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Posttranscriptional regulation of gene expression is an elaborate and intricate process, constituting an important mechanism for the control of protein expression. During its existence, mRNA is escorted by proteins and other RNAs, which control the maturation, transportation, localization, translational efficiency, and ultimately its degradation. Without changes at the transcription level, mRNA steady-state levels can vary dramatically by just small changes in mRNA stability. By influencing the metabolism of specific mRNAs, the abundance of specific mRNAs can be controlled in organisms from bacteria to mammals. In eukaryotic cells, the control of mRNA stability is exerted through specific cis-acting elements (sequence-specific control elements) and trans-acting factors (mRNA binding proteins and some miRNAs). mRNA stability appears to be a key regulator in controlling the expression of many proteins. Dysregulation of mRNA stability has been associated with human diseases, including cancer, inflammatory disease, and Alzheimer's. These observations suggest that modulating the stability of specific mRNAs may represent a viable strategy for pharmaceutical intervention. The literature already describes several compounds that influence mRNA stability. Measuring mRNA stability by conventional methods is labor intensive and time-consuming. However, several systems have been described that can be used to screen for modulators of mRNA levels in a high-throughput format. Thus, these assay systems offer a novel approach for screening targets that at present appear to be poorly "drugable." This review describes the utility of mRNA stability as a novel approach to drug discovery, focusing on assay methods and tool compounds available to monitor mRNA stability. The authors describe mRNA stability assays and issues related to this approach.

The AU-rich element (ARE) is a stability determinant found in the 3' UTR of a number of short-lived mRNAs. The best characterized ARE is the Shaw-Kamen (SK) box or AUUUA motif. Previously, a fungal metabolite, radicicol analog A (RAA), was shown to destabilize SK box-containing mRNAs based on 16 mRNAs examined [T. Kastelic et al., Cytokine 8 (1996) 751-761]. Using serial analysis of gene expression (SAGE) to examine the global effect of RAA on mRNA expression in interferon-gamma/lipopolysaccharide-stimulated THP-1 human monocytes, we observed that the expression level of greater than 99% of the SAGE tags was unchanged by RAA treatment and only 34 of the 17,608 unique tags annotated were reduced (p< or =0.0001). RAA destabilized approximately half of the down-regulated transcripts. Whereas all the destabilized mRNAs possessed at least one SK box, for transcripts not destabilized but nonetheless down-regulated, RAA appears to function by a SK box-independent mechanism not currently understood.

A radicicol analogue (analogue A) was found to inhibit interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha) secretion from THP-1 cells. If added to cells activated by interferon gamma and lipopolysaccharide, radicicol analogue A not only inhibited the secretion of IL-1 beta but also induced an extremely rapid degradation of IL-1 beta, IL-6 and TNF-alpha mRNA to undetectable levels within 5-8 h. This degradation is independent of translation and of the signal inducing transcription. The common feature of these genes is the inclusion of one or more copies of the mRNA-instability sequence, AUUUA, in the 3' untranslated region. Indeed, no destabilizing effect of radicicol analogue A could be observed on mRNA derived from the expression of an IL-1 beta construct lacking the AUUUA motifs of the 3'UTR. The effect of radicicol analogue A on protein/mRNA interaction and on post-translational modifications of cytoplasmic proteins is described. This class of compound constitutes a valuable tool for the further elucidation of the mechanism of mRNA degradation of cytokines and proto-oncogenes.

We identified the earliest events in autophosphorylation of the insulin receptor after insulin addition. Insulin-stimulated autophosphorylation at specific sites in the tyrosine kinase domain of the receptor's beta-subunit is correlated kinetically with activation of kinase-catalyzed phosphorylation of a model substrate (reduced and carboxyamidomethylated lysozyme; RCAM-lysozyme). To identify these sites, the deduced amino acid sequence of the 3T3-L1 adipocyte insulin receptor of the mouse was determined. Insulin-induced activation of substrate phosphorylation was shown to require autophosphorylation of three neighboring tyrosines (Tyr1148, Tyr1152, and Tyr1153) in the mouse receptor. A search for cellular substrates of the receptor kinase revealed that insulin causes accumulation of a 15,000-Mr phosphorylated (on tyrosine) cytosolic protein (pp15) in 3T3-L1 adipocytes treated with oxophenylarsine (PAO). PAO blocks turnover of the phosphoryl group of pp15, causing its accumulation, and thereby appears to interrupt signal transmission from the receptor to the glucose-transport system. Two membrane-bound protein phosphotyrosine phosphatases that are inhibited by PAO and are apparently responsible for the turnover of the pp15 phosphoryl group have been purified from 3T3-L1 adipocytes and characterized. These and other results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin-receptor tyrosine kinase action, couples signal transmission to the glucose-transport system. [32P]pp15 was purified to homogeneity from 3T3-L1 adipocytes. Amino acid and radiochemical sequence analysis of the purified tryptic [32P]phosphopeptide revealed that pp15 is the phosphorylation product of 422(aP2) protein, a 15,000-Mr adipocyte protein whose cDNA we previously cloned and sequenced. 422(aP2) protein was found to bind fatty acids. When exposed to a free fatty acid, notably oleic acid, 422(aP2) protein becomes an excellent substrate of the isolated insulin-receptor tyrosine kinase. Compelling evidence indicates that on binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19 becomes accessible to the receptor tyrosine kinase and undergoes O-phosphorylation. Adipose tissue and skeletal and heart muscle, which exhibit insulin-stimulated glucose uptake, express a specific insulin-responsive glucose transporter. A cDNA (GT2) that encodes this protein was isolated from a mouse 3T3-L1 adipocyte library and sequenced. We also isolated and characterized the corresponding mouse gene GLUT4. DNase I footprinting with nuclear extracts from 3T3-L1 cells revealed that a differentiation-specific nuclear factor binds to the GLUT4 promoter. The purified transcription factor C/EBP binds at the same position.(ABSTRACT TRUNCATED AT 400 WORDS)