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Isolation of translationally controlled mRNAs by differential screening
W Mikulits1, B Pradet-Balade, B Habermann
1Institute of Molecular Biology, Institute of Molecular Pathology, Vienna Biocenter, University of Vienna, Dr. Bohr-Gasse, A-1030 Vienna, Austria.
Abstract:
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.
Insights
This study identifies translationally controlled genes in human T cells during immune responses. The novel method efficiently detects changes in messenger RNA (mRNA) loading onto ribosomes, revealing new insights into gene expression regulation.
Area of Science:
- Molecular Biology
- Immunology
- Gene Expression Regulation
Background:
- Translational regulation is a key mechanism controlling gene expression, primarily through alterations in translation initiation rates and subsequent messenger RNA (mRNA) loading onto ribosomes.
- Differential mobilization of mRNAs onto polyribosomes serves as a direct indicator of translational control during cellular activation.
- Understanding translational control in immune cells like T lymphocytes is crucial for deciphering immune responses.
Purpose of the Study:
- To develop and validate a direct screening method for identifying translationally controlled messenger RNAs (mRNAs).
- To discover novel translationally regulated transcripts in primary human T lymphocytes upon antigenic stimulation.
- To assess the efficiency of differential screening using cDNA arrays for analyzing both transcriptional and translational regulation.
Main Methods:
- Preparation of ribosome-free and polysome-bound mRNAs from quiescent and activated human T lymphocytes.
- Synthesis of complementary DNA (cDNA) pools from isolated mRNAs to serve as hybridization probes.
- Differential screening of a T cell cDNA library and cDNA arrays using synthesized cDNA probes to identify translationally regulated genes.
Main Results:
- Identification of 52 translationally controlled cDNAs through cDNA array screening and 12 additional cDNAs via library screening.
- Validation of the method by confirming known translationally controlled mRNAs and discovering novel ones.
- Experimental verification of translational control for selected transcripts by demonstrating mRNA redistribution on polysome gradients post-T cell activation.
Conclusions:
- The developed differential screening strategy provides an efficient tool for directly isolating and identifying translationally controlled mRNAs in various physiological contexts.
- This approach enables simultaneous analysis of transcriptional and translational regulation, significantly enhancing gene expression analysis capabilities.
- The findings expand the understanding of translational control in T lymphocyte activation and offer a powerful platform for future gene expression studies.