Messenger RNA translation state: The second dimension of high-throughput expression screening

Technological advances over the past 10 years have generated powerful tools for parallel analysis of complex biological problems, Among these new tech nologies, DNA arrays have provided an important experimental approach for i dentifying changes in the levels of individual mRNA molecules during import ant cellular transitions. However, cellular behavior is dictated not by mRN A levels, but by the proteins translated from the individual mRNA species. We report a high-throughput method for simultaneously monitoring the transl ation state and level of individual mRNA species, Messenger RNAs from resti ng and mitogenically activated fibroblasts were separated, according to deg ree of ribosome loading, into well-translated and under-translated pools, c DNA probes generated from these fractions were used to interrogate cDNA arr ays. Among approximately 1,200 genes analyzed, less than 1% were found to b e translationally regulated in response to mitogenic activation, demonstrat ing the strong selectivity of this regulatory mechanism. This high-throughp ut approach is shown to be an effective tool for superimposing translation profile on mRNA level for large numbers of genes, as well as for identifyin g translationally regulated genes for further study.