Angiotensinogen messenger RNA stabilization by angiotensin II.
OBJECTIVE
To further characterize the molecular mechanism whereby angiotensin II stabilizes the angiotensinogen messenger (m)RNA through binding studies of the previously isolated polysomal stabilizing protein to partial and mutagenized sequences of the 3' untranslated region of the gene and to explore its importance to rodent genetic hypertension.


METHODS
Analysis of angiotensinogen mRNA mutants for half-life and binding to a polysomal protein with a molecular weight of 12000.


METHODS
Protein/RNA interactions were determined in band shift assays employing radiolabelled 3' untranslated region of angiotensinogen mRNA. Measurement of the mRNA half-life used a cell-free incubation system and 3' untranslated region DNA sequences were polymerase chain reaction (PCR) cloned and sequenced. Sequences of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rat (SHR) strains were compared. Point mutations were introduced by site directed mutagenesis.


RESULTS
The angiotensinogen 3' untranslated region exhibited specific binding to the polysomal 12000 molecular weight protein which, in an in vitro incubation system, increased 10-fold the half-life of full-length angiotensinogen mRNA; no effect was observed with 3' deleted angiotensinogen mRNA indicating a regulatory function of protein at the 3' untranslated region. Sequence analysis of PCR amplified DNA fragments identified a (G-->C) point mutation in the La Jolla colony SHR. Following introduction of this point mutation into wild-type 3' untranslated regions, protein binding significantly increased (wild-type binding constant, 19 mumol/l; mutant binding constant 3.5 mumol/l), indicating that this point mutation affects 3' untranslated region secondary structure, binding of the RNA stabilizing protein and, consequently, the half-life of angiotensinogen mRNA. Deletion of a U-rich region (position 1609-1613, UCCUU) expressed twice in the 3' untranslated region almost completely abolished protein binding suggesting this sequence as one part of the putative binding motif in the 3' untranslated region.


CONCLUSIONS
Angiotensin II regulates hepatic angiotensinogen synthesis and secretion by inhibiting degradation of angiotensinogen mRNA by the action of a polysomal protein. Mutations in the 3' untranslated region mRNA coding sequence alter binding and half-life and may significantly affect the half-life of angiotensinogen mRNA thereby altering the secretion rate of angiotensinogen.
Klett
C P
CP
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Printz
M P
MP
Bader
M
M
Ganten
D
D
Eggena
P
P
D000804:Angiotensin II / Q000494:pharmacology*; D000808:Angiotensinogen / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002469:Cell Separation; D004355:Drug Stability; D016678:Genome; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011132:Polyribosomes / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011918:Rats, Inbred SHR / Q000235:genetics; D011921:Rats, Inbred WKY / Q000235:genetics; D017207:Rats, Sprague-Dawley
D012333:RNA, Messenger; D000808:Angiotensinogen; D000804:Angiotensin II