Ribosome Distribution in HeLa Cells during the Cell Cycle

Yuan-Jhih, Tsai; Lee, Hsing-I; Lin, Alan

doi:10.1371/journal.pone.0032820

Cited by 7 publications

(8 citation statements)

References 28 publications

(51 reference statements)

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“…As controls, cryptopleurine (CRY), which is known to bind the 40S subunit of the ribosome [ 48 ], did not disrupt cellular staining by F-DAP. The observed staining is consistent with ribosomes in HeLa cells [ 49 ].…”

Section: Discussionsupporting

confidence: 76%

Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humans

et al. 2016

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BackgroundDaptomycin is a recently introduced, last-resort antibiotic that displays a unique mode of action against Gram-positive bacteria that is not fully understood. Several bacterial targets have been proposed but no human binding partner is known.MethodsIn the present study we tested daptomycin in cell viability and proliferation assays against six human cell lines, describe the synthesis of biotinylated and fluorescently labeled analogues of daptomycin. Biotinylated daptomycin was used as bait to isolate the human binding partner by the application of reverse chemical proteomics using T7 phage display of five human tumor cDNA libraries. The interaction between the rescued protein and daptomycin was validated via siRNA knockdown, DARTS assay and immunocytochemistry.ResultsWe have found that daptomycin possesses selective growth inhibition of some cancer cell lines, especially MCF7. The unbiased interrogation of human cDNA libraries, displayed on bacteriophage T7, revealed a single human target of daptomycin; ribosomal protein S19. Using a drug affinity responsive target stability (DARTS) assay in vitro, we show that daptomycin stabilizes RPS19 toward pronase. Fluorescently labeled daptomycin stained specific structures in HeLa cells and co-localized with a RPS19 antibody.ConclusionThis study provides, for the first time, a human protein target of daptomycin and identifies RPS19 as a possible anticancer drug target for the development of new pharmacological applications and research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-017-0124-2) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 76%

Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humans

et al. 2016

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“…Rpl7 isoforms participate in the earliest steps of 60S precursor rRNA processing ( Jakovljevic et al 2012 ), and bind to 25S and 5S rRNAs in the mature 60S ribosomal subunit ( Ben-Shem et al 2011 ). Five of the 244 amino acid residues in Rpl7a and Rpl7b are divergent, with four amino acid substitutions in Rpl7b relative to Rpl7a (A2S, A3T, S16T, and V26I) being in the conserved N-terminal domain that is predicted to be on the surface of the ribosome ( Lin 1991 ; Tsai et al 2012 ). The possibility that Rpl7a and Rpl7b have unique functions as components of heterogeneous ribosomes was suggested by the involvement of different ribosome biogenesis factors in assembly of ribosomes containing Rpl7a vs. Rpl7b ( Komili et al 2007 ).…”

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confidence: 99%

Paralog-Specific Functions ofRPL7AandRPL7BMediated by Ribosomal Protein or snoRNA Dosage inSaccharomyces cerevisiae

Palumbo

Fuchs

Lutz

et al. 2017

G3 Genes|Genomes|Genetics

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Most ribosomal proteins in Saccharomyces cerevisiae are encoded by two paralogs that additively produce the optimal protein level for cell growth. Nonetheless, deleting one paralog of most ribosomal protein gene pairs results in a variety of phenotypes not observed when the other paralog is deleted. To determine whether paralog-specific phenotypes associated with deleting RPL7A or RPL7B stem from distinct functions or different levels of the encoded isoforms, the coding region and introns of one paralog, including an intron-embedded snoRNA (small nucleolar RNA) gene, were exchanged with that of the other paralog. Among mutants harboring a single native or chimeric RPL7 allele, expression from the RPL7A locus exceeded that from the RPL7B locus, and more Rpl7a was expressed from either locus than Rpl7b. Phenotypic differences in tunicamycin sensitivity, ASH1 mRNA localization, and mobility of the Ty1 retrotransposon were strongly correlated with Rpl7 and ribosome levels, but not with the Rpl7 or snoRNA isoform expressed. Although Ty1 RNA is cotranslationally localized, depletion of Rpl7 minimally affected synthesis of Ty1 Gag protein, but strongly influenced Ty1 RNA localization. Unlike the other processes studied, Ty1 cDNA accumulation was influenced by both the level and isoform of Rpl7 or snoRNA expressed. These cellular processes had different minimal threshold values for Rpl7 and ribosome levels, but all were functional when isoforms of either paralog were expressed from the RPL7A locus or both RPL7 loci. This study illustrates the broad range of phenotypes that can result from depleting ribosomes to different levels.

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“…Protein synthesis in cells is taking place at ribosomes which are not always homogenously distributed in the cell, e.g., they show changing distribution patterns in dependence of the cell cycle phase [ 23 ]. We therefore tried to use the biotin GA methyl ester 4b to show the putative co-localization with eIF2α in the ribosomes of a cultured cell population.…”

Section: Resultsmentioning

confidence: 99%

Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria

et al. 2018

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The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.

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Ribosome Distribution in HeLa Cells during the Cell Cycle

Cited by 7 publications

References 28 publications

Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humans

Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humans

Paralog-Specific Functions ofRPL7AandRPL7BMediated by Ribosomal Protein or snoRNA Dosage inSaccharomyces cerevisiae

Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria

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