Elongation Inhibitors do not Prevent the Release of Puromycylated Nascent Polypeptide Chains from Ribosomes

Hobson, Benjamin D.; Kong, Linghao; Hartwick, Erik W.; Gonzalez, Ruben L.; Sims, Peter A.

doi:10.1101/2020.06.15.152488

Cited by 6 publications

(6 citation statements)

References 50 publications

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“…We treated animals/cells with CHX/PMY, isolated ribosomes from cell lysates on sucrose gradients in monosome and polysome fractions, blotted fractions onto nitrocellulose and stained with antibodies against RPL7 or PMY. The robust PMY signal shows that, contrary to recent claims 38,39 , PMY does not completely release nascent chains when ribosomes are previously exposed to CHX (Figure 5A).…”

Section: Contribution Of Monosomes Vs Polysomes To T Cell Translationcontrasting

confidence: 76%

Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

Seedhom¹,

Dersh²,

Hollý³

et al. 2022

Preprint

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Rapid lymphocyte cell division places enormous demands on the protein synthesis machinery. Flow cytometric measurement of puromycylated ribosome-associated nascent chains after treating cells or mice with translation initiation inhibitors reveals that ribosomes in resting lymphocytes in vitro and in vivo elongate at typical rates for mammalian cells. Intriguingly, elongation rates can be increased up to 30% by activation in vivo or fever temperature in vitro. Resting and activated lymphocytes possess abundant monosome populations, most of which actively translate in vivo, while in vitro, nearly all can be stalled prior to activation. Quantitating lymphocyte protein mass and translating ribosomes reveals a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner. Our findings demonstrate the importance of a global understanding of protein synthesis in lymphocytes and other rapidly dividing immune cells.

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Section: Contribution Of Monosomes Vs Polysomes To T Cell Translationcontrasting

confidence: 76%

Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

Seedhom¹,

Dersh²,

Hollý³

et al. 2022

Preprint

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“…Recent studies showed that puromycylated peptides are released and diffuse quickly from the site of translation in cultured cell lines, leading to inaccuracy in identification of translation sites (Enam et al, 2020;Hobson et al, 2020) . It is likely that the dense environment of the myofiber restricts diffusion of labeled peptides away from the site of synthesis (Papadopoulos et al, 2000) , avoiding the pitfalls of this technique observed in dividing cells.…”

Section: Spatially Dispersed Translation In the Cytoplasm Requires Intact Microtubulesmentioning

confidence: 99%

Microtubule-based Transport is Essential to Distribute RNA and Nascent Protein in Skeletal Muscle

Denes

Kelley

Wang

2021

Preprint

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While the importance of RNA localization in highly differentiated cells is well appreciated, basic principles of RNA localization in skeletal muscle remain poorly characterized. Here, we develop a method to detect single RNA molecules and quantify localization patterns in skeletal myofibers, and we uncover a critical and general role for directed transport of RNPs in muscle. We find that RNAs are localized and translated along cytoskeletal filaments, and we identify the Z-disk as a biological hub for RNA localization and protein synthesis. We show that muscle development triggers complete reliance on the lattice-like microtubule network to transport RNAs and that disruption of microtubules leads to striking accumulation of RNPs and nascent protein around myonuclei. Our observations suggest that active transport may be globally required to distribute RNAs in highly differentiated cells and reveal fundamental mechanisms relevant to myopathies caused by perturbations to RNPs, microtubules, and the nuclear envelope.

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“…Treating the cells with puromycin in a com bination with cycloheximide leads to the accumulation of ribosomes exclusively on start codons, which facilitates their identification by ribosome profiling [127]. On the other hand, the combined effect of puromycin and other antibiotics depends on the ratio and concentrations of these compounds and cannot always be predicted, which may lead to artifacts [128,129]. The activity of puromycin is unique; for example, structurally similar antibiotic A201A (see above) does not act as a peptide bond accep tor and only inhibits the peptidyl transferase reac tion [13,40].…”

Section: Inhibitors Of Eukaryotic Ribosomementioning

confidence: 99%

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

Dmitriev

Vladimirov

Lashkevich

2020

Biochemistry Moscow

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Eukaryotic ribosome and cap-dependent translation are attractive targets in the antitumor, antiviral, anti-inflammatory, and antiparasitic therapies. Currently, a broad array of small-molecule drugs is known that specifically inhibit protein synthesis in eukaryotic cells. Many of them are well-studied ribosome-targeting antibiotics that block translocation, the peptidyl transferase center or the polypeptide exit tunnel, modulate the binding of translation machinery components to the ribosome, and induce miscoding, premature termination or stop codon readthrough. Such inhibitors are widely used as anticancer, anthelmintic and antifungal agents in medicine, as well as fungicides in agriculture. Chemicals that affect the accuracy of stop codon recognition are promising drugs for the nonsense suppression therapy of hereditary diseases and restoration of tumor suppressor function in cancer cells. Other compounds inhibit aminoacyl-tRNA synthetases, translation factors, and components of translation-associated signaling pathways, including mTOR kinase. Some of them have antidepressant, immunosuppressive and geroprotective properties. Translation inhibitors are also used in research for gene expression analysis by ribosome profiling, as well as in cell culture techniques. In this article, we review well-studied and less known inhibitors of eukaryotic protein synthesis (with the exception of mitochondrial and plastid translation) classified by their targets and briefly describe the action mechanisms of these compounds. We also present a continuously updated database (http://eupsic.belozersky.msu.ru/) that currently contains information on 370 inhibitors of eukaryotic protein synthesis.

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Elongation Inhibitors do not Prevent the Release of Puromycylated Nascent Polypeptide Chains from Ribosomes

Cited by 6 publications

References 50 publications

Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

Microtubule-based Transport is Essential to Distribute RNA and Nascent Protein in Skeletal Muscle

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

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