Angiotensin (Ang II) receptors (R), which are widely distributed, play a major role in blood pressure regulation and electrolyte balance. The 5' leader sequence (5'LS) of the mRNA coding for the type 1 Ang II R (AT/1 R) shares distinctive features with other mRNAs that are partially controlled at the level of translation, including the likelihood of highly stable secondary structure which can play a critical role in RNA- protein recognition. Consistent with this possibility, we have discovered cytosolic proteins (BP) in rat (r) tissues that bind to the 5'LS of the rat/1a R mRNA and inhibit translation. Deletion and completion experiments indicate that RNA BP specifically interact with a 65 nt RNA cis element with the 282 nt rAT/1a 5'LS. This proposal focuses on several questions related to these intriguing preliminary findings.
In Aim 1, we propose to fully characterize the mRNA BP which interact with RNA cis elements in the rAT/1a 5'LS. The BP cis element will be determined by a combination of approaches including Tl RNase and RNase H mapping experiments as well as electromobility shift assays (EMSA) using a series of 5'LS mutant RNAs. The specificity of 5'LS-BP and the optimal sequence of RNA cis elements will be determined with various RNAs in EMSA competition experiments. Finally, the 5'LS-BP will be purified and cloned.
In Aim 2, the role of 5'LS-BP in regulating translation will be examined: in vitro, using a rabbit reticulocyte lysate preparation and in vivo, in intact cells using vascular smooth muscle cells. The dose dependence of 5'LS-BP on inhibition of rAT/1a R translation will be determined as well as the effect of competitor RNAs on abrogation of this inhibition. The role of 5'LS-AT/1a R. In the latter studies, the effect of 5'ls mutants on Rat/1A R protein expression and mRNA translation rates will be determined.
In Aim 3, we will assess the role of 5'LS-BP in regulation of rAT/1A R in an animal model in order to determine the physiological significance of 5'LS-BP in R up- and down-regulation. By comparing changes in R expression with changes in 5'LS-BP activity, mRNA translation, and transcription rates, we will determine the relative importance of translational control of AT/1a R expression by 5'LS-BP activity, mRNA translation, and transcription rates, we will determine the relative importance of translational control of AT/1a R expression by 5'LS-BP in mediating physiological changes in R changes. These proposed experiments will therefore evaluate the general hypothesis that mRNA BP acting upon cis elements in the 5'LS regulate AT/1a R expression by inhibiting R translation. Thus, this grant will establish the foundation for investigation of post-transcriptional regulatory mechanisms involving the 5'LS in modulating the activity of the renin angiotensin system, and serve as a paradigm for the study of the involvement of translational regulation of other G-protein coupled receptor mRNAs.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL057502-03
Application #
6389612
Study Section
Cardiovascular and Renal Study Section (CVB)
Program Officer
Lin, Michael
Project Start
1999-04-01
Project End
2003-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
3
Fiscal Year
2001
Total Cost
$231,730
Indirect Cost
Name
Georgetown University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Sandberg, Kathryn; Ji, Hong; Einstein, Gillian et al. (2016) Is immune system-related hypertension associated with ovarian hormone deficiency? Exp Physiol 101:368-74
Ji, Hong; Zheng, Wei; Wu, Xie et al. (2016) Aging-related impairment of urine-concentrating mechanisms correlates with dysregulation of adrenocortical angiotensin type 1 receptors in male Fischer rats. Am J Physiol Regul Integr Comp Physiol 310:R513-21
Sandberg, Kathryn; Ji, Hong (2013) Is ? the ? dog in estrogen receptor-mediated protection from hypertension? Hypertension 61:1153-4
Sandberg, Kathryn; Samson, Willis K; Ji, Hong (2013) Decoding noncoding RNA: da Vinci redux? Circ Res 113:240-1
Ji, Hong; Zheng, Wei; Wu, Xie et al. (2010) Sex chromosome effects unmasked in angiotensin II-induced hypertension. Hypertension 55:1275-82
Ji, Hong; Menini, Stefano; Zheng, Wei et al. (2008) Role of angiotensin-converting enzyme 2 and angiotensin(1-7) in 17beta-oestradiol regulation of renal pathology in renal wrap hypertension in rats. Exp Physiol 93:648-57
Sandberg, Kathryn; Ji, Hong (2008) Why can't a woman be more like a man?: Is the angiotensin type 2 receptor to blame or to thank? Hypertension 52:615-7
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Hassan, Ali; Ji, Hong; Zhang, Yinghua et al. (2006) Splice variant-specific silencing of angiotensin II type 1a receptor messenger RNA by RNA interference in vascular smooth muscle cells. Biochem Biophys Res Commun 339:499-505
Lee, Sunghou; Wu, Zheng; Sandberg, Kathryn et al. (2006) Posttranscriptional mechanisms contribute to osmotic regulation of ANG type 1 receptors in cultured rat renomedullary interstitial cells. Am J Physiol Regul Integr Comp Physiol 290:R44-9

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