Isolation of translationally controlled mRNAs by differential screening

• Published in: The FASEB journal 2000-08, Vol.14 (11), p.1641-1652
• Main Authors: Mikulits, W, Pradet-Balade, B, Habermann, B, Beug, H, Garcia-Sanz, J A, Müllner, E W
• Format: Article
• Language: English
• Subjects: Cells, Cultured; Centrifugation, Density Gradient; Cloning, Molecular - methods; DNA, Complementary - genetics; Flow Cytometry; Gene Expression Regulation; Gene Library; Genes; Humans; Lymphocyte Activation - genetics; Lymphocyte Activation - immunology; Oligonucleotide Array Sequence Analysis; Polyribosomes - genetics; Polyribosomes - metabolism; Protein Biosynthesis - physiology; RNA, Messenger - genetics; RNA, Messenger - metabolism; T-Lymphocytes - cytology; T-Lymphocytes - immunology; T-Lymphocytes - metabolism
• ISSN: 0892-6638; 1530-6860; 1530-6860
• DOI: 10.1096/fj.14.11.1641

Translationalregulation plays an important role in the control of gene expression. Changes in translation initiation rates are the most common translation-regulating mechanisms, resulting in alterations in mRNA loading of ribosomes. This differential mobilization of mRNAs onto polyribosomes was used in differential screening to directly identify cDNAs whose transcripts are translationally controlled during antigenic stimulation of primary human T lymphocytes. Ribosome-free and polysome-bound mRNAs were prepared from quiescent and activated T cells and used as templates to synthesize four cDNA pools. These in turn were used as probes to hybridize four identical replicas of a T cell library or, alternatively, four cDNA arrays. Translational activation was indicated by redistribution of the hybridization signals from the ribosome-free fraction in resting T cells to the polysome-associated fraction in activated T cells. Translational repression corresponded to the opposite hybridization pattern. Fifty-two cDNAs were identified as translationally 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 representative examples by demonstrating the redistribution of the corresponding mRNAs on polysome gradients in response to T cell activation. Our strategy therefore provides an efficient tool to directly isolate or identify translationally controlled mRNAs in a variety of physiological situations. Moreover, differential screening using arrays enables simultaneous analysis of both transcriptional and translational regulation, further enhancing the power of gene expression analysis.