Isolation of translationally controlled mRNAs by differential screening

Translational regulation plays an important role in the control of gene exp ression, Changes in translation initiation rates are the most common transl ation-regulating mechanisms, resulting in alterations in mRNA loading of ri bosomes. This differential mobilization of mRNAs onto polyribosomes tvas us ed in differential screening to directly identify cDNAs whose transcripts a re translationally controlled during antigenic stimulation of primary human T lymphocytes. Ribosome-free and polysome-bound mRNAs were prepared from q uiescent and activated T cells and used as templates to synthesize four cDN A pools, These in turn were used as probes to hybridize four identical repl icas of a T cell library or, alternatively, four cDNA arrays. Translational activation was indicated by redistribution of the hybridization signals fr om the ribosome-free fraction in resting T cells to the polysome-associated fraction in activated T cells. Translational repression corresponded to th e opposite hybridization pattern. Fifty-two cDNAs were identified as transl ationally controlled by screening 472 genes in a cDNA array; 12 additional ones were obtained by screening a cDNA library. Several of the transcripts corresponded to mRNAs previously reported to be translationally controlled, thus validating the method. For the majority, however, such regulation had not yet been described, Translational control was verified for representat ive examples by demonstrating the redistribution of the corresponding mRNAs on polysome gradients in response to T cell activation, Our strategy there fore provides an efficient tool to directly isolate or identify translation ally controlled mRNAs in a variety of physiological situations, Moreover, d ifferential screening using arrays enables simultaneous analysis of both tr anscriptional and translational regulation, further enhancing the power of gene expression analysis.