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Regulation of Angiotensin Receptor (AT1aR) Translation By Premature Termination
Author(s) -
Fabucci Maria Elena,
Sandberg Kathryn
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1191-a
Subject(s) - translation (biology) , luciferase , exon , messenger rna , rna splicing , splice , stop codon , start codon , transfection , microbiology and biotechnology , eukaryotic translation , hek 293 cells , open reading frame , mutant , alternative splicing , biology , chemistry , gene , rna , genetics , peptide sequence
Objective Determine the role of termination codons within the 5′leader sequence (5′LS) of the rat AT 1a R mRNA in mRNA translation. Methods We have developed a novel in vitro assay to study the role of cis ‐acting elements within the AT 1a R 5′LS in translation. There are two splice variants created by alternative splicing of exon 2 (5′LS E1,2,3 and 5′LS E1,3). These two splice variants were subcloned in frame with the luciferase gene to create 5′LS E1,3‐Luc and 5′LS E1,2,3‐Luc. Two mutants were also generated: ‐‐‐/T −93 AA, in which the TAA codon at position in −57 was deleted; T −57 AA/‐‐‐‐, in which the TAA codon at position in −93 was deleted. Constructs were transiently transfected in 293 human embryonic kidney cells (HEK) before luciferase activity was determined. Results The presence of E2 markedly inhibited luciferase activity by 43% at 48h, 39% at 72h and 43% at 96h after transfection. The mutant ‐‐‐/T −93 AA relieved the inhibitory effect of E2 on translation by 48% at 48h while T −57 AA/‐‐‐‐ had minimal effects. Conclusions We previously showed that disruption of two AUGs within E2 markedly relieved E2 inhibition of AT 1a R translation. Our new finding that the E2 inhibitory effect on translation is abrogated by deletion of the −57 termination codon in frame with these 2 upstream AUGs suggests that AT 1a Rs can be regulated by premature translation initiation and termination via alternative splicing. Supported by NKF grant