and after fractionation on sucrose gradients strongly suggest that one or both proteins are part of RNA-dependent complexes. Cold treatment of cell cultures or solubilization of mitochondria in the presence of MgCl(2) favored the detection of high-molecular-mass complexes. This study paves the way for detailed analysis of high-molecular-mass complexes in mitochondria of higher plants.

Purified human SUV3p exhibits multiple-substrate unwinding activity upon conformational change.

Suv3 of Saccharomyces cerevisiae has been classified as a mitochondrial RNA helicase. However, the helicase domain in both yeast and human SUV3 varies considerably from the typical RNA helicase motifs. To investigate its enzymatic activities, a homogeneously purified preparation of SUV3 is required. Expression of a processed form of human SUV3 carrying an N-terminal deletion of 46 amino acids (SUV3DeltaN46) in a yeast suv3 null mutant, which otherwise fails to grow in a nonfermentable carbon source and forms petite colonies in glucose medium, rescues the null phenotype. Through a five-step chromatographic procedure, an 83 kDa SUV3DeltaN46 protein (SUV3-83) and a partially degraded 70 kDa product (SUV3-70) containing amino acids 68-685 were purified to homogeneity. Single- or double-stranded DNA and RNA stimulated ATPase activity of both proteins. SUV3-70, which retains core catalytic domains, can bind and unwind multiple duplex substrates of RNA and DNA with a 5'-3' directionality over a wide range of pH, while SUV3-83 has helicase activity at only acidic pH. ATP, but not nonhydrolyzable ATP, is essential for the unwinding activity, suggesting the requirement of the energy derived from ATP hydrolysis. Consistent with this notion, suv3 mutants containing alanine (A) or arginine (R) substitutions at the conserved lysine residue in the ATP binding site (K213) lost ATPase activity and also failed to unwind the substrates. Importantly, circular dichroism (CD) spectral analysis showed that SUV3-83, at pH 5.0, adopts a conformation similar to that of SUV3-70, suggesting a conformational change in SUV3-83 is required for its helicase activity. The physiological relevance of the multiple-substrate helicase activity of human SUV3 is discussed.

Seeking for ageing-associated gene expression in cerebral tissue of senescence-accelerated mouse (SAM)

In this study, the ageing-specific expression genes of the murine cerebrum were investigated by employing differential-display reverse transcription polymerase chain reaction (DDRT-PCR) in three senescence-accelerated mouse (SAM) strains: SAMP8/Ta, SAMP10/Ta and SAMR1TA. In the comparison of gene expression profile in different strain mice, 16 differential fragments have been detected, 3, 6 and 7 of them belong to SAMP10/Ta, SAMP8/Ta and SAMR1TA, respectively. Sequencing data indicated that those fragments are homologous with heat shock protein cognate protein 70, ATP-dependent mitochondrial RNA helicase, Dleu2 mRNA, Mouse DNA sequence from clone RP23-334C3 on chromosome X, ubiquinol-cytochrome c reductase complex (7.2 kDa), 60S ribosomal protein L21, FIS, phenylalkylamine Ca 2+ antagonist (emopamil) binding protein, fucosyltransferase 9, glial cell line derived neurotrophic factor family receptor alpha 1, endonuclease/reverse transcriptase, PER1 interacting protein of the suprachiamatic nucleus homolog, centrosomal protein CG-NAP, ferritin heavy chain gene, NID-2 gene, and prkdc gene for DNA-dependent protein kinase catalytic subunit.

A novel mitochondrial DEAD box protein (Mrh4) required for maintenance of mtDNA inSaccharomyces cerevisiae

In a screen of nuclear genes that assist splicing of mitochondrial localized group II introns in yeast we isolated low-copy number suppressors of splicing and respiratory-deficient point mutants of intron aI5γ, the last intron of the gene encoding cytochrome c oxidase subunit I. One of the genes found contains the open reading frame (ORF) YGL064c that has previously been proposed to encode a putative RNA helicase of the DEAD box family. Deletion of the ORF gives rise to 100% cytoplasmic petites, indicating that the protein plays an essential role in the mitochondrial RNA metabolism. Overexpression of YGL064c–GFP fusions clearly revealed a mitochondrial localization of the protein. The gene encodes the fourth putative RNA helicase of Saccharomyces cerevisiae implicated in a mitochondrial function and was therefore termed MRH4 (for mitochondrial RNA helicase).

A novel mitochondrial DEAD box protein (Mrh4) required for maintenance of mtDNA in Saccharomyces cerevisiae.

In a screen of nuclear genes that assist splicing of mitochondrial localized group II introns in yeast we isolated low-copy number suppressors of splicing and respiratory-deficient point mutants of intron aI5gamma, the last intron of the gene encoding cytochrome c oxidase subunit I. One of the genes found contains the open reading frame (ORF) YGL064c that has previously been proposed to encode a putative RNA helicase of the DEAD box family. Deletion of the ORF gives rise to 100% cytoplasmic petites, indicating that the protein plays an essential role in the mitochondrial RNA metabolism. Overexpression of YGL064c-GFP fusions clearly revealed a mitochondrial localization of the protein. The gene encodes the fourth putative RNA helicase of Saccharomyces cerevisiae implicated in a mitochondrial function and was therefore termed MRH4 (for mitochondrial RNA helicase).