29 research outputs found
Characterization of chondrogenic progenitor cells using mass spectrometry and multidimensional separation approaches
Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes
The mammalian respiratory chain complexes I, III 2, and IV (CI, CIII 2, and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI-CIII 2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and the regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knockin mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in mice but raise questions about their alternate functions, such as those relating to the regulation of protein stability and prevention of age-associated protein aggregation
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C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis.
In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain-an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process
A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps
BACKGROUND: The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs. RESULTS: Here, we present a simple, flexible and efficient PCR-fusion/Gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps. Results from selected applications of the procedure which include ORF fusion, introduction of Cys>Ser mutations, insertion of StrepII tag sequence and domain swaps for Arabidopsis secondary cell wall AtCesA genes are demonstrated. CONCLUSION: The PCR-fusion/Gateway cloning procedure described provides an elegant, simple and efficient solution for a wide range of diverse and complicated cloning tasks. Through streamlined cloning of sets of gene fusions and modification variants into binary vectors for systematic functional studies of gene families, our method allows for efficient utilization of the growing sequence and expression data
Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1
The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1. The glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting enzyme in the hexosamine pathway producing uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), an essential glycosylation precursor. Here, the authors dissect the mechanisms of GFAT-1 regulation by protein kinase A (PKA)-mediated phosphorylation
IMPACT ON MITOCHONDRIAL FUNCTION BY INCREASED LEVELS OF MITOCHONDRIAL RNA POLYMERASE
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Genetic diversity and relationships of indigenous and newly bred Bulgarian grape cultivars assessed by nuclear and chloroplast markers
Aim: Assessment of the genetic diversity and relationships in a group of 31 Bulgarian grape accessions through microsatellite markers.
Methods and results: Thirty-one accessions, including 20 old rare local and 11 newly bred varieties were characterized through 13 nuclear and 5 chloroplast microsatellite loci. The genetic diversity (0.81±0.01) obtained for the investigated group of cultivars was comparable to those reported for other grapevine germplasms. The low PI value (1.0x10-16) allowed proper genetic identification and determination of synonyms. Microsatellite analysis of the 31 accessions resulted in 26 unique genotypes and 2 groups of synonyms. Four cases of supposed synonymy with local Bulgarian and foreign cultivars were rejected. Three chlorotypes, B, C and D, were defined among the studied cultivars, with a prevalence of chlorotype C (62%).
Conclusion: The high genetic diversity found in the set of old rare grapevines demonstrated their importance as a rich source of alleles for breeding. The pattern of chlorotype distribution observed among local varieties confirmed the previous results and supports the hypothesis of an Eastern origin of local Bulgarian cultivars.
Significance and impact of the study: The obtained results provide an important support for the preservation of grape biodiversity in Bulgaria as well as for the clarification of genetic relationships between local and foreign cultivars
NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin
Mitochondria are multidimensional organelles whose activities are essential to cellular vitality and organismal longevity, yet underlying regulatory mechanisms spanning these different levels of organization remain elusive(1-5). Here we show that Caenorhabditis elegans nuclear transcription factor Y, beta subunit (NFYB-1), a subunit of the NF-Y transcriptional complex(6-8), is a crucial regulator of mitochondrial function. Identified in RNA interference (RNAi) screens, NFYB-1 loss leads to perturbed mitochondrial gene expression, reduced oxygen consumption, mitochondrial fragmentation, disruption of mitochondrial stress pathways, decreased mitochondrial cardiolipin levels and abolition of organismal longevity triggered by mitochondrial impairment. Multi-omics analysis reveals that NFYB-1 is a potent repressor of lysosomal prosaposin, a regulator of glycosphingolipid metabolism. Limiting prosaposin expression unexpectedly restores cardiolipin production, mitochondrial function and longevity in the nfyb-1 background. Similarly, cardiolipin supplementation rescues nfyb-1 phenotypes. These findings suggest that the NFYB-1-prosaposin axis coordinates lysosomal to mitochondria signalling via lipid pools to enhance cellular mitochondrial function and organismal health