MRS2 Gene Research Articles

Molecular cloning and functional characterization of mitochondrial RNA splicing 2 in fish Megalobrama amblycephala, and its potential roles in magnesium homeostasis and mitochondrial function

Mitochondrial function can be regulated by ion channels. Mitochondrial RNA splicing 2 (Mrs2) is a magnesium ion (Mg2+) channel located in the inner mitochondrial membrane, thereby mediating the Mg2+ influx into the mitochondrial matrix. However, its potential role in regulating the Mg homeostasis and mitochondrial function in aquatic species is still unclear. This study molecularly characterizes the gene encoding Mrs2 in fish M. amblycephala with its functions in maintaining the Mg homeostasis and mitochondrial function verified. The mrs2 gene is 2133 bp long incorporating a 1269 bp open reading frame, which encodes 422 amino acids. The Mrs2 protein includes two transmembrane domains and a conserved tripeptide Gly-Met-Asn, and has a high homology (65.92–97.64%) with those of most vertebrates. The transcript of mrs2 was relatively high in the white muscle, liver and kidney. The inhibition of mrs2 reduces the expressions of Mg2+ influx/efflux-related proteins, mitochondrial Mg content, and the activities of mitochondrial complex I and V in hepatocytes. However, the over-expression of mrs2 increases the expressions of Mg2+ influx/efflux-related proteins, mitochondrial Mg content, and the complex V activity, but decreases the activities of mitochondrial complex III and IV and citrate synthase in hepatocytes. Collectively, Mrs2 is highly conserved among different species, and is prerequisite for maintaining Mg homeostasis and mitochondrial function in fish.

Downregulation of aspartoacylase during the progression of myelin breakdown in the dmy mutant rat with mitochondrial magnesium channel MRS2 defect

ObjectiveThe dmy rat is an autosomal recessive mutant that exhibits severe rapid myelin breakdown throughout the central nervous system at 7–8 weeks of age. The dmy rat has a point mutation in Mrs2 gene, which encodes an essential component of the major electrophoretic Mg2+ influx system in the mitochondria. However, it remains unknown how mitochondrial dysfunction leads to the myelin breakdown. MethodsWe focused on the aspartoacylase (ASPA) and mitochondrion-related metabolites to clarify the mechanism of myelin pathology in dmy rats. Aspa mRNA was significantly decreased in both the gray matter and the ventral white matter of spinal cord in the dmy rats from 4 to 8 weeks of age. Very faint immunohistochemical expression for ASPA was noted in the gray and white matter of the affected dmy rats at 8 weeks. Liquid chromatography mass spectrometry revealed no different amount of N-acetylaspartate (NAA), which is synthesized from aspartate and acetyl-coenzyme A (CoA) in neurons, in the brain and spinal cord between the dmy and control rats. ConclusionOur results indicated that the pyruvate dehydrogenase activity might be reduced due to the loss of Mg2+ transport activity in the mitochondria of the dmy rats, suggesting acetyl CoA production might be reduced. The number of oligodendrocytes was well preserved until 7 weeks. It is intriguing that prior to the myelin destruction at 7–8 weeks, disrupted expression of Aspa mRNA and ASPA protein undergoes from early stage of myelinogenesis. These data indicate that ASPA expression would be a useful index to evaluate a function of oligodendrocyte in the dmy rat.

Critical Issues in the Study of Magnesium Transport Systems and Magnesium Deficiency Symptoms in Plants.

Magnesium (Mg) is the second most abundant cation in living cells. Over 300 enzymes are known to be Mg-dependent, and changes in the Mg concentration significantly affects the membrane potential. As Mg becomes deficient, starch accumulation and chlorosis, bridged by the generation of reactive oxygen species, are commonly found in Mg-deficient young mature leaves. These defects further cause the inhibition of photosynthesis and finally decrease the biomass. Recently, transcriptome analysis has indicated the transcriptinal downregulation of chlorophyll apparatus at the earlier stages of Mg deficiency, and also the potential involvement of complicated networks relating to hormonal signaling and circadian oscillation. However, the processes of the common symptoms as well as the networks between Mg deficiency and signaling are not yet fully understood. Here, for the purpose of defining the missing pieces, several problems are considered and explained by providing an introduction to recent reports on physiological and transcriptional responses to Mg deficiency. In addition, it has long been unclear whether the Mg deficiency response involves the modulation of Mg2+ transport system. In this review, the current status of research on Mg2+ transport and the relating transporters are also summarized. Especially, the rapid progress in physiological characterization of the plant MRS2 gene family as well as the fundamental investigation about the molecular mechanism of the action of bacterial CorA proteins are described.

Exome sequencing in familial corticobasal degeneration

BackgroundCorticobasal degeneration (CBD) is a neurodegenerative, sporadic disorder of unknown cause. Few familial cases have been described. ObjectiveWe aim to characterize the clinical, imaging, pathological and genetic features of two familial cases of CBD. MethodsWe describe two first cousins with CBD associated with atypical MRI findings. We performed exome sequencing in both subjects and in an unaffected first cousin of similar age. ResultsThe cases include a 79-year-old woman and a 72-year-old man of Native American and British origin. The onset of the neurological manifestations was 74 and 68 years respectively. Both patients presented with a combination of asymmetric parkinsonism, apraxia, myoclonic tremor, cortical sensory syndrome, and gait disturbance. The female subject developed left side fixed dystonia. The manifestations were unresponsive to high doses of levodopa in both cases. Extensive bilateral T1-W hyperintensities and T2-W hypointensities in basal ganglia and thalamus were observed in the female patient; whereas these findings were more subtle in the male subject. Postmortem examination of both patients was consistent with corticobasal degeneration; the female patient had additional findings consistent with mild Alzheimer's disease. No Lewy bodies were found in either case. Exome sequencing showed mutations leading to possible structural changes in MRS2 and ZHX2 genes, which appear to have the same upstream regulator miR-4277. ConclusionsCorticobasal degeneration can have a familial presentation; the role of MRS2 and ZHX2 gene products in CBD should be further investigated.

Analysis of in vivo binding of yeast heat shock factor to promoter DNA

The in vivo binding of yeast heat shock factor (HSF) to promoter domains of stress-inducible genes was examined using chromatin immunoprecipitation (ChIP) combined with PCR. The promoter-specfic PCR amplification of DNA immunoprecipitated with antibodies against HSF showed that yeast HSF was bound constitutively to the promoters of GTT2, YLL058W, COQ6, GND2 and YGR066C genes, induciblely to the MRS2 gene. HSF-binding consensus sites prediction indicated the promoter sequences of the stress-inducible genes contain heat shock elements (HSEs). These results suggest the genes are potential HSF-regulated targets.

Mitochondrial Mg(2+) homeostasis is critical for group II intron splicing in vivo.

The product of the nuclear MRS2 gene, Mrs2p, is the only candidate splicing factor essential for all group II introns in mitochondria of the yeast Saccharomyces cerevisiae. It has been shown to be an integral protein of the inner mitochondrial membrane, structurally and functionally related to the bacterial CorA Mg(2+) transporter. Here we show that mutant alleles of the MRS2 gene as well as overexpression of this gene both increase intramitochondrial Mg(2+) concentrations and compensate for splicing defects of group II introns in mit(-) mutants M1301 and B-loop. Yet, covariation of Mg(2+) concentrations and splicing is similarly seen when some other genes affecting mitochondrial Mg(2+) concentrations are overexpressed in an mrs2Delta mutant, indicating that not the Mrs2 protein per se but certain Mg(2+) concentrations are essential for group II intron splicing. This critical role of Mg(2+) concentrations for splicing is further documented by our observation that pre-mRNAs, accumulated in mitochondria isolated from mutants, efficiently undergo splicing in organello when these mitochondria are incubated in the presence of 10 mM external Mg(2+) (mit(-) M1301) and an ionophore (mrs2Delta). This finding of an exceptional sensitivity of group II intron splicing toward Mg(2+) concentrations in vivo is unprecedented and raises the question of the role of Mg(2+) in other RNA-catalyzed reactions in vivo. It explains finally why protein factors modulating Mg(2+) homeostasis had been identified in genetic screens for bona fide RNA splicing factors.

The Human Mitochondrial Mrs2 Protein Functionally Substitutes for Its Yeast Homologue, A Candidate Magnesium Transporter

We report here on the human MRS2 gene that encodes a protein, hsaMrs2p, the first molecularly characterized candidate for a magnesium transporter in metazoa. The protein, like the yeast mitochondrial Mrs2 and Lpe10 proteins, contains two predicted transmembrane domains in its carboxyl-terminus, the first of which terminates with the conserved motif F/Y-G-M-N. These are typical features of the CorA family of magnesium transporters. Expression of hsaMrs2p in mrs2-1 knock-out mutant yeast partly restores mitochondrial magnesium concentrations that are significantly reduced in this mutant. It also alleviates other defects of this mutant, which may be secondary to the reduction in magnesium concentrations. These findings suggest that hsaMrs2p and yMrs2p are functional homologues. Like its yeast homologues, hsaMrs2p has been localized in mitochondria. The hsaMRS2 gene is located on chromosome 6 (6p22.1–p22.3) and is composed of 11 exons. A low level of the transcript is detected in various mouse tissues.

The Bacterial Magnesium Transporter CorA Can Functionally Substitute for Its Putative Homologue Mrs2p in the Yeast Inner Mitochondrial Membrane

The yeast nuclear gene MRS2 encodes a protein of 54 kDa, the presence of which has been shown to be essential for the splicing of group II intron RNA in mitochondria and, independently, for the maintenance of a functional respiratory system. Here we show that the MRS2 gene product (Mrs2p) is an integral protein of the inner mitochondrial membrane. It appears to be inserted into this membrane by virtue of two neighboring membrane spanning domains in its carboxyl-terminal half. A large amino-terminal and a shorter carboxyl-terminal part are likely to be exposed to the matrix space. Structural features and a short sequence motif indicate that Mrs2p may be related to the bacterial CorA Mg2+ transporter. In fact, overexpression of the CorA gene in yeast partially suppresses the pet- phenotype of an mrs2 disrupted yeast strain. Disruption of the MRS2 gene leads to a significant decrease in total magnesium content of mitochondria which is compensated for by the overexpression of the CorA gene. Mutants lacking or overproducing Mrs2p exhibit phenotypes consistent with the involvement of Mrs2p in mitochondrial Mg2+ homeostasis.

Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the catalytic core of a mitochondrial group II intron

Previous studies show that some yeast strains carrying point mutations of domain 5 that block splicing of a mitochondrial group II intron yield spontaneous revertants in which splicing is partially restored by dominant mutations of nuclear genes. Here we cloned and sequenced the suppressor allele of one such gene, and found it to be a missense mutation of the MRS2 gene (MRS2-L232F). The MRS2 gene was first implicated in group II intron splicing by the finding that overexpression of the wild-type gene weakly suppresses the splicing defect of a mutation of another intron. Tetrad analysis showed that independently isolated suppressors of two other domain 5 mutations are also allelles of the MRS2 gene and DNA sequencing identified a new missense mutation in each strain (MRS2-T230I and MRS2-L213M). All three suppressor mutations cause a temperature-sensitive respiration defect that is dominant negative in heterozygous diploids, but those strains splice the mutant intron at the elevated temperature. The three mutations are in a domain of the protein that is likely to be a helix-turn-helix region, so that effects of the mutations on protein-protein interactions may contribute to these phenotypes. These mutations suppress the splicing defect of many, but not all, of the available splicing defective mutations of aI5γ, including mutations of several intron domains. Protein and RNA blot experiments show that the level of the protein encoded by the MRS2 gene, but not the mRNA, is elevated by these mutations. Interestingly, overexpression of the wild-type protein restores much lower levels of splicing than were obtained with similar elevated levels of the mutated Mrs2 proteins. The splicing phenotypes of these strains suggest a direct role for Mrs2 protein on group II intron splicing, but an indirect effect is not yet ruled out.

Overexpression of a novel member of the mitochondrial carrier family rescues defects in both DNA and RNA metabolism in yeast mitochondria.

The PIF1 and MRS2 gene products have previously been shown to be essential for mitochondrial DNA maintenance at elevated temperatures and mitochondrial group II intron splicing, respectively, in the yeast Saccharomyces cerevisiae. A multicopy suppressor capable of rescuing the respiratory deficient phenotype associated with null alleles of either gene has been isolated. This suppressor is a nuclear gene that was called RIM2/MRS12. The RIM2/MRS12 gene encodes a predicted protein of 377 amino acids that is essential for mitochondrial DNA metabolism and proper cell growth. Inactivation of this gene causes the total loss of mitochondrial DNA and, compared to wild-type rhoo controls, a slow-growth phenotype on media containing glucose. Analysis of the RIM2/MRS12 protein sequence suggests that RIM2/MRS12 encodes a novel member of the mitochondrial carrier family. In particular, a typical triplicate structure, where each repeat consists of two putative transmembrane segments separated by a hydrophilic loop, can be deduced from amino acid sequence comparisons and the hydropathy profile of RIM2/MRS12. Antibodies directed against the aminoterminus of RIM2/MRS12 detect this protein in mitochondria. The function of the RIM2/MRS12 protein and the substrates it might transport are discussed.

A multitude of suppressors of group II intron-splicing defects in yeast.

Disruption of the nuclear MRS2 gene (mrs2-1 mutation) causes a strong pet- phenotype in strains with mitochondrial group II introns, and a leaky pet- phenotype in strains without group II introns. MRS3 and MRS4, the genes for two mitochondrial-solute carrier proteins, can suppress both phenotypes when present in high-copy-number plasmids. In order to search for further multicopy suppressors of the mrs2-1 mutant phenotype, an yeast genomic DNA library, MW90, was constructed in YEp351 from a strain deleted for the MRS2, MRS3 and MRS4 genes. Ten different Sau3A DNA fragments that act as multicopy suppressors of the mrs2-1 respiratory-deficient phenotype were isolated from this library. Some of the newly isolated genes suppress the pet- phenotypes of mrs2-1 cells in strains with and without mitochondrial group II introns. Other genes, however, are suppressors only for the mitochondrial intron-less strains. This supports the notion that the MRS2 gene product is bifunctional i.e., it is essential for the splicing of group II introns and is also involved in processes of mitochondrial biogenesis other than RNA splicing.

The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.