The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level

Abstract

While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding detection of newly synthesized proteins released from ribosomes. RPM visualizes actively translating ribosomes in cells via standard immunofluorescence microscopy in fixed and permeabilized cells using a puromycin-specific monoclonal antibody to detect puromycylated nascent chains trapped on ribosomes treated with a chain elongation inhibitor.

Keywords: Nascent chain; Puromycin; Ribopuromycylation; Ribosome; Translation site.

Figure 1.. Schematic representation of RPM (from David et al., 2012b ).

Following freezing of polysome with an elongation inhibitor (step 1), PMY is added (step 2) to living cells and nascent chains become puromycylated through ribosome catalysis (step 3). Anti-PMY monoclonal antibodies detect puromycylated nascent chains via indirect immunofluorescence (step 4). Reproduced from David et al. (2012b) with permission of the publisher and of Dr. Yewdell.

Figure 2.. Deconvolved images of HeLa cell labeled with RPM (from David et al., 2011 ).

HeLa cells were pulsed with PMY + CHX to label translating ribosomes and extracted with Dig to remove free PMY and cytosolic components. Cells were then fixed, permeabilized and stained for KRS, PMY or ribosomal P proteins. Co-localization was estimated using ImageJ (NIH) and JACoP plugin that compiles general co-localization indicators such as Pearson’s coefficient ( Manders et al., 1992 ) and Van Steensel’s CCF (Van Steensel et al., 1996 ). KRS and RPM demonstrate extensive co-localization as quantitated by Van Steensel’s CCF greater than 0.75 and Pearson’s coefficient (R) greater than 0.5. Bar scales, 10 μm, 5 μm for Z1. Reproduced from David et al., 2011 with permission of the publisher and of Dr. Yewdell.