The human glucocorticoid receptor as an RNA-binding protein: global analysis of glucocorticoid receptor-associated transcripts and identification of a target RNA motif.

Posttranscriptional regulation is emerging as a key factor in glucocorticoid (GC)-mediated gene regulation. We investigated the role of the human GC receptor (GR) as an RNA-binding protein and its effect on mRNA turnover in human airway epithelial cells. Cell treatment with the potent GC budesonide accelerated the decay of CCL2 mRNA (t(1/2) = 8 ± 1 min versus 62 ± 17 min in DMSO-treated cells) and CCL7 mRNA (t(1/2) = 15 ± 4 min versus 114 ± 37 min), but not that of CCL5 mRNA (t(1/2)=231 ± 8 min versus 266 ± 5 min) in the BEAS-2B cell line. This effect was inhibited by preincubation with an anti-GR Ab, indicating that GR itself plays a role in the turnover of these transcripts. Coimmunoprecipitation and biotin pulldown experiments showed that GR associates with CCL2 and CCL7 mRNAs, but not CCL5 mRNA. These methods confirmed CCL2 mRNA targeting by GR in human primary airway epithelial cells. Association of the GR was localized to the 5' untranslated region of CCL2 mRNA and further mapped to nt 44-60. The collection of transcripts associated with GR, identified by immunoprecipitation of GR-mRNA complexes followed by microarray analysis, revealed 479 transcripts that associated with GR. Computational analysis of the primary sequence and secondary structures of these transcripts yielded a GC-rich motif, which was shown to bind to GR in vitro. This motif was used to predict binding of GR to an additional 7889 transcripts. These results indicate that cytoplasmic GR interacts with a subset of mRNA through specific sequences and can regulate turnover rates, suggesting a novel posttranscriptional role for GR as an RNA-binding protein

The Metalloprotease Meprinβ Processes E-Cadherin and Weakens Intercellular Adhesion

BACKGROUND: Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used MDCK and Caco-2 cells stably transfected with meprin alpha and or meprin beta to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprin beta, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins beta-catenin and plakoglobin were processed by an intracellular protease, whereas alpha-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprin beta and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprin beta-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates. CONCLUSIONS/SIGNIFICANCE: By identifying E-cadherin as a substrate for meprin beta in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprin beta in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression

Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms

Role of the RNA-Binding Protein Tristetraprolin in Glucocorticoid-Mediated Gene Regulation

Glucocorticoids (GCs) are the mainstay of anti-inflammatory therapy. Modulation of posttranscriptional regulation (PTR) of gene expression by GCs is a relevant yet poorly characterized mechanism of their action. The RNA-binding protein tristetraprolin (TTP) plays a central role in PTR by binding to AU-rich elements in the 3'-untranslated region of proinflammatory transcripts and accelerating their decay. We found that GCs induce TTP expression in primary and immortalized human bronchial epithelial cells. To investigate the importance of PTR and the role of TTP in GC function, we compared the effect of GC treatment on genome-wide gene expression using mouse embryonic fibroblasts (MEFs) obtained from wild-type and TTP(-/-) mice. We confirmed that GCs induce TTP in MEFs and observed in TTP(-/-) MEFs a striking loss of up to 85% of GC-mediated gene expression. Gene regulation by TNF-alpha was similarly affected, as was the antagonistic effect of GC on TNF-alpha-induced response. Inflammatory genes, including cytokines and chemokines, were among the genes whose sensitivity to GCs was affected by lack of TTP. Silencing of TTP in WT MEFs by small interfering RNA confirmed loss of GC response in selected targets. Immunoprecipitation of ribonucleoprotein complexes revealed binding of TTP to several validated transcripts. Changes in the rate of transcript degradation studied by actinomycin D were documented for only a subset of transcripts bound to TTP. These results reveal a strong and previously unrecognized contribution of PTR to the anti-inflammatory action of GCs and point at TTP as a key factor mediating this process through a complex mechanism of action