Experimental layout and data analysis for translation profiling. (a) Four equal mRNA fractions were each competitively hybridized to DNA microarrays against genomic DNA as common reference. Polysome profiles were prepared from unstressed control cells and from cells exposed to stress for 5, 15 and 60 minutes. (b) Comparison of polysome profiles for medium-resolution translation profiling with high-resolution profiling applied before . The graphs show polysome profiles of the 10% mRNAs with the lowest ribosome occupancy (green) and the 10% mRNAs with the highest ribosome occupancy (red), measured by medium-resolution (left graph) and by high-resolution translation profiling (right graph). The profiles represent the average from three independent biological repeats. Ribosome occupancy was determined based on previous high-resolution translation profiling . (c) Two complementary data analysis approaches to uncover translationally regulated mRNAs (Materials and methods). Left: the total difference for a given mRNA between the translation profile under stress and the translation profile in the control was calculated by summing up the differences of each fraction (indicated by arrows). Right: scores for each mRNA in each condition were calculated as described in Materials and methods. A translation ratio was then obtained by dividing the score of a given mRNA in a stress condition by the score of the same mRNA in the control condition (see Additional file 1 for data from translational profiling analysis). The combined lists from both approaches were then visually inspected to generate high-confidence, curated lists of translationally regulated genes (Table 1). (d) Left graph: translation profiles for sod1 mRNA before and after exposure to 39°C. Right graph: translation profiles for sks2 mRNA before and after exposure to H2O2. Blue lines, control (unstressed) samples; orange lines, 15 minutes after stress; red lines, 60 minutes after stress. Multiple lines of the same color represent translation profiles from independent biological repeats.
Lackner et al. Genome Biology 2012 13:R25 doi:10.1186/gb-2012-13-4-r25