Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (⌬F508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ⌬F508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ⌬F508 CFTR mRNA in the vicinity of the mutation. The consequence of ⌬F508 CFTR mRNA "misfolding" is decreased translational rate. A synonymous single nucleotide variant of the ⌬F508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ⌬F508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ⌬F508 CFTR misfolding. Therefore, we propose that mRNA "misfolding" contributes to ⌬F508 CFTR protein misfolding and consequently to the severity of the human ⌬F508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ⌬F508 homozygous CF patients.
The unfolded protein response (UPR) aids cellular recovery by increasing the capacity and decreasing the protein load of the endoplasmic reticulum (ER). Although the main pathways of the UPR are known, the mechanisms of UPR-associated transcriptional repression have not been explored in mammalian cells. Previous studies indicate that endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency decrease when the UPR is activated. In the present study, we demonstrate that inhibition of CFTR expression under ER stress leads to reduced cAMP-activated chloride secretion in epithelial monolayers, an indication of diminished CFTR function. Moreover, ER stress and the UPR obliterate endogenous ⌬F508 CFTR mRNA expression in CFPAC-1 cells without affecting recombinant ⌬F508 CFTR mRNA levels or mRNA half-life. These results emphasize that transcriptional repression of CFTR under ER stress, in concert with decreased CFTR maturation efficiency, leads to diminished function. Using human CFTR promoter reporter constructs, we confined the ER stress-associated CFTR transcriptional repression to the minimal promoter. Chromatin immunoprecipitation assays established the binding of the UPR-activated ATF6 transcription factor to this region during ER stress, which links the repression to the UPR. Methylation-specific PCR (MSP) revealed hypermethylation of CpG sites inside and in the vicinity of the MAZ transcription factor binding region of CFTR, demonstrating methylation-dependent repression. Using pharmacological inhibitors, we show that both DNA methylation and histone deacetylation contribute to CFTR transcriptional inhibition. These studies provide novel insight into the mechanism of gene repression during the mammalian UPR.In eukaryotic cells, the endoplasmic reticulum (ER) 3 is the site of protein folding and assembly. The unfolded protein response (UPR) can result from ER stress brought on by any number of insults (1-3), including depletion of ER Ca 2ϩ stores (1), proteasome blockade (4), increase in the concentration of reactive oxygen species (5, 6), inflammation (7), overexpression of secretory proteins (2, 8), or altered glycosylation (9). In addition to increasing the capacity of the ER by enhancing the synthesis of membrane components and chaperones (10), the UPR also decreases the ER protein load by enhancing ERAD (11) and to some extent by inhibiting transcription and translation (10,12). Although the principal mechanisms of the UPR have been studied extensively, only limited information is available regarding the extent and specificity of transcriptional repression during the UPR. In yeast, the limited number of genes that are transcriptionally repressed by the UPR encode secreted or cell surface proteins (6). Importantly, neither the extent nor the mechanisms of UPRassociated transcriptional repression have been investigated in mammalian cells.The cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane glycoprotein expressed in the ap...
Zoonotic transmission of Salmonella infections causes an estimated 11% of salmonellosis annually in the United States. This report describes the epidemiologic, traceback and laboratory investigations conducted in the United States as part of four multistate outbreaks of Salmonella infections linked to small turtles. Salmonella isolates indistinguishable from the outbreak strains were isolated from a total of 143 ill people in the United States, pet turtles, and pond water samples collected from turtle farm A, as well as ill people from Chile and Luxembourg. Almost half (45%) of infections occurred in children aged <5 years, underscoring the importance of the Centers for Disease Control and Prevention recommendation to keep pet turtles and other reptiles out of homes and childcare settings with young children. Although only 43% of the ill people who reported turtle exposure provided purchase information, most small turtles were purchased from flea markets or street vendors, which made it difficult to locate the vendor, trace the turtles to a farm of origin, provide education and enforce the United States federal ban on the sale and distribution of small turtles. These outbreaks highlight the importance of improving public awareness and education about the risk of Salmonella from small turtles not only in the United States but also worldwide.
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