Structural basis of translation termination, rescue, and recycling in mammalian mitochondria.

The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondrial release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present structural models detailing the process of mitochondrial ribosome recycling to explain how a dedicated elongation factor, mitochondrial EFG2 (mtEFG2), has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process.

Rescuing stalled mammalian mitoribosomes - what can we learn from bacteria?

In the canonical process of translation, newly completed proteins escape from the ribosome following cleavage of the ester bond that anchors the polypeptide to the P-site tRNA, after which the ribosome can be recycled to initiate a new round of translation. Not all protein synthesis runs to completion as various factors can impede the progression of ribosomes. Rescuing of stalled ribosomes in mammalian mitochondria, however, does not share the same mechanisms that many bacteria use. The classic method for rescuing bacterial ribosomes is trans-translation. The key components of this system are absent from mammalian mitochondria; however, four members of a translation termination factor family are present, with some evidence of homology to members of a bacterial back-up rescue system. To date, there is no definitive demonstration of any other member of this family functioning in mitoribosome rescue. Here, we provide an overview of the processes and key players of canonical translation termination in both bacteria and mammalian mitochondria, followed by a perspective of the bacterial systems used to rescue stalled ribosomes. We highlight any similarities or differences with the mitochondrial translation release factors, and suggest potential roles for these proteins in ribosome rescue in mammalian mitochondria.

Immature colon carcinoma transcript-1 promotes proliferation of gastric cancer cells.

Gastric cancer is the fourth most common malignant tumor and has been considered as one of the leading causes of cancer-related death worldwide. The identification of the molecular mechanism during gastric cancer progression is urgently needed, which will help to develop more effective treatment strategies. As a component of the human mitoribosome, immature colon carcinoma transcript-1 (ICT1) might be involved in tumor formation and progression. However, its biological function and the corresponding mechanism in gastric cancer have been poorly characterized. To study the mechanism of ICT1 in gastric cancer, we first investigated the mRNA levels of ICT1 in human normal and gastric cancer tissues using datasets from the publicly available Oncomine database. The results showed that ICT1 is overexpressed in gastric cancer tissues. Then in order to study the role of ICT1 in gastric cancer, two shRNAs were used to silence ICT1 in MGC80-3 and AGS cells. Functional analysis showed ICT1 knockdown significantly inhibited the proliferation of gastric cancer cells and induced apoptosis. Further, mechanistic study demonstrated that ICT1 silencing induced cell-cycle arrest at G2/M phase via the suppression of cyclin A2 and cyclin B1. In addition, ICT1 silencing also increased cleaved caspase-3 and activated PARP in gastric cancer cells. These findings suggest that ICT1 may play a crucial role in promoting gastric cancer proliferation in vitro.

ICT1 deficiency leads to reduced oxygen resistance due to the cell wall damage in S. cerevisiae.

BACKGROUND: Disruption of ICT1 has been known to cause a significant deficiency in the phospholipid composition which is necessary for cell stress adaptation. However, the effects of ICT1 deletion on antioxidant research are not clear. OBJECTIVE: Construct a knockout strain to investigate the efficacy of ICT1 on antioxidant activity. METHODS: The antioxidant-related genes and phospholipid-related genes were determined by RT-PCR, the cell wall shape was observed by TME, and CWI pathway phosphorylation experiments were also analyzed by HPLC. RESULTS: The expression of antioxidant related genes and phospholipid-related genes has a slight reduction compared to the wild type. The cell wall was observed impaired with apparent CWI pathway phosphorylation weakening in the mutant. CONCLUSION: These findings indicate the role of ICT1 on antioxidant activity because it not only directly affects phospholipid composition but also further leads to the activation of CWI.

MiR-1301-3p inhibits human breast cancer cell proliferation by regulating cell cycle progression and apoptosis through directly targeting ICT1.

BACKGROUND: MiRNAs regulate a variety of biological processes, such as cell proliferation and apoptosis and play critical roles in cancer progression. Accumulating studies have demonstrated that miR-1301-3p could regulate the development and progression of multiple cancers, but its biological behaviors in breast cancer (BC) are still elusive. METHODS: The expression of miR-1301-3p was determined in BC tissues and cell lines using quantitative real-time PCR analysis. The effects of miR-1301-3p on BC cell growth, proliferation, cell cycle distribution, and apoptosis were also explored in vitro using MTT, colony formation and Flow cytometry assays. The potential target gene of miR-1301-3p was determined by dual-luciferase reporter assay and verified by quantitative real-time PCR and western blot analysis. RESULTS: We found the expression of miR-1301-3p was observably significantly down-regulated in BC tissues and cell lines. MiR-1301-3p expression in BC tissues was significantly associated with tumor size and clinical stage. Gain-of-function assays demonstrated that miR-1301-3p inhibited the cell growth and proliferation in breast cancer cell lines, MCF-7 and T-47D. Moreover, up-regulation of miR-1301-3p induced cell cycle G0/G1 phase arrest and apoptosis. Mechanistically, up-regulation of miR-1301-3p reduced the expression of CDK4, Cyclin D1, Bcl-2, but elevated the expression of p21, Bad and Bax. ICT1 was confirmed as a direct target of miR-1301-3p. Furthermore, ICT1 overexpression could partially reverse the effects of miR-1301-3p on BC cell proliferation, cell cycle progression and apoptosis. CONCLUSION: Our observations suggested that miR-1301-3p inhibits cell proliferation via inducing cell cycle arrest and apoptosis through targeting ICT1, and might be a therapeutic target for BC.

Knockdown of Immature Colon Carcinoma Transcript 1 Inhibits Proliferation and Promotes Apoptosis of Non-Small Cell Lung Cancer Cells.

Non-small cell lung cancer, as the most frequent type lung cancer, has lower survival rate of 5 years, despite improvements in surgery and chemotherapy. Previous studies showed immature colon carcinoma transcript 1 is closely related to tumorigenesis of human cancer cells. In the present study, we found immature colon carcinoma transcript 1 was overexpressed in lung cancer tissues using Oncomine database mining, and the biological effect of immature colon carcinoma transcript 1 was investigated in non-small cell lung cancer cell lines 95D and A549. Lentivirus-mediated RNA interference was used to knock down immature colon carcinoma transcript 1 expression in 95D and A549 cells in vitro, and the knockdown efficiency was determined using quantitative real-time polymerase chain reaction and Western blot assay. Knockdown of immature colon carcinoma transcript 1 significantly suppressed non-small cell lung cancer cell proliferation and colony formation ability confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay. Flow cytometry was applied to measure cell cycle arrest, and the result showed the cell cycle arrested in G2/M phase in 95D cells and arrested in G0/G1 phase in A549 cells. Furthermore, we measured the levels of cell cycle-associated proteins by Western blot analysis and found immature colon carcinoma transcript 1 -mediated cell proliferation inhibition appeared due to downregulation of cell cycle activator cyclin D1 and upregulation of cell cycle inhibitor p21. In addition, immature colon carcinoma transcript 1 silencing significantly induced non-small cell lung cancer cell apoptosis by annexin V/7-amino-actinomycin D double-staining assay. All our data suggest that immature colon carcinoma transcript 1 may play an important role for non-small cell lung cancer cell proliferation and could be a potential molecular target for diagnosing and treating human non-small cell lung cancer.

miR-205 regulation of ICT1 has an oncogenic potential via promoting the migration and invasion of gastric cancer cells.

Immature colon carcinoma transcript-1 (ICT1) is a newly identified oncogene, which regulates mobility, apoptosis, cell cycle progression and proliferation of cancer cells. Nevertheless, the role of ICT1 and its clinical significance in gastric cancer (GC) is largely uncovered. Here, we found that ICT1 displayed higher expression in GC tissues compared to corresponding tumor-adjacent tissues. Further investigation confirmed ICT1 overexpression in GC cell lines. Clinical data disclosed that high ICT1 expression correlated with distant metastasis and advanced tumor-node-metastasis (TNM) stage. The Cancer Genome Atlas (TCGA) data further demonstrated that GC tissues with metastasis showed a significant higher level of ICT1 compared to those without metastasis. Furthermore, ICT1 overexpression notably predicted poor prognosis of GC patients. Functionally, we demonstrated that ICT1 knockdown suppressed invasion and migration of MGC-803 and BGC-823 cells in vitro. ICT1 overexpression promoted the mobility of SGC-7901 cells. Mechanistically, microRNA-205 (miR-205) was recognized as a direct down-regulator and inversely modulated ICT1 abundance in GC cells. miR-205 expression was down-regulated and negatively associated with ICT1 level in GC tissues. Underexpression of miR-205 indicated an obvious shorter survival of GC patients. miR-205 overexpression inhibited migration and invasion of MGC-803 cells, while these inhibitory effects were reversed by ICT1 restoration. Taken together, we have the earliest evidence that miR-205 regulation of ICT1 functions as an oncogene and prognostic biomarker in GC.

ICT1 predicts a poor survival and correlated with cell proliferation in diffuse large B-cell lymphoma.

Immature colon carcinoma transcript-1 (ICT1) is a crucial member of the large mitoribosomal subunit in mitochondrial ribosome, which has been shown to be closely related to tumorigenesis. Its expression and function in human diffuse large B-cell lymphoma (DLBCL), however, remained elusive. In this study, analysis of public available Oncomine database suggested that the expression levels of ICT1 mRNA was significantly upregulated in DLBCL tissues. Consistently, we described ICT1 was remarkably upregulated in fresh DLBCL samples compared with the corresponding normal tissues using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. Moreover, ICT1 overexpression was associated with the poor overall survival (OS) of DLBCL patients. Finally, we used DLBCL cell lines to further probe the potential mechanisms, and found shRNA-mediated knockdown of ICT1 significantly suppressed DLBCL cell proliferation, induced cell cycle arrest at G0/G1 phase and apoptosis in vitro. Further verification showed that inhibition of ICT1 gene expression caused the upregulation of the p21, Bad and caspase-3, and downregulation of PCNA, Survivin, CDK4, CDK6 and Cyclin D1. Taken together, this study suggested that ICT1 may play an oncogenic role in human DLBCL by promoting cell proliferation and it might be a biomarker of unfavorable prognosis in DLBCL patients.

Immature Colon Carcinoma Transcript 1 Is Essential for Prostate Cancer Cell Viability and Proliferation.

Prostate cancer is the second leading cause of cancer-related death among men in the United States. More recently, immature colon carcinoma transcript 1 (ICT1) has been reported to be overexpressed in various kinds of cancer cells. However, the role of ICT1 in human prostate cancer has not yet been determined. The authors selected two ICT1-specific short hairpin RNA (shRNA) sequences to block its endogenous expression in human androgen-independent prostate cancer cell lines DU145 and PC-3. Decreased ICT1 expression by either specific shRNA significantly inhibited cell viability and proliferation. Moreover, compared to controls, ICT1-silenced cells were more inclined to redistribute in the G2/M phase, leading to cell cycle arrest. Flow cytometry and Annexin V-APC/7-AAD double staining confirmed that knockdown of ICT1 increased late apoptotic cells. Furthermore, they found that ICT1 knockdown restricting G2-M transition may be partly through suppression of CDK1 and Cyclin B1. Knockdown of ICT1 induced apoptosis through activation of poly ADP-ribose polymerase and caspase 3, upregulation of Bax expression, and downregulation of Bcl-2 expression in DU145 cells. In conclusion, this study highlights the crucial role of ICT1 in promoting prostate cancer cell proliferation in vitro. The depletion of ICT1 by lentivirus-mediated shRNA or small molecular inhibitor may provide a novel therapeutic approach for the treatment of prostate cancer.

Overcoming stalled translation in human mitochondria.

Protein synthesis is central to life and maintaining a highly accurate and efficient mechanism is essential. What happens when a translating ribosome stalls on a messenger RNA? Many highly intricate processes have been documented in the cytosol of numerous species, but how does organellar protein synthesis resolve this stalling issue? Mammalian mitochondria synthesize just thirteen highly hydrophobic polypeptides. These proteins are all integral components of the machinery that couples oxidative phosphorylation. Consequently, it is essential that stalled mitochondrial ribosomes can be efficiently recycled. To date, there is no evidence to support any particular molecular mechanism to resolve this problem. However, here we discuss the observation that there are four predicted members of the mitochondrial translation release factor family and that only one member, mtRF1a, is necessary to terminate the translation of all thirteen open reading frames in the mitochondrion. Could the other members be involved in the process of recycling stalled mitochondrial ribosomes?

Identification and characterization of CHCHD1, AURKAIP1, and CRIF1 as new members of the mammalian mitochondrial ribosome.

Defects in mitochondrial ribosomal proteins (MRPs) cause various diseases in humans. Because of the essential role of MRPs in synthesizing the essential subunits of oxidative phosphorylation (OXPHOS) complexes, identifying all of the protein components involved in the mitochondrial translational machinery is critical. Initially, we identified 79 MRPs; however, identifying MRPs with no clear homologs in bacteria and yeast mitochondria was challenging, due to limited availability of expressed sequence tags (ESTs) in the databases available at that time. With the improvement in genome sequencing and increased sensitivity of mass spectrometry (MS)-based technologies, we have established four previously known proteins as MRPs and have confirmed the identification of ICT1 (MRP58) as a ribosomal protein. The newly identified MRPs are MRPS37 (Coiled-coil-helix-coiled-coil-helix domain containing protein 1-CHCHD1), MRPS38 (Aurora kinase A interacting protein1, AURKAIP1), MRPS39 (Pentatricopeptide repeat-containing protein 3, PTCD3), in the small subunit and MRPL59 (CR-6 interacting factor 1, CRIF1) in the large subunit. Furthermore, we have demonstrated the essential roles of CHCHD1, AURKAIP1, and CRIF1in mitochondrial protein synthesis by siRNA knock-down studies, which had significant effects on the expression of mitochondrially encoded proteins.