Nonsense-mediated mRNA decay in health and disease.
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All eukaryotes possess the ability to detect and degrade transcripts harboring premature signals for the termination of translation. Despite the ubiquitous nature of nonsense-mediated mRNA decay (NMD) and its demonstrated role in the modulation of phenotypes resulting from selected nonsense alleles, very little is known regarding its basic mechanism or the selective pressure for complete evolutionary conservation of this function. This review will present the current models of NMD that have been generated during the study of model organisms and mammalian cells. The physiological burden of nonsense transcripts and the emerging view that NMD plays a broad and critical role in the regulation of gene expression will also be discussed. Such issues are relevant to the proposal that pharmacological manipulation of NMD will find therapeutic application. (+info)
The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism.
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Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline-controlled expression system to determine the half-lives of interleukin (IL)-6 and IL-8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal-induced stabilization was transferred to the stable beta-globin mRNA by a 161-nucleotide fragment of IL-8 mRNA which contains an AU-rich region, as well as by defined AU-rich elements (AREs) of the c-fos and GM-CSF mRNAs. Of the different MEKK1-activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF-kappaB pathways. Selective activation of the p38 MAP kinase (=SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant-negative mutant of p38 MAP kinase interfered with MEKK1 and also IL-1-induced stabilization. Furthermore, an active form of the p38 MAP kinase-activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6-induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress-induced gene expression by stabilizing mRNAs through an MK2-dependent, ARE-targeted mechanism. (+info)
In vivo evidence for back and forth oscillations of the transcription elongation complex.
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We have used a combination of DNA and RNA footprinting experiments to analyze the structural rearrangements experienced by a transcription elongation complex that was halted in vivo by a protein readblock. We show that the complex readblocked within an (ATC/TAG)(n) sequence is in a dynamic equilibrium between upstream- and downstream- translocated conformers. By increasing the strength of the putative RNA-DNA hybrid, the ternary complex is readily trapped in the downstream-translocated conformation, where the melted DNA region is limited to 8 bp. The shift of the equilibrium towards the downstream location is also achieved by introducing within the 5' end of the message an RNA sequence that can pair with a segment of the transcript in the vicinity of the halted ternary complex. Our results demonstrate that within certain template DNA sequences, the back and forth oscillations of the ternary complex actually occur in a multipolymerase system and inside the cell. Furthermore, the cis-acting effect of the upstream RNA sequence underscores an important phenomenon in gene regulation where a transcript may regulate its own elongation. (+info)
Identification of a novel dexamethasone-sensitive RNA-destabilizing region on rat monocyte chemoattractant protein 1 mRNA.
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Glucocorticoids are potent anti-inflammatory agents widely used in the treatment of human disease. We have previously shown that the inflammatory cytokine monocyte chemoattractant protein 1 (MCP-1) is regulated posttranscriptionally by glucocorticoids in arterial smooth muscle cells (SMC). To elucidate the mechanism mediating this effect, in vitro-transcribed radiolabeled MCP-1 mRNA was incubated with cytoplasmic extracts from SMC and analyzed by gel electrophoresis. Extracts from SMC treated with platelet-derived growth factor (PDGF) did not degrade the transcripts for up to 3 h. In contrast, extracts from cells treated with 1 microM dexamethasone (Dex) alone or in combination with PDGF degraded the probe with a half-life of approximately 15 min. Dex had maximal effect at concentrations above 0.01 microM and was effective on both rat and human MCP-1 transcripts. By deletion analysis, the Dex-sensitive region of the MCP-1 mRNA was localized to the initial 224 nucleotides (nt) at the 5' end and did not involve an AU-rich sequence in the 3' untranslated end. The 224-nt region conferred Dex sensitivity to heterologous mRNA. These studies provide new insights into the molecular mechanisms underlying the effect of glucocorticoids on gene expression. (+info)
Yeast Upf proteins required for RNA surveillance affect global expression of the yeast transcriptome.
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mRNAs are monitored for errors in gene expression by RNA surveillance, in which mRNAs that cannot be fully translated are degraded by the nonsense-mediated mRNA decay pathway (NMD). RNA surveillance ensures that potentially deleterious truncated proteins are seldom made. NMD pathways that promote surveillance have been found in a wide range of eukaryotes. In Saccharomyces cerevisiae, the proteins encoded by the UPF1, UPF2, and UPF3 genes catalyze steps in NMD and are required for RNA surveillance. In this report, we show that the Upf proteins are also required to control the total accumulation of a large number of mRNAs in addition to their role in RNA surveillance. High-density oligonucleotide arrays were used to monitor global changes in the yeast transcriptome caused by loss of UPF gene function. Null mutations in the UPF genes caused altered accumulation of hundreds of mRNAs. The majority were increased in abundance, but some were decreased. The same mRNAs were affected regardless of which of the three UPF gene was inactivated. The proteins encoded by UPF-dependent mRNAs were broadly distributed by function but were underrepresented in two MIPS (Munich Information Center for Protein Sequences) categories: protein synthesis and protein destination. In a UPF(+) strain, the average level of expression of UPF-dependent mRNAs was threefold lower than the average level of expression of all mRNAs in the transcriptome, suggesting that highly abundant mRNAs were underrepresented. We suggest a model for how the abundance of hundreds of mRNAs might be controlled by the Upf proteins. (+info)
Induction of interleukin-8 synthesis integrates effects on transcription and mRNA degradation from at least three different cytokine- or stress-activated signal transduction pathways.
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A hallmark of inflammation is the burst-like formation of certain proteins, initiated by cellular stress and proinflammatory cytokines like interleukin 1 (IL-1) and tumor necrosis factor, stimuli which simultaneously activate different mitogen-activated protein (MAP) kinases and NF-kappaB. Cooperation of these signaling pathways to induce formation of IL-8, a prototype chemokine which causes leukocyte migration and activation, was investigated by expressing active and inactive forms of protein kinases. Constitutively active MAP kinase kinase 7 (MKK7), an activator of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway, induced IL-8 synthesis and transcription from a minimal IL-8 promoter. Furthermore, MKK7 synergized in both effects with NF-kappaB-inducing kinase (NIK). Activation of the IL-8 promoter by either of the kinases required functional NF-kappaB and AP-1 sites. While NIK and MKK7 did not affect degradation of IL-8 mRNA, an active form of MKK6, which selectively activates p38 MAP kinase, induced marked stabilization of the transcript and further increased IL-8 protein formation induced by NIK plus MKK7. Consistently, the MAP kinase kinase kinase MEKK1, which can activate NF-kappaB, SAPK/JNK, and p38 MAP kinases, most potently induced IL-8 formation. These results provide evidence that maximal IL-8 gene expression requires the coordinate action of at least three different signal transduction pathways which cooperate to induce mRNA synthesis and suppress mRNA degradation. (+info)
Mutation analysis of the 5' untranslated region of the cold shock cspA mRNA of Escherichia coli.
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The mRNA for CspA, a major cold shock protein in Escherichia coli, contains an unusually long (159 bases) 5' untranslated region (5'-UTR), and its stability has been shown to play a major role in cold shock induction of CspA. The 5'-UTR of the cspA mRNA has a negative effect on its expression at 37 degrees C but has a positive effect upon cold shock. In this report, a series of cspA-lacZ fusions having a 26- to 32-base deletion in the 5'-UTR were constructed to examine the roles of specific regions within the 5'-UTR in cspA expression. It was found that none of the deletion mutations had significant effects on the stability of mRNA at both 37 and 15 degrees C. However, two mutations (Delta56-86 and Delta86-117) caused a substantial increase of beta-galactosidase activity at 37 degrees C, indicating that the deleted regions contain a negative cis element(s) for translation. A mutation (Delta2-27) deleting the highly conserved cold box sequence had little effect on cold shock induction of beta-galactosidase. Interestingly, three mutations (Delta28-55, Delta86-117, and Delta118-143) caused poor cold shock induction of beta-galactosidase. In particular, the Delta118-143 mutation reduced the translation efficiency of the cspA mRNA to less than 10% of that of the wild-type construct. The deleted region contains a 13-base sequence named upstream box (bases 123 to 135), which is highly conserved in cspA, cspB, cspG, and cspI, and is located 11 bases upstream of the Shine-Dalgarno (SD) sequence. The upstream box might be another cis element involved in translation efficiency of the cspA mRNA in addition to the SD sequence and the downstream box sequence. The relationship between the mRNA secondary structure and translation efficiency is discussed. (+info)
Downregulation of the human taurine transporter by glucose in cultured retinal pigment epithelial cells.
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In diabetes, activation of the aldose reductase (AR) pathway and alterations of glucose-sensitive signal transduction pathways have been implicated in depletion of intracellular taurine, an endogenous antioxidant and compatible osmolyte. Cellular taurine accumulation occurs by an osmotically induced, protein kinase C (PKC)-regulated Na(+)-taurine cotransporter (hTT). The effects of ambient glucose on taurine content, hTT activity, and hTT gene expression were therefore evaluated in low and high AR-expressing human retinal pigment epithelial cell lines. In low AR-expressing cells, 20 mM glucose decreased taurine content, hTT transporter activity, and mRNA levels, and these effects were unaffected by AR inhibition (ARI). In these cells, the inhibitory effects of high glucose on hTT appeared to be posttranscriptionally mediated, because 20 mM glucose decreased hTT mRNA stability without affecting hTT transcriptional rate. Inhibition of PKC overcame the decrease in hTT activity in high glucose-exposed cells. In high AR-expressing cells, prolonged exposure to 20 mM glucose resulted in intracellular taurine depletion, which paralleled sorbitol accumulation and was prevented by ARI. In these cells exposed to 5 mM glucose, hTT mRNA abundance was decreased and declined further in 20 mM glucose but was corrected by ARI. In 5 mM glucose, hTT transcriptional rate was markedly decreased in high AR-expressing cells, did not decline further in 20 mM glucose, but was increased by ARI to levels above those observed in low AR-expressing cells. Therefore, glucose rapidly and specifically decreases taurine content, hTT activity, and mRNA abundance by AR-unrelated and AR-related posttranscriptional and transcriptional mechanisms. (+info)