Translation initiation in eukaryotes is accomplished through the coordinated and orderly action of a large number of proteins, including the eIF4 initiation factors. Herein, we report that pateamine A (PatA), a potent antiproliferative and proapoptotic marine natural product, inhibits cap-dependent eukaryotic translation initiation. PatA bound to and enhanced the intrinsic enzymatic activities of eIF4A, yet it inhibited eIF4A-eIF4G association and promoted the formation of a stable ternary complex between eIF4A and eIF4B. These changes in eIF4A affinity for its partner proteins upon binding to PatA caused the stalling of initiation complexes on mRNA in vitro and induced stress granule formation in vivo. These results suggest that PatA will be a valuable molecular probe for future studies of eukaryotic translation initiation and may serve as a lead compound for the development of anticancer agents.
Stress granules are aggregates of small ribosomal subunits, mRNA, and numerous associated RNA-binding proteins that include several translation initiation factors. Stress granule assembly occurs in the cytoplasm of higher eukaryotic cells under a wide variety of stress conditions, including heat shock, UV irradiation, hypoxia, and exposure to arsenite. Thus far, a unifying principle of eukaryotic initiation factor 2␣ phosphorylation prior to stress granule formation has been observed from the majority of experimental evidence. Pateamine A, a natural product isolated from marine sponge, was recently reported to inhibit eukaryotic translation initiation and induce the formation of stress granules. In this report, the protein composition and fundamental progression of stress granule formation and disassembly induced by pateamine A was found to be similar to that for arsenite. However, pateamine A-induced stress granules were more stable and less prone to disassembly than those formed in the presence of arsenite. Most significantly, pateamine A induced stress granules independent of eukaryotic initiation factor 2␣ phosphorylation, suggesting an alternative mechanism of formation from that previously described for other cellular stresses. Taking into account the known inhibitory effect of pateamine A on eukaryotic translation initiation, a model is proposed to account for the induction of stress granules by pateamine A as well as other stress conditions through perturbation of any steps prior to the rejoining of the 60S ribosomal subunit during the entire translation initiation process. Stress granules (SGs)2 were first observed as cellular bodies visible by microscopy in tomato cells subjected to heat shock (1-3). Subsequently, SGs were identified in mammalian cells exposed to a variety of stress conditions, including oxidative stress, energy depletion, UV irradiation, and hypoxia (4). SG assembly is part of an adaptive response that recruits selected mRNAs and associated proteins for storage or triage to processing bodies (PBs) (5) that are sites of mRNA decay, allowing survival under adverse conditions. Sequestration of these components may help cells to recover post-stress by replenishing the cellular pool of mRNA without the need for new transcription. The physiological relevance of SGs is underscored by the presence of SGs in tissues of animals under stress (4), and SGs have been implicated in radioresistance of tumor cells (6) and tumor necrosis factor ␣ signaling (7). The study of SGs, their mechanism of formation, and biological role is a relatively new field in cell biology. Thus, a deeper understanding of the mechanism of SG formation and cellular functions may be clinically relevant.A critical step in SG formation shared by most stress conditions is phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2) (8), which is a component of the eIF2-GTPtRNAi Met ternary complex. The ternary complex is part of the 43S complex (40S particle, eIF3, and ternary complex) that is recruited to mRNA by ...
Transcriptional profiling is a useful strategy to study development and disease. Approaches to isolate RNA from specific cell types, or from specific cellular compartments, would extend the power of this strategy. Previous work has shown that isolation of genetically tagged ribosomes (translating ribosome affinity purification; TRAP) is an effective means to isolate ribosome-bound RNA selectively from transgene-expressing cells. However, widespread application of this technology has been limited by available transgenic mouse lines. Here we characterize a TRAP allele (Rosa26 fsTRAP ) that makes this approach more widely accessible. We show that endothelium-specific activation of Rosa26 fsTRAP identifies endothelial cell-enriched transcripts, and that cardiomyocyte-restricted TRAP is a useful means to identify genes that are differentially expressed in cardiomyocytes in a disease model. Furthermore, we show that TRAP is an effective means for studying translational regulation, and that several nuclear-encoded mitochondrial genes are under strong translational control. Our analysis of ribosome-bound transcripts also shows that a subset of long intergenic noncoding RNAs are weakly ribosome-bound, but that the majority of noncoding RNAs, including most long intergenic noncoding RNAs, are ribosome-bound to the same extent as coding transcripts. Together, these data show that the TRAP strategy and the Rosa26 fsTRAP allele will be useful tools to probe cell type-specific transcriptomes, study translational regulation, and probe ribosome binding of noncoding RNAs.heart | pressure overload G enome-wide and unbiased measurement of RNA transcript levels using microarrays and RNA-seq (1) has powered fundamental advances in biology over the past decade. However, when used to study tissues composed of multiple cell types, RNA expression profiling faces two fundamental limitations. First, whole-tissue transcript levels represent the average of the distinct cell lineages in the tissue, and this averaging process can result in loss of important information or misassignment of gene expression changes in one type of cell to another. Second, RNA profiling measures transcript abundance, but translational regulation is also an important determinant of gene expression (2, 3).To overcome these limitations, approaches have been developed to isolate RNAs from selected cell types and/or selected transcript fractions (4-7). Often these approaches involve tissue dissociation followed by FACS, but this is slow and the dissociation procedure itself likely alters expression profiles. Translating ribosome affinity purification (TRAP) permits isolation of transcripts from selected cell types of intact tissues, without dissociation (4). In this approach, ribosomes of selected tissues are genetically labeled by transgenic expression of GFP fused to L10a, an integral component of the 60S ribosomal subunit. To collect RNA from the transgene-expressing subpopulation of a tissue, whole-cell lysates are prepared under conditions that stabilize ribosomes...
We report here that des-methyl, des-amino pateamine A (DMDA-PatA), a structurally simplified analogue of the marine natural product pateamine A, has potent antiproliferative activity against a wide variety of human cancer cell lines while showing relatively low cytotoxicity against nonproliferating, quiescent human fibroblasts. DMDAPatA retains almost full in vitro potency in P-glycoprotein-overexpressing MES-SA/Dx5-Rx1 human uterine sarcoma cells that are significantly resistant to paclitaxel, suggesting that DMDA-PatA is not a substrate for P-glycoprotein-mediated drug efflux. Treatment of proliferating cells with DMDA-PatA leads to rapid shutdown of DNA synthesis in the S phase of the cell cycle. Cell-free studies show that DMDA-PatA directly inhibits DNA polymerases α and γ in vitro albeit at concentrations considerably higher than those that inhibit cell proliferation. DMDA-PatA shows potent anticancer activity in several human cancer xenograft models in nude mice, including significant regressions observed in the LOX and MDA-MB-435 melanoma models. DMDA-PatA thus represents a promising natural product-based anticancer agent that warrants further investigation.
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