Tristetraprolin mediates interferon-gamma mRNA decay.

Tristetraprolin (TTP) regulates expression at the level of mRNA decay of several cytokines, including the T cell-specific cytokine, interleukin-2. We performed experiments to determine whether another T cell-specific cytokine, interferon-gamma (IFN-gamma), is also regulated by TTP and found that T cell receptor-activated T cells from TTP knock-out mice overproduced IFN-gamma mRNA and protein compared with activated T cells from wild-type mice. The half-life of IFN-gamma mRNA was 23 min in anti-CD3-stimulated T cells from wild-type mice, whereas it was 51 min in anti-CD3-stimulated T cells from TTP knock-out mice, suggesting that the overexpression of IFN-gamma mRNA in TTP knock-out mice was due to stabilization of IFN-gamma mRNA. Insertion of a 70-nucleotide AU-rich sequence from the murine IFN-gamma 3'-untranslated region, which contained a high affinity binding site for TTP, into the 3'-untranslated region of a beta-globin reporter transcript conferred TTP-dependent destabilization on the beta-globin transcript. Together these results suggest that TTP binds to a functional AU-rich element in the 3'-untranslated region of IFN-gamma mRNA and mediates rapid degradation of the IFN-gamma transcript. Thus, TTP plays an important role in turning off IFN-gamma expression at the appropriate time during an immune response.

Tristetraprolin recruits functional mRNA decay complexes to ARE sequences.

AU-rich elements (AREs) in the 3' untranslated region (UTR) of numerous mammalian transcripts function as instability elements that promote rapid mRNA degradation. Tristetraprolin (TTP) is an ARE-binding protein that promotes rapid mRNA decay through mechanisms that are poorly understood. A 31 nucleotide ARE sequences from the TNF-alpha 3' UTR promoted TTP-dependent mRNA decay when it was inserted into the 3' UTR of a beta-globin reporter transcript, indicating that this short sequence was sufficient for TTP function. We used a gel shift assay to identify a TTP-containing complex in cytoplasmic extracts from TTP-transfected HeLa cells that bound specifically to short ARE sequences. This TTP-containing complex also contained the 5'-3' exonuclease Xrn1 and the exosome component PM-scl75 because it was super-shifted with anti-Xrn1 or anti-PMscl75 antibodies. RNA affinity purification verified that these proteins associated specifically with ARE sequences in a TTP-dependent manner. Using a competition binding assay, we found that the TTP-containing complex bound with high affinity to short ARE sequences from GM-CSF, IL-3, TNF-alpha, IL-2, and c-fos, but did not bind to a U-rich sequence from c-myc, a 22 nucleotide poly U sequence or a mutated GM-CSF control sequence. High affinity binding by the TTP-containing complex correlated with TTP-dependent deadenylation and decay of capped, polyadenylated transcripts in a cell-free mRNA decay assay, suggesting that the TTP-containing complex was functional. These data support a model whereby TTP functions to enhance mRNA decay by recruiting components of the cellular mRNA decay machinery to the transcript.

Treatment of biofilm infections on implants with low-frequency ultrasound and antibiotics.

Medical implants are sometimes colonized by biofilm-forming bacteria, which are very difficult to treat effectively. The combination of gentamicin and ultrasonic exposure for 24 hours was previously shown to reduce the viability of Escherichia coli biofilms in vivo. This article shows that such treatment for 48 hours reduced viable E coli bacteria to nearly undetectable levels. However, when Pseudomonas aeruginosa biofilms were implanted and treated for 24 and 48 hours, no significant ultrasonic-enhanced reduction of viable bacteria was observed. The difference in response of these 2 organisms is attributed to greater impermeability and stability of the outer membrane of P aeruginosa.

Tristetraprolin down-regulates IL-2 gene expression through AU-rich element-mediated mRNA decay.

Posttranscriptional regulation of IL-2 gene expression at the level of mRNA decay is mediated by an AU-rich element (ARE) found in the 3'-untranslated region. We hypothesized that the ARE-binding protein tristetraprolin (TTP) regulates T lymphocyte IL-2 mRNA decay by interacting with the IL-2 ARE and targeting the transcript for decay. rTTP protein expressed in HeLa cells bound specifically to the IL-2 ARE with high affinity in a gel shift assay. In primary human T lymphocytes, TTP mRNA and protein expression were induced by TCR and CD28 coreceptor stimulation. Using a gel shift assay, we identified a cytoplasmic RNA-binding activity that was induced by TCR and CD28 coreceptor stimulation and bound specifically to the IL-2 ARE sequence. Using anti-TTP Abs, we showed by supershift that this inducible activity contained TTP. We also showed that insertion of the IL-2 ARE sequence into the 3'-untranslated region of a beta-globin reporter construct conferred TTP-dependent mRNA destabilization on the beta-globin reporter. To determine whether TTP also regulates IL-2 gene expression in vivo, we examined IL-2 expression in primary cells from wild-type and TTP knockout mice. Compared with their wild-type counterparts, TCR- and CD28-activated splenocytes and T cells from TTP knockout mice overexpressed IL-2 mRNA and protein. Also, IL-2 mRNA was more stable in activated splenocytes from TTP knockout mice compared with wild-type mice. Taken together, these data suggest that TTP functions to down-regulate IL-2 gene expression through ARE-mediated mRNA decay.

Patterns of coordinate down-regulation of ARE-containing transcripts following immune cell activation.

We evaluated the expression of over 900 AU-rich element (ARE)-containing transcripts in primary human T lymphocytes following stimulation with anti-CD3 and anti-CD28 antibodies and found that approximately 48% of these transcripts were regulated following T cell activation. We identified approximately 145 ARE-containing transcripts that were rapidly induced and then rapidly disappeared within 1 h after activation. Another 250 ARE-containing transcripts expressed in resting T cells were rapidly turned off within 30 min after activation. The rates of transcript disappearance correlated well with rapid mRNA decay measured following transcriptional arrest with actinomycin D. We identified a subset of ARE-containing transcripts that were rapidly induced following T cell activation that were also induced following lipopolysaccharide stimulation of THP-1 monocytes, and these transcripts exhibited rapid decay in both cell types. Our results suggest that ARE-mediated mRNA decay plays an important role in the precisely coordinated down-regulation of gene expression following immune cell activation.

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

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.