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

Abstract

Technological advances over the past 10 years have generated powerful tools for parallel analysis of complex biological problems. Among these new technologies, DNA arrays have provided an important experimental approach for identifying changes in the levels of individual mRNA molecules during important cellular transitions. However, cellular behavior is dictated not by mRNA levels, but by the proteins translated from the individual mRNA species. We report a high-throughput method for simultaneously monitoring the translation state and level of individual mRNA species. Messenger RNAs from resting and mitogenically activated fibroblasts were separated, according to degree of ribosome loading, into well-translated and under-translated pools. cDNA probes generated from these fractions were used to interrogate cDNA arrays. Among approximately 1,200 genes analyzed, less than 1% were found to be translationally regulated in response to mitogenic activation, demonstrating the strong selectivity of this regulatory mechanism. This high-throughput approach is shown to be an effective tool for superimposing translation profile on mRNA level for large numbers of genes, as well as for identifying translationally regulated genes for further study.