Inhibition of vasopressin V1b receptor translation by upstream open reading frames in the 5'-untranslated region.
The 5'-UTR of the vasopressin V1b receptor (V1bR) mRNA contains small open reading frames (ORF) located upstream (u) of the main ORF encoding the V1bR. The ability of the three proximal uORFs to be translated into peptides and their influence on V1bR translation was examined using fusion constructs of uORFs and V5 epitope, or ATG/ATA uORF mutations in the V1bR cDNA. In vitro translation and western blot analysis after transfection of uORF1-V5 or uORF2-V5 into cells revealed that uORF1 can be translated. As predicted by computer analysis, in vitro translation using a rabbit reticulocyte/canine microsome system, immunohistochemistry and western blot in membranes of transfected cells with uORF1-V5 revealed translocation of the uORF1 peptide into membrane fractions. In vitro translation of V1bR cDNA with mutations of the two uORFs proximal to the initiating methionine, uORFs 1 and 2 (Mut 1-2), or uORF2 (Mut 2) showed significantly increased translation of a 46 kDa band corresponding to the V1bR, compared with wild-type (WT) V1bR, an effect that was attenuated by cotranslation of uORF1-V5. Consistently, VP-induced inositol phosphate formation was higher in Chinese hamster ovay cells transfected with Mut 1-2 than with WT V1bR. Immunohistochemical and western blot analysis, using an antibody against uORF1, revealed peptide immunoreactivity in rat pituitary but not in liver. Pituitary uORF immunoreactivity increased following glucocorticoid administration. The present study shows that uORFs in the 5'-UTR of the V1bR mRNA inhibit V1bR translation, and suggests that translation of a 38-amino acid membrane peptide encoded by uORF1 exerts tonic inhibition of V1bR translation.
Rabadan-Diehl
C
C
Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Martínez
A
A
Volpi
S
S
Subburaju
S
S
Aguilera
G
G
D024506:5' Flanking Region / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017483:Receptors, Vasopressin / Q000235:genetics* / Q000378:metabolism
D017483:Receptors, Vasopressin