Translational activation and repression by distinct elements within the 5'-UTR of ENaC alpha-subunit mRNA.
The rat amiloride-sensitive epithelial Na(+) channel (rENaC), the rate-limiting step in epithelial Na(+) transport, consists of three subunits, alpha, beta, and gamma. We hypothesized that alpha-rENaC translation is regulated via its 5'-untranslated region (UTR). Transient transfections of alpha-rENaC promoter-reporter constructs in representative epithelial cell lines demonstrated up to fivefold differences in activity among constructs containing different amounts of the alpha-rENaC 5'-UTR sequence. Differences in reporter protein activity did not parallel differences in reporter mRNA, demonstrating that 5'-UTR regulation must be at the level of translation. Specifically, translation was enhanced by a region extending from +53 to +211 bp downstream from the transcription start site and repressed by the region between +367 and +499 bp. Examination of the 5'-UTR sequence revealed an out-of-frame initiation codon within the repressive region, 43 bp upstream from the start of the alpha-rENaC open reading frame. Mutational analysis of this upstream start codon indicated that it plays, at most, a minor role in impeding translation both in vitro and in vivo, suggesting that additional mechanisms of translational regulation are operative.
Otulakowski
G
G
Department of Paediatrics, University of Toronto, Ontario M5G 1X8, Canada. gotulak@sickkids.on.ca
Freywald
T
T
Wen
Y
Y
O'Brodovich
H
H
D020121:5' Untranslated Regions / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018387:Codon, Initiator; D004847:Epithelial Cells / Q000378:metabolism; D053503:Epithelial Sodium Channels; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D021122:Protein Subunits; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D015222:Sodium Channels / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D020121:5' Untranslated Regions; D018387:Codon, Initiator; D053503:Epithelial Sodium Channels; D021122:Protein Subunits; D012333:RNA, Messenger; D015222:Sodium Channels