Loss-of-function mutation in DDX53 associated with hereditary spastic paraplegia-like disorder.

DEAD-box helicase 53 (DDX53) is a member of the DEAD-box protein family of RNA helicases. Unlike other family members that are responsible for RNA metabolism, the biological function of DDX53 and its impact on the human condition are unclear. Herein, we found a full-length DDX53 deletion mutation in a hereditary spastic paraplegia-like (HSP-like) patient with lower extremity spasticity, walking disorder, visual impairment, and lateral ventricular white matter lesions. Bioinformatic analysis revealed that DDX53 was mainly expressed in the cerebellar cortex and may function as a tissue-specific RNA helicase. Transcriptome analysis showed that the expression of multiple brain-associated genes involved in synapse organization, neuron function, and neuromuscular junctions was affected by DDX53 depletion. Moreover, RNA immunoprecipitation sequencing (RIP-seq) analysis showed that DDX53 interacted with 176 genes, and 96 of these genes were associated with the execution of neurofunction, particularly in the regulation of cell projection organization and nervous system development. Collectively, although a more specified cell or animal model is required to fully understand the functional role of DDX53 in the human brain, we report for the first time that the patient with DDX53 defects exhibits HSP-like symptoms and that DDX53 is essential for maintaining neuronal function, with loss-of-function mutation in DDX53 potentially leading to HSP due to impaired RNA metabolism in the nervous system. KEY MESSAGES: DDX53 deficiency was first reported to be associated with HSP disorder. DDX53 exhibited minimal impact on mitochondrial function. DDX53 impaired RNA metabolism in the nervous system.

YY1 promotes pancreatic cancer cell proliferation by enhancing mitochondrial respiration.

KRAS-driven metabolic reprogramming is a known peculiarity features of pancreatic ductal adenocarcinoma (PDAC) cells. However, the metabolic roles of other oncogenic genes, such as YY1, in PDAC development are still unclear. In this study, we observed significantly elevated expression of YY1 in human PDAC tissues, which positively correlated with a poor disease progression. Furthermore, in vitro studies confirmed that YY1 deletion inhibited PDAC cell proliferation and tumorigenicity. Moreover, YY1 deletion led to impaired mitochondrial RNA expression, which further inhibited mitochondrial oxidative phosphorylation (OXPHOS) complex assembly and altered cellular nucleotide homeostasis. Mechanistically, the impairment of mitochondrial OXPHOS function reduced the generation of aspartate, an output of the tricarboxylic acid cycle (TCA), and resulted in the inhibition of cell proliferation owing to unavailability of aspartate-associated nucleotides. Conversely, exogenous supplementation with aspartate fully restored PDAC cell proliferation. Our findings suggest that YY1 promotes PDAC cell proliferation by enhancing mitochondrial respiration and the TCA, which favors aspartate-associated nucleotide synthesis. Thus, targeting nucleotide biosynthesis is a promising strategy for PDAC treatment.

SERAC1 is a component of the mitochondrial serine transporter complex required for the maintenance of mitochondrial DNA.

SERAC1 deficiency is associated with the mitochondrial 3-methylglutaconic aciduria with deafness, (hepatopathy), encephalopathy, and Leigh-like disease [MEGD(H)EL] syndrome, but the role of SERAC1 in mitochondrial physiology remains unknown. Here, we generated Serac1-/- mice that mimic the major diagnostic clinical and biochemical phenotypes of the MEGD(H)EL syndrome. We found that SERAC1 localizes to the outer mitochondrial membrane and is a protein component of the one-carbon cycle. By interacting with the mitochondrial serine transporter protein SFXN1, SERAC1 facilitated and was required for SFXN1-mediated serine transport from the cytosol to the mitochondria. Loss of SERAC1 impaired the one-carbon cycle and disrupted the balance of the nucleotide pool, which led to primary mitochondrial DNA (mtDNA) depletion in mice, HEK293T cells, and patient-derived immortalized lymphocyte cells due to insufficient supply of nucleotides. Moreover, both in vitro and in vivo supplementation of nucleosides/nucleotides restored mtDNA content and mitochondrial function. Collectively, our findings suggest that MEGD(H)EL syndrome shares both clinical and molecular features with the mtDNA depletion syndrome, and nucleotide supplementation may be an effective therapeutic strategy for MEGD(H)EL syndrome.

Determining Mitochondrial 3243A>G Heteroplasmy Using an ARMS-ddPCR Strategy.

OBJECTIVES: Determining mitochondrial DNA (mtDNA) A-to-G substitution at nucleotide 3243 (m.3243A>G) heteroplasmy is essential for both precision diagnosis of m.3243A>G-associated mitochondrial disease and genetic counseling. Precise determination of m.3243A>G heteroplasmy is challenging, however, without appropriate strategies to accommodate heteroplasmic levels ranging from 1% to 100% in samples carrying thousands to millions of mtDNA copies. METHODS: We used a combined strategy of amplification-refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR) and droplet digital PCR (ddPCR) to determine m.3243A>G heteroplasmy. Primers were specifically designed and screened for both ARMS-qPCR and ddPCR to determine m.3243A>G heteroplasmy. An optimized ARMS-qPCR-ddPCR-based strategy was established using artificial standards, with different mixtures of m.3243A-containing and m.3243G-containing plasmids and further tested using clinical samples containing the m.3243A>G mutation. RESULTS: One of 20 primer pairs designed in the study was omitted for ARMS-qPCR-ddPCR strategy application according to criteria of 85% to 110%, R2> 0.98 amplification efficiency, melt curve with a single clear peak, and specificity for m.3243A and m.3243G artificial standards (|CtWt-CtMut|max). Using plasmid standards with various m.3243A>G heteroplasmy (1%-100%) at low, mid, and high copy numbers (3,000, 104, and 105-107, respectively) and DNA from the blood of 20 patients carrying m.3243A>G with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes, we found that ARMS-qPCR was reliable for determining m.3243A>G at 3% to 100% for low copy number and 1% to 100% for mid to high copy number samples. Meanwhile, ddPCR was reliable for determining m.3243A>G at 1% to 100% at low to mid copy number samples. CONCLUSIONS: An ARMS-qPCR-ddPCR-based strategy was successfully established for precise determination of m.3243A>G heteroplasmy in complex clinical samples.