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Puromycin reactivity does not accurately localize translation at the subcellular level.

ABSTRACT: Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.

SUBMITTER: Enam SU

PROVIDER: S-EPMC7490009 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Puromycin reactivity does not accurately localize translation at the subcellular level.

Enam Syed Usman SU Zinshteyn Boris B Goldman Daniel H DH Cassani Madeline M Livingston Nathan M NM Seydoux Geraldine G Green Rachel R

eLife 20200826

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosom ...[more]

PMID: 32844748

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Project description:To compare accuracy and interobserver variability in the detection and localization of recurrent ovarian cancer with contrast material-enhanced (CE) computed tomography (CT) and positron emission tomography (PET)/CT and determine whether imaging findings can be used to predict survival.Waiving informed consent, the institutional review board approved this HIPAA-compliant, retrospective study of 35 women (median age, 54.4 years) with histopathologically proven recurrent ovarian carcinoma who underwent CE CT and PET/CT before exploratory surgery. All CE CT and PET/CT scans were independently analyzed. Tumor presence, number of lesions, and the size and maximum standardized uptake value (SUV(max)) of the largest lesion were recorded for patient and region. Surgical histopathologic findings constituted the reference standard. Areas under the receiver operating characteristic curves (AUCs), κ statistics, and hazard ratios were calculated.Readers' AUCs in detection of recurrence for region were 0.85 (95% confidence interval [CI]: 0.81, 0.90) and 0.78 (95% CI: 0.72, 0.83) for CE CT and 0.84 (95% CI: 0.79, 0.89) and 0.74 (95% CI: 0.67, 0.81) for PET/CT (P = .76); 12 patients died. At PET/CT, size, number, and SUV(max) of peritoneal deposits were significantly associated with poor survival for readers 1 and 2 (P ≤ .01and ≤ .05, respectively), as were long- and short-axis diameters, number, and SUV(max) of distant lymph nodes for reader 1 (P ≤ .001). With CE CT, size (reader 1) and number (readers 1 and 3) of peritoneal deposits were significantly associated with poor survival (P ≤ .01), as were long- and short-axis diameters and number of distant lymph nodes for reader 1 (P ≤ .01). Interobserver agreement ranged from fair (patient, κ = 0.30) to moderate (region, κ = 0.55) for CE CT and fair (patient, κ = 0.24) to substantial (region, κ = 0.63) for PET/CT.Preliminary data suggest that CE CT and PET/CT may have similar accuracy in detection of recurrent ovarian cancer. Tumor size, number, and SUV(max) may have potential as prognostic biomarkers for patients with recurrent ovarian cancer.

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Puromycin reactivity does not accurately localize translation at the subcellular level.

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Puromycin reactivity does not accurately localize translation at the subcellular level.

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