Translational Control of Cytochrome c by RNA-Binding Proteins TIA-1 and HuR (original) (raw)
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Elucidation of a C-Rich Signature Motif in Target mRNAs of RNA-Binding Protein TIAR
Molecular and Cellular Biology, 2007
The RNA-binding protein TIAR (related to TIA-1 [T-cell-restricted intracellular antigen 1]) was shown to associate with subsets of mRNAs bearing U-rich sequences in their 3 untranslated regions. TIAR can function as a translational repressor, particularly in response to cytotoxic agents. Using unstressed colon cancer cells, collections of mRNAs associated with TIAR were isolated by immunoprecipitation (IP) of (TIAR-RNA) ribonucleoprotein (RNP) complexes, identified by microarray analysis, and used to elucidate a common signature motif present among TIAR target transcripts. The predicted TIAR motif was an unexpectedly cytosine-rich, 28-to 32-nucleotide-long element forming a stem and a loop of variable size with an additional side loop. The ability of TIAR to bind an RNA oligonucleotide with a representative C-rich TIAR motif sequence was verified in vitro using surface plasmon resonance. By this analysis, TIAR containing two or three RNA recognition domains (TIAR12 and TIAR123) showed low but significant binding to the C-rich sequence. In vivo, insertion of the C-rich motif into a heterologous reporter strongly suppressed its translation in cultured cells. Using this signature motif, an additional ϳ2,209 UniGene targets were identified (2.0% of the total UniGene database). A subset of specific mRNAs were validated by RNP IP analysis. Interestingly, in response to treatment with short-wavelength UV light (UVC), a stress agent causing DNA damage, each of these target mRNAs bearing C-rich motifs dissociated from TIAR. In turn, expression of the encoded proteins was elevated in a TIAR-dependent manner. In sum, we report the identification of a C-rich signature motif present in TIAR target mRNAs whose association with TIAR decreases following exposure to a stress-causing agent.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2018
Cytochrome c (Cyt c) plays a vital role in the mitochondrial electron transport chain (ETC). In addition, it is a key regulator of apoptosis. Cyt c has multiple other functions including ROS production and scavenging, cardiolipin peroxidation, and mitochondrial protein import. Cyt c is tightly regulated by allosteric mechanisms, tissue-specific isoforms, and post-translational modifications (PTMs). Distinct residues of Cyt c are modified by PTMs, primarily phosphorylations, in a highly tissue-specific manner. These modifications downregulate mitochondrial ETC flux and adjust the mitochondrial membrane potential (ΔΨ), to minimize reactive oxygen species (ROS) production under normal conditions. In pathologic and acute stress conditions, such as ischemia-reperfusion, phosphorylations are lost, leading to maximum ETC flux, ΔΨ hyperpolarization, excessive ROS generation, and the release of Cyt c. It is also the dephosphorylated form of the protein that leads to maximum caspase activatio...
Extensive Translatome Remodeling during ER Stress Response in Mammalian Cells
PLoS ONE, 2012
In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ,10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ,800-900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ,50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000-1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 59UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 59UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed.
Free Radical Biology and Medicine, 2012
Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegen-erative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H 2 O 2 or by the Fe(II)/ascorbate/H 2 O 2 system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H 2 O 2 relative to the Fe(II)/ascorbate/H 2 O 2 system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H 2 O 2 concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H 2 O 2 , it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA-cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress.
Journal of Biological Chemistry, 2012
Background: HuR regulates expression of many oncogenic proteins by modulating mRNA stability. Results: Glycolysis inhibition facilitates HuR degradation through a novel -TrCP-mediated mechanism. Conclusion: This mechanism underlies the complexity in the regulation of HuR turnover under different stress stimuli. Significance: The ability of glycolysis inhibitors to target expression of oncogenic proteins by promoting HuR degradation might foster novel strategies for cancer therapy.
Translational control in stress and apoptosis
Nature Reviews Molecular Cell Biology, 2005
Cells respond to stress stimuli through coordinated changes in gene expression. The regulation of translation is often used under these circumstances because it allows immediate and selective changes in protein levels. There are many examples of translational control in response to stress. Here we examine two representative models, the regulation of eukaryotic initiation factor-2α by phosphorylation and internal ribosome initiation through the internal ribosome-entry site, which illustrate the importance of translational control in the cellular stress response and apoptosis. ENDOPLASMIC RETICULUM (ER) STRESS Perturbations of the ER function that are caused by the accumulation of misfolded proteins, oxidative stress, inhibition of glycosylation or alteration in Ca 2+ homeostasis.
Gastroenterology, 2007
Cyclooxygenase-2 (COX-2) expression is regulated at the levels of messenger RNA (mRNA) stability and translation by AU-rich elements (ARE) located in its 3= untranslated region (3=UTR). Although structurally homologous RNA binding proteins HuR and CUGBP2 stabilize COX-2 mRNA, HuR induces whereas CUGBP2 inhibits COX-2 mRNA translation. This study aimed to determine the antagonism between these proteins on COX-2 expression. Methods: COX-2 ARE binding activity was determined by nitrocellulose filter binding and UV cross-linking assays using recombinant glutathione S-transferase (GST)/HuR and GST/CUGBP2. Protein:protein interactions were determined by GST pull-down, yeast 2-hybrid, and immunocytochemistry assays. Nucleocytoplasmic shutting was determined by heterokaryon analyses. The effect of CUGBP2 and HuR on COX-2 ARE-dependent translation was shown by a chimeric luciferase mRNA containing COX-2 3=UTR. HT-29 cells were subjected to 12 Gy ␥-irradiation in a cesium irradiator. Results: CUGBP2 and HuR bind with similar affinities to COX-2 ARE, but CUGBP2 competes with HuR for binding. In vitro, HuR and CUGBP2 heterodimerize. Furthermore, FLAGtagged HuR and myc-tagged CUGBP2 colocalize in the nucleus of HCT-116 cells. Moreover, both proteins shuttled between the nucleus and cytoplasm. In vitro, HuR enhanced whereas CUGBP2 inhibited translation of the chimeric luciferase COX-2 3=UTR mRNA. Furthermore, CUGBP2 competitively inhibited HuR-mediated translation of the transcript. In HT-29 cells transfected with HuR and CUGBP2, a switch in COX-2 mRNA binding from predominantly HuR to CUGBP2 occurred after radiation treatment, which was coupled with increased silencing of the COX-2 mRNA. Conclusions: CUGBP2 overrides HuR and suppresses COX-2 mRNA translation.