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.
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