analysis-expression-complexes-assembly-mitochondrial-respiratory

Analysis of the Expression and Complexes Assembly of the Mitochondrial Respiratory Chain Proteins in the Fission Yeast <em>Schizosaccharomyces pombe</em>

The mitochondrial respiratory chain is crucial for cellular energy metabolism, serving as the core of oxidative phosphorylation. The mitochondrial ...

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To clarify the significance of circulating tumor cells (CTC) undergoing epithelial-mesenchymal transition (EMT) in patients with hepatocellular carcinoma (HCC), we used an advanced CanPatrol CTC-enrichment technique and  hybridization to enrich and classify CTC from blood samples. One hundred and one of 112 (90.18%) patients with HCC were CTC positive, even with early-stage disease. CTCs were also detected in 2 of 12 patients with hepatitis B virus (HBV), both of whom had small HCC tumors detected within 5 months. CTC count ≥16 and mesenchymal-CTC (M-CTC) percentage ≥2% prior to resection were significantly associated with early recurrence, multi-intrahepatic recurrence, and lung metastasis. Postoperative CTC monitoring in 10 patients found that most had an increased CTC count and M-CTC percentage before clinically detectable recurrence nodules appeared. Analysis of HCC with high CTC count and high M-CTC percentage identified 67 differentially expressed cancer-related genes involved in cancer-related biological pathways (e.g., cell adhesion and migration, tumor angiogenesis, and apoptosis). One of the identified genes, BCAT1, was significantly upregulated, and knockdown in Hepg2, Hep3B, and Huh7 cells reduced cell proliferation, migration, and invasion while promoting apoptosis. A concomitant increase in epithelial marker expression (EpCAM and E-cadherin) and reduced mesenchymal marker expression (vimentin and Twist) suggest that BCAT1 may trigger the EMT process. Overall, CTCs were highly correlated with HCC characteristics, representing a novel marker for early diagnosis and a prognostic factor for early recurrence. BCAT1 overexpression may induce CTC release by triggering EMT and may be an important biomarker of HCC metastasis. In liver cancer, CTC examination may represent an important "liquid biopsy" tool to detect both early disease and recurrent or metastatic disease, providing cues for early intervention or adjuvant therapy. .

Circulating Tumor Cells Undergoing EMT Provide a Metric for Diagnosis and Prognosis of Patients with Hepatocellular Carcinoma.

Mitochondrial gene expression is largely controlled through post-transcriptional processes including mitochondrial RNA (mt-RNA) processing, modification, decay, and quality control. Defective mitochondrial gene expression results in mitochondrial oxidative phosphorylation (OXPHOS) deficiency and has been implicated in human disease. To fully understand mitochondrial transcription and RNA processing, we performed RNA-seq analyses of mt-RNAs from the fission yeast  RNA-seq analyses show that the abundance of mt-RNAs vary greatly. Analysis of data also reveals mt-RNA processing sites including an unusual RNA cleavage event by mitochondrial tRNA (mt-tRNA) 5'-end processing enzyme RNase P. Additionally, this analysis reveals previously unknown mitochondrial transcripts including the -derived fragment, mitochondrial small RNAs (mitosRNAs) such as mt-tRNA-derived fragments (mt-tRFs) and mt-tRNA halves, and mt-tRNAs marked with 3'-CCACCA/CCACC in  Finally, RNA-seq reveals that inactivation of  encoding  mitochondrial tRNA 3'-end processing enzyme globally impairs mt-tRNA 3'-end processing, inhibits mt-mRNA 5'-end processing, and causes accumulation of unprocessed transcripts, demonstrating the feasibility of using RNA-seq to examine the protein known or predicted to be involved in mt-RNA processing in  Our work uncovers the complexity of a fungal mitochondrial transcriptome and provides a framework for future studies of mitochondrial gene expression using  as a model system.

The  mitochondrial transcriptome.

The fission yeast Schizosaccharomyces pombe has two tRNase Z genes (trz1 and trz2) involved in nuclear and mitochondrial tRNA 3'-end processing, respectively. Overexpression of trz2 but not trz1 is toxic to cells. In the present work, we showed that trz2 overexpression led to apoptotic cell death, as revealed by DAPI and Annexin V-FITC staining. Overexpression of trz2 also caused a loss of mitochondrial membrane potential and an increased reactive oxygen species (ROS) formation. These effects required mitochondrial localization but not its catalytic activity. RNA sequencing (RNA-seq) analysis revealed increased expression levels of genes involved in iron uptake and/or iron homeostasis, suggesting an elevated level of intracellular iron in the trz2-overexpressing cells. Indeed, we showed that overexpressing trz2 increased the level of intracellular iron by ∼2-fold. We further showed that the iron chelator, bathophenanthroline disulfonic acid (BPS) nearly restored the viability of trz2-overexpression cells and reduced ROS levels in the cells. These results suggest that trz2 overexpression may cause mitochondrial dysfunction, which is likely to lead to perturbation of iron homeostasis, ROS accumulation and induction of apoptotic cell death in S. pombe.

Overexpression of Schizosaccharomyces pombe tRNA 3'-end processing enzyme Trz2 leads to an increased cellular iron level and apoptotic cell death.

Autophagy is a degradation system in the cell, involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. Autophagy is induced by nutrient starvation, in which cytoplasmic components and organelles are digested  vacuoles/lysosomes. In this study, by using electron microscopy, we observed that hypovirus CHV1-EP713 infection of , the causative agent of chestnut blight disease, caused proliferation of autophagic-like vesicles. This phenomenon could be mimicked by treating the wild-type strain of the fungus EP155 with the autophagy induction drug rapamycin. Some of the hypovirulence-associated traits, including reduced pigmentation and conidiation, were also observed in the rapamycin-treated EP155. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that genes involved in autophagy were up-regulated in expression. Deletion of , a gene encoding a homolog of ATG8 in , resulted in attenuation of virulence and reduction in sporulation, as well as accumulation of the double-stranded viral RNA. Furthermore, virus-encoded p29 protein was found to co-localize with CpATG8, implying that the viral protein may interfere with the function of CpATG8. Taken together, these findings show that  can be regulated by the hypovirus and is required for virulence and development of the fungus and accumulation of viral dsRNA in chestnut blight fungus.

CpATG8, a Homolog of Yeast Autophagy Protein ATG8, Is Required for Pathogenesis and Hypovirus Accumulation in the Chest Blight Fungus.

Mitochondrial division and fusion play critical roles in maintaining functional mitochondria. Fzo1 is an outer mitochondrial membrane GTPase that played an essential role in mitochondrial fusion in budding yeast Saccharomyces cerevisiae. Here, we report the characterization of the Schizosaccharomyces pombe homologue of S. cerevisiae Fzo1p, Fzo1. Disruption of the fzo1 gene in S. pombe results in a fragmented mitochondrial morphology and a dramatically reduced growth on glycerol medium phenotype, indicating that deletion of fzo1 compromises respiratory function. Fluorescence microscopy shows that Fzo1p is located in the mitochondria. Overexpressing Fzo1 from a heterologous promoter induces mitochondrial aggregation. We also find that dnm1 mutations could both block mitochondrial fragmentation and rescue respiration growth defect in Δfzo1 single mutant cells. Our results proposed that a genetic interaction between fzo1 and a balance between division- and fusion-controlled mitochondrial shape and function in S. pombe. This study represents the first report of Fzo1 mediator of mitochondrial fusion in S. pombe.

dnm1 deletion blocks mitochondrial fragmentation in Δfzo1 cells.

Portal vein tumor thrombus (PVTT) and microvascular invasion (MVI) are types of intrahepatic vascular metastasis of hepatocellular carcinoma (HCC) and are highly correlated with poor prognosis. However, the underlying biomarkers of PVTT and MVI are unclear.

S100P as a novel biomarker of microvascular invasion and portal vein tumor thrombus in hepatocellular carcinoma.

In this study, we provide a global view of population and processing of mitochondrial tRNAs-derived fragments (mt-tRFs) in fission yeast Schizosaccharomyces pombe. Here, mt-tRFs of 15-30 nucleotides were retrieved from S. pombe small RNA libraries obtained from unstressed, stress, and during stationary phase conditions. We demonstrate that production of these fragments increase during heat stress and stationary phase conditions in S. pombe, especially (most notably) in stationary phase. Analysis of data also reveals depending on the tRNA, either 5'-mt-tRF or 3'-mt-tRF was found and major mt-tRNA processing sites have been precisely identified. Furthermore, RNA-seq reveals that inactivation of trz2 encoding S. pombe mitochondrial tRNase Z globally impairs mt-tRF processing. Finally, our result showed mt-tRFs were predicted to target mitochondrial genome mapping mtDNA-encoded protein gene. These observations suggest that mitochondrial tRFs may play an important regulatory role in response to stress and development.

Global view of dynamic expression and precise mapping of mitochondrial tRNAs-derived fragments during stressed conditions in S. pombe.

The multiplicity of hepatocellular carcinoma (HCC) recurrence patterns is the most important determinant of patients' postsurgical survival. A systematic HCC recurrence classification is needed to help prevent and treat postoperative HCC recurrence in the era of precision medicine.

Clinical implications and biological features of a novel postoperative recurrent HCC classification: A multi-centre study.

The RING (Really Interesting New Gene) finger proteins are a large diverse group of Zinc finger proteins. Many determined RING finger proteins are ubiquitin-protein E3 ligases and RING E3s are the most abundant type of ubiquitin ligase. RING finger and RING finger E3s have been discovered in many organisms where they play various functions, including DNA repair, ubiquitination and mitochondrial protein quality control. In this study, we identified a novel mitochondrial protein (SPBC16G5.03) with predicted RING finger domain within an N-terminal 21-60 amino acids and named it Mrz1 (mitochondrial RING finger protein). Our results showed that Mrz1 is localized in the mitochondrial outer membrane. Deletion of mrz1 did not affect cell growth in an unstressed state, but increases sensitivity to selenite. We showed that Mrz1 was degraded during the stationary phase and blocked by addition proteasome inhibitor MG132. We further showed that the E2 enzyme Ubc13 was identified among 8 candidate proteins as the ubiquitin-conjugating enzyme in this system. These data suggested that the Mrz1 was degraded likely through the ubiquitin-proteasome system.

Mrz1, a Novel Mitochondrial Outer Membrane RING Finger Protein, is Degraded Through the Ubiquitin-Proteasome Pathway in Schizosaccharomyces pombe.

Pentatricopeptide repeat proteins are involved in mitochondrial both transcriptional and posttranscriptional regulation. Schizosaccharomyces pombe Ppr2 is a general mitochondrial translation factor that plays a critical role in the synthesis of all mitochondrial DNA-encoded oxidative phosphorylation subunits, which are essential for mitochondrial respiration. Our previous analysis showed that ppr2 deletion resulted in increased expression of iron uptake genes and caused ferroptosis-like cell death in S. pombe. In the present work, we showed that deletion of ppr2 reduced viability on glycerol- and galactose-containing media.Php4 is a transcription repressor that regulates iron homeostasis in fission yeast. We found that in the ppr2 deletion strain, Php4 was constitutively active and accumulated in the nucleus in the stationary phase. We also found that deletion of ppr2 decreased the ferroptosis-related protein Gpx1 in the mitochondria. Overexpression of Gpx1 improves the viability of Δppr2 cells. We showed that the deletion of ppr2 increased the production of ROS, downregulated heme synthesis and iron-sulfur cluster proteins, and induced stress proteins. Finally, we observed the nuclear accumulation of Pap1-GFP and Sty1-GFP, suggesting that Sty1 and Pap1 in response to cellular stress in the ppr2 deletion strain. These results suggest thatppr2 deletion may cause mitochondrial dysfunction, which is likely to lead to iron-sensing defect and iron starvation response, resulting in perturbation of iron homeostasis and increased hydroxyl radical production. The increased hydroxyl radical production triggers cellular responses in theppr2 deletion strain.

The deletion of ppr2 interferes iron sensing and leads to oxidative stress response in Schizosaccharomyces pombe.

Canonical oncohistones are histone H3 mutations in the N-terminal tail associated with tumors and affect gene expression by altering H3 post-translational modifications (PTMs) and the epigenetic landscape. Noncanonical oncohistone mutations occur in both tails and globular domains of all four core histones and alter gene expression by perturbing chromatin remodeling. However, the effects and mechanisms of noncanonical oncohistones remain largely unknown. Here we characterized 16 noncanonical H2B oncohistones in the fission yeast Schizosaccharomyces pombe. We found that seven of them exhibited temperature sensitivities and 11 exhibited genotoxic sensitivities. A detailed study of two of these onco-mutants H2BG52D and H2BP102L revealed that they were defective in homologous recombination (HR) repair with compromised histone eviction and Rad51 recruitment. Interestingly, their genotoxic sensitivities and HR defects were rescued by the inactivation of the H2BK119 deubiquitination function of Ubp8 in the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. The levels of H2BK119 monoubiquitination (H2Bub) in the H2BG52D and H2BP102L mutants are reduced in global genome and at local DNA break sites presumably due to enhanced recruitment of Ubp8 onto nucleosomes and are recovered upon loss of H2B deubiquitination function of the SAGA complex. Moreover, H2BG52D and H2BP102L heterozygotes exhibit genotoxic sensitivities and reduced H2Bub in cis. We therefore conclude that H2BG52D and H2BP102L oncohistones affect HR repair and genome stability via the reduction of H2Bub and propose that other noncanonical oncohistones may also affect histone PTMs to cause diseases.

H2B oncohistones cause homologous recombination defect and genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe.

Serine-arginine protein kinases (SRPKs) play important roles in diverse biological processes such as alternative splicing and cell cycle. However, the functions of SRPKs in DNA damage response remain unclear. Here we characterized the function of SRPKs homolog Dsk1 in regulating DNA repair in the fission yeast Schizosaccharomyces pombe. We demonstrated that Dsk1 defective mutants of loss of the gene, spacer domain, and kinase activity as well as its overexpression mutant exhibited sensitivities of replication stress. Genetic analysis revealed that the loss of dsk1 compromised the efficiency of homologous recombination (HR) repair, and Dsk1 was probably involved in the Rad52- and Rad51-dependent HR repair pathways. Interestingly, Dsk1 translocated into the nucleus upon replication stress and directly interacted with Rad51-mediator Rad52 and phosphorylated Rad52-Ser365 residue. The Rad52-Ser365 phosphorylation-defective mutant was slightly sensitive to replication stress, and the phosphorylation-mimicking mutants exhibited more sensitivities, which were partially correlated with phenotypes of the loss- and gain-of-function of dsk1. This study uncovers a potential HR repair regulator Dsk1 in response to replication stress and implies that its homolog SRPKs may have the conserved targets and functions in higher eukaryotes.

Article	Year
Circulating Tumor Cells Undergoing EMT Provide a Metric for Diagnosis and Prognosis of Patients with Hepatocellular Carcinoma. Cancer research\| PubMed ID: 29915159	2018
The mitochondrial transcriptome. RNA (New York, N.Y.)\| PubMed ID: 29954949	2018
Overexpression of Schizosaccharomyces pombe tRNA 3'-end processing enzyme Trz2 leads to an increased cellular iron level and apoptotic cell death. Fungal genetics and biology : FG & B\| PubMed ID: 30393157	2019
CpATG8, a Homolog of Yeast Autophagy Protein ATG8, Is Required for Pathogenesis and Hypovirus Accumulation in the Chest Blight Fungus. Frontiers in cellular and infection microbiology\| PubMed ID: 31355148	2019
dnm1 deletion blocks mitochondrial fragmentation in Δfzo1 cells. Yeast (Chichester, England)\| PubMed ID: 33125774	2021
S100P as a novel biomarker of microvascular invasion and portal vein tumor thrombus in hepatocellular carcinoma. Hepatology international\| PubMed ID: 33495903	2021
Global view of dynamic expression and precise mapping of mitochondrial tRNAs-derived fragments during stressed conditions in S. pombe. Mitochondrion\| PubMed ID: 34428580	2021
Clinical implications and biological features of a novel postoperative recurrent HCC classification: A multi-centre study. Liver international : official journal of the International Association for the Study of the Liver\| PubMed ID: 35810457	2022
Mrz1, a Novel Mitochondrial Outer Membrane RING Finger Protein, is Degraded Through the Ubiquitin-Proteasome Pathway in Schizosaccharomyces pombe. Current microbiology\| PubMed ID: 36088506	2022
The deletion of ppr2 interferes iron sensing and leads to oxidative stress response in Schizosaccharomyces pombe. Mitochondrion\| PubMed ID: 38499131	2024
H2B oncohistones cause homologous recombination defect and genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe. The Journal of biological chemistry\| PubMed ID: 38718864	2024
SRPKs Homolog Dsk1 Regulates Homologous Recombination Repair in Schizosaccharomyces pombe. Genes to cells : devoted to molecular & cellular mechanisms\| PubMed ID: 39789818	2025

Jinjie Shang

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