Posttranscriptional regulation of gene networks by GU-rich elements and CELF proteins.

GU-rich elements found in pre-mRNA and mRNA transcripts play diverse roles in the control of gene expression by regulating mRNA stability, translation and pre-mRNA processing. Regulatory GU-rich elements are highly conserved throughout evolution, and play major roles in development in diverse species from worms to mammals. The conservation of the GU-rich element allowed it to be identified as a sequence that was enriched in the 3' UTR of human transcripts that exhibited rapid mRNA decay. This element functions, at least in part, as a molecular target for members of the CELF family of RNA-binding proteins, which recruit other components of the cellular posttranscriptional gene regulatory machinery to the transcript. Depending on the context, binding to GU-rich sequences by CELF proteins direct a variety of posttranscriptional regulatory events, including deadenylation, mRNA decay, translation or pre-mRNA processing. Thus, GU-rich elements and CELF proteins serve multiple functions in gene expression regulation and define an important evolutionarily conserved posttranscriptional regulatory network.

Analysis of CUGBP1 targets identifies GU-repeat sequences that mediate rapid mRNA decay.

CUG-repeat binding protein 1 (CUGBP1) mediates selective mRNA decay by binding to GU-rich elements (GREs) containing the sequence UGUUUGUUUGU found in the 3' untranslated region (UTR) of short-lived transcripts. We used an anti-CUGBP1 antibody to immunoprecipitate CUGBP1 from HeLa cytoplasmic extracts and analyzed the associated transcripts using oligonucleotide microarrays. We identified 613 putative mRNA targets of CUGBP1 and found that the UGUUUGUUUGU GRE sequence and a GU-repeat sequence were both highly enriched in the 3' UTRs of these targets. We showed that CUGBP1 bound specifically to the GU-repeat sequence and that insertion of this sequence into the 3' UTR of a beta-globin reporter transcript conferred instability to the transcript. Based on these results, we redefined the GRE to include this GU-repeat sequence. Our results suggest that CUGBP1 coordinately regulates the mRNA decay of a network of transcripts involved in cell growth, cell motility, and apoptosis.

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.

Conserved GU-rich elements mediate mRNA decay by binding to CUG-binding protein 1.

We used computational algorithms to find conserved sequences in the 3' untranslated region (UTR) of transcripts that exhibited rapid decay in primary human T cells and found that the consensus sequence UGUUUGUUUGU, which we have termed a GU-rich element (GRE), was enriched in short-lived transcripts. Using a tet-off reporter system, we showed that insertion of GRE-containing sequences from c-jun, jun B, or TNF receptor 1B, but not mutated GRE sequences, into the 3'UTR of a beta-globin transcript conferred instability on the otherwise stable beta-globin transcript. CUG-binding protein 1 (CUGBP1) was identified as the major GRE-binding activity in cytoplasmic extracts from primary human T cells based on supershift and immunoprecipitation assays. siRNA-mediated knockdown of CUGBP1 in HeLa cells caused stabilization of GRE-containing transcripts, suggesting that CUGBP1 is a mediator of GRE-dependent mRNA decay. Overall, our results suggest that the GRE mediates coordinated mRNA decay by binding to CUGBP1.

Three functional variants of IFN regulatory factor 5 (IRF5) define risk and protective haplotypes for human lupus.

Systematic genome-wide studies to map genomic regions associated with human diseases are becoming more practical. Increasingly, efforts will be focused on the identification of the specific functional variants responsible for the disease. The challenges of identifying causal variants include the need for complete ascertainment of genetic variants and the need to consider the possibility of multiple causal alleles. We recently reported that risk of systemic lupus erythematosus (SLE) is strongly associated with a common SNP in IFN regulatory factor 5 (IRF5), and that this variant altered spicing in a way that might provide a functional explanation for the reproducible association to SLE risk. Here, by resequencing and genotyping in patients with SLE, we find evidence for three functional alleles of IRF5: the previously described exon 1B splice site variant, a 30-bp in-frame insertion/deletion variant of exon 6 that alters a proline-, glutamic acid-, serine- and threonine-rich domain region, and a variant in a conserved polyA+ signal sequence that alters the length of the 3' UTR and stability of IRF5 mRNAs. Haplotypes of these three variants define at least three distinct levels of risk to SLE. Understanding how combinations of variants influence IRF5 function may offer etiological and therapeutic insights in SLE; more generally, IRF5 and SLE illustrates how multiple common variants of the same gene can together influence risk of common disease.

Coordinate stabilization of growth-regulatory transcripts in T cell malignancies.

We used microarray technology to compare mRNA decay rates of approximately 7000 transcripts in normal purified human T lymphocytes or the malignant T cell lines Jurkat and H9 following transcriptional arrest with actinomycin D. We found that over 2000 transcripts were expressed at abnormal levels in malignant T cells, including approximately 100 transcripts that were overexpressed and exhibited abnormally stable mRNA. Seventeen transcripts that encoded components of the ubiquitin-proteasome system were coordinately overexpressed and stabilized in both malignant cell lines. This pathway plays an important role in regulating cell growth and the development of malignancy. Numerous additional transcripts that encode proteins involved in growth regulation, damage repair and stress responses, posttranscriptional gene expression, and mitochondrial metabolism were also coordinately up-regulated and stabilized. Overall, our results suggest that abnormal mRNA stabilization in malignancy can lead to the overexpression of growth-regulatory genes and contribute to the malignant phenotype.

Particular Features of Immune Response after Invasion of Different Species of Human Demodex Mites.

The purpose of the present study was to find peculiarities of the immune response to the invasion of Demodex folliculorum and Demodex brevis mites. Sixty-six patients with human demodicosis were included in this study. The Demodex mites' density was more than 5 per 1 cm(2). The immune response was evaluated by the identification of membrane markers of the different immune cells by monoclonal antibodies. A significant decrease of the percentage of various parameters was found: the decrease for CD5(+) cells was in 1.4, for CD3(+) in 1.7, for CD4(+) in 1.5, for CD8(+) in 1.5, for CD25(+) in 1.3, for CD71(+) in 1.5, for CD20(+) in 1.2, for IgM in 1.2 and for IgG in 1.1 times. On the other hand, some parameters appeared to be elevated: for CD25(+) B cells in 1.5, for IgA in 1.9 and for circulating immune complexes in 1.4 times in patients with D. brevis infection in comparison with patients with D. folliculorum. The development of two variants of the effector response to the invasion of two different Demodex mites' species was shown: the classic expansion of the humoral reaction with the production of IgM and IgG in the case of D. folliculorum, and the activation of non-specific defense cells in the case of D. brevis. The particular capability of D. brevis to suppress T-cell compartment of immunity was distinctly observed in our comparative study. Thus, there are sharp immune distinctions during an invasion of the two species of human Demodex mites.

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.