Emetine optimally facilitates nascent chain puromycylation and potentiates the ribopuromycylation method (RPM) applied to inert cells

Abstract

We previously described the ribopuromyclation method (RPM) to visualize and quantitate translating ribosomes in fixed and permeabilized cells by standard immunofluorescence. RPM is based on puromycylation of nascent chains bound to translating ribosomes followed by detection of puromycylated nascent chains with a puromycin-specific mAb. We now demonstrate that emetine optimally enhances nascent chain puromycylation, and describe a modified RPM protocol for identifying ribosome-bound nascent chains in metabolically inert permeabilized cells.

Figure 1

A. Immunoblotting of total cell lysates demonstrates energy independence of puromycylation and also the temperature dependence (37°C vs 4°C) when intact cells are labeled with PMY. B. Immunoblotting of NP-40 extracts form HeLa labeled at 37 °C in culture or at 4 °C after digitonin treatment. Pre-incubation of cells with emetine prior to digitonin treatment greatly increases puromycylation.

Figure 2

A. HeLa cells were incubated or not for 15 min with emetine, were washed with PBS supplemented with CHX, permeabilized with digitonin and labeled with PMY in the presence of CHX. Cells were then fixed and stained for PMY (green) and large ribosomal subunit (white). Co-localization was quantitated using Van Steensel’s cross-correlation coefficient (graph) and Pearson’s coefficient (r; 1=complete co-localization, −1 = complete non-colocalization). Bar scale: 10 m. B. HeLa cells pre-treated for 15 min with the inhibitor indicated were labeled as in A. For each condition, multiple fields were acquired and the mean fluorescence ratio of PMY/ribosome staining for each field was quantitated using ImageJ. Values are plotted on the right. Statistical analyses, two-tailed unpaired t-test; ^***, P < 0.0001. C. As in B, but with the treatment times indicated D. As in B with CHX and emetine pre-treatments. Statistical analyses, two-tailed unpaired t-test; ^*, P < 0.05. E. Direct comparison of RPM labeling using the 4 °C or 37 °C protocol. Statistical analyses, two-tailed unpaired t-test; ^***, P < 0.0001.