Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes - PubMed (original) (raw)

Comparative Study

. 2002 Dec 15;30(24):5529-38.

doi: 10.1093/nar/gkf682.

Affiliations

Comparative Study

Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes

Arvind Raghavan et al. Nucleic Acids Res. 2002.

Abstract

We used microarray technology to measure mRNA decay rates in resting and activated T lymphocytes in order to better understand the role of mRNA decay in regulating gene expression. Purified human T lymphocytes were stimulated for 3 h with medium alone, with an anti-CD3 antibody, or with a combination of anti-CD3 and anti-CD28 antibodies. Actinomycin D was added to arrest transcription, and total cellular RNA was collected at discrete time points over a 2 h period. RNA from each point was analyzed using Affymetrix oligonucleotide arrays and a first order decay model was used to determine the half-lives of approximately 6000 expressed transcripts. We identified hundreds of short-lived transcripts encoding important regulatory proteins including cytokines, cell surface receptors, signal transduction regulators, transcription factors, cell cycle regulators and regulators of apoptosis. Approximately 100 of these short-lived transcripts contained ARE-like sequences. We also identified numerous transcripts that exhibited stimulus-dependent changes in mRNA decay. In particular, we identified hundreds of transcripts whose steady-state levels were repressed following T cell activation and were either unstable in the resting state or destabilized following cellular activation. Thus, rapid mRNA degradation appears to be an important mechanism for turning gene expression off in an activation-dependent manner.

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Figures

Figure 1

Figure 1

Comparison of transcript half-lives determined by northern blot or microarrays. Purified human T lymphocytes were stimulated for 3 h with medium or αCD3+αCD28. Act D was added and total cellular RNA was then isolated at the 0, 45, 90 and 120 min time points. Expression of TNFSF14, MAD-3 and p27kip1 was evaluated by northern blot. Each plot was also probed for GAPDH expression. The blots were quantified using a phosphorimager and the intensity of each band was normalized to the intensity of the GAPDH band. mRNA decay curves were derived for each transcript and were used to calculate transcript half-lives. Transcript half-life values derived using microarrays are also shown. The Affymetrix probe IDs for TNFSF14, MAD-3 and p27kip1 are 31724_at, 1461_at and 33847_s_at, respectively.

Figure 2

Figure 2

Profile of T lymphocyte transcript half-lives. (A) Purified human T lymphocytes were stimulated for 3 h with medium, αCD3 or αCD3+αCD28. Act D was added and total cellular RNA was isolated at discrete time points over a 2 h period. This RNA was used to probe Affymetrix microarrays in order to calculate mRNA half-lives. Transcripts with an Affymetrix ‘present’ call in at least three of four experiments under each stimulation condition were categorized by their median half-life value into five intervals. The median half-life values were calculated based on data from four independent experiments. The data is shown as a percentage of transcripts expressed under each stimulation condition. (B) The subset of transcripts that exhibited 5-fold or greater induction upon stimulation with αCD3 and αCD3+αCD28 were profiled by median half-life values.

Figure 3

Figure 3

Short-lived transcripts whose steady-state levels were induced or repressed upon T cell activation. Purified human T lymphocytes were stimulated for 3 h with medium or αCD3+αCD28. Act D was added and total cellular RNA was isolated at the 0, 45, 90 and 120 min time points. This RNA was used to probe Affymetrix microarrays. The data shown is from an individual experiment and shows raw hybridization intensity (AD) data for 200 short-lived transcripts that were induced or repressed following αCD3+αCD28 stimulation. The intensity data is represented by a color scale, showing low intensity in green and high intensity in red.

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