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Post-transcriptional regulation of the AT1 receptor mRNA. Identification of the mRNA binding motif and functional characterization¹

Klinik und Poliklinik Innere Medizin III, Universität des Saarlandes, 66421 Homburg, Germany

³Correspondence: Klinik und Poliklinik Innere Medizin III, Universität des Saarlandes, 66421 Homburg, Germany. E-mail: nickenig{at}med-in.uni-sb.de

SPECIFIC AIMS

The expression of the angiotensin AT1 receptor that plays a pivotal role in cardiovascular pathophysiology is regulated predominately via post-transcriptional mechanisms. This study investigated interactions of polysomal proteins with the 3'-untranslated region of the AT1 receptor mRNA that are potentially involved in post-transcriptional regulation.

PRINCIPAL FINDINGS

1. Destabilization of AT1 receptor mRNA
Angiotensin II reduces the AT1 receptor mRNA half-life from 6 to 2 h, as assessed by experiments under transcriptional blockade with 5,6-dichlorobenzimidazole in rat aortic smooth muscle cells in culture (VSMC). In vitro mRNA decay assays in isolated polysomes confirmed the destabilizing effect of angiotensin II on the AT1 receptor mRNA.

2. Interaction of polysomal proteins with the AT1 receptor mRNA
Polysomal proteins isolated from VSMC were incubated with various AT1 receptor mRNA transcripts, cross-linked by UV light, and separated by SDS-polyacrylamide electrophoresis. The analysis of multiple experiments led to the identification of at least six proteins that specifically bind to the 3'-untranslated region of the AT1 receptor mRNA from bases 1864–2213. The relative molecular masses were 100, 63, 60, 47, 43, and 22 kDa, respectively. Mutational studies narrowed the binding region to the region bases 2175–2195 at the very 3' end of the 3'-untranslated region compromising the nucleotides 5'-AAGUAAUUUUAUUGUAAUGU-3'. Figure 1 displays a representative experiments using the AT1 receptor riboprobe base 1864–2213 and RNA competitors base 2175–2195 (5'-AAGUAAUUUUAUUGUAAUGU-3') and base 2196–2213 (5'-AAAAAAAAAAAAAAAAA-3'). In addition, competition with the AT1 receptor mRNA transcript base 1864–2213 served as positive control and competition with a 30- and 50-fold excess of a nonspecific competitor Xen.elongation factor 1 served as negative control.

View larger version (97K): [in this window] [in a new window]   Figure 1. UV cross-link assay of polysomal proteins to the AT1 receptor mRNA. Representative autoradiogram of the AT1 receptor mRNA base 1864–2213 cross-linked to polysomal proteins isolated from VSMC. As indicated at the top, some reaction included 30- and 10-fold excess of unlabeled AT1 receptor mRNA competitors base 1864–2213, 2175–2195, 2196–2213, and an unspecific RNA (Xen.E.factor 1) in a 30- and 50-fold excess. Autoradiographs are representative of 3–5 separate experiments.

The nonspecific competitor and the poly(A)-tail (base 2196–2213) showed no competition, whereas the AT1 receptor mRNA bases 1864–2213 and 2175–2195 displayed effective competition for the binding proteins.

Polysomal proteins from VSMC did not bind to GAPDH mRNA, showed a different binding pattern with the eNOS mRNA, and revealed no interaction with an AT1 receptor mRNA transcript bases 1864–2213 lacking bases 2175–2195. Binding of polysomal proteins from bovine endothelial cells and cos-7 cells to the AT1 receptor mRNA was essentially different or absent, respectively.

3. Inducible binding of polysomal proteins to the AT1 receptor mRNA
Angiotensin II destabilizes AT1 receptor mRNA. Binding of a 50 and 60 kDa polysomal protein to the AT1 receptor mRNA was induced in VSMC preincubated for 2 h with 100 nmol/l angiotensin II.

4. Functional effect of binding sequences on AT1 receptor mRNA expression
Overexpression of AT1 receptor mRNA fragments should lead to intracellular competition for binding of the polysomal proteins to the AT1 receptor wild-type mRNA. Therefore, the AT1 receptor mRNA transcripts bases 1–400 (control, no binding activity), 1864–2213, 2175–2213, 2175–2195 were overexpressed in VSMC, followed by quantification of AT1 receptor mRNA expression. A representative Northern hybridization of RNA from cells transfected with a vector containing the 2175–2196 AT1 receptor mRNA fragment and a summarized densitometric analysis of three separate experiments for various AT1 receptor mRNA transcripts are shown in Fig. 2 . The transfected AT1 receptor mRNA fragments bases 1864–2213, 2175–2213, 2175–2196 led to a significantly increased expression of the wild-type AT1 receptor mRNA compared with basal levels whereas the insertless vector as well as the AT1 receptor mRNA fragment base 1–400 originated from the 5'-untranslated region had no influence on AT1 receptor mRNA levels.

View larger version (46K): [in this window] [in a new window]   Figure 2. Overexpression of AT1 receptor mRNA transcripts in VSMC. A) Representative Northern blot of the total RNA isolated from VSMC transfected with the empty vector (control) and the expression vector harboring the AT1 receptor transcript bases 2175–2195. Membranes were hybridized with an AT1 receptor cDNA probe 368–846 bp. B) Summary of densitometric analysis of AT1 receptor mRNA expression in VSMC transfected with various AT1 receptor mRNA transcripts as depicted. n = 3–5, mean ± SE, *P < 005.

Data were confirmed under the condition of an in vitro decay assay. The AT1 receptor mRNA transcript 2175–2195 was added to the reaction containing polysomal protein leading to significant stabilization of the AT1 receptor mRNA, suggesting that binding of polysomal proteins to this region of the AT1 receptor mRNA act as destabilizing factors.

Angiotensin II-induced AT1 receptor down-regulation was inhibited by forced overexpression of the AT1 receptor mRNA bases 2175–2195.

CONCLUSIONS

The modulation of AT1 receptor expression is influenced by various agonists, of which most (if not all) induce profound alterations in AT1 receptor mRNA turnover. This mRNA processing islocated in the polyribosomal compartment and involves the interaction of mRNA binding proteins with the 3'-untranslated region of the AT1 receptor mRNA. Our data demonstrate that protein binding is located at the very 3' end of the 3'-untranslated region of the AT1 receptor mRNA, immediately upstream of the poly(A) tract. This cognate sequence contains an AUUUUA hexamer that reveals similarities to the ß₂-adrenergic receptor mRNA, although the flanking region of this RNA binding region differs slightly between genes. Nevertheless, the bases 2175–2195 of the AT1 receptor mRNA are almost completely composed of A and U nucleotides (except for three G), a feature that has been shown for most mRNA binding sequences residing in the 3'-untranslated region, although nucleotide sequences differ in various degrees between genes. Secondary and tertiary structures of the RNA influence the interaction with proteins. Hairpins or stem loops formed by the RNA region of interest may interact with neighboring proteins. In the case of the AT1 receptor, the identified AT1 receptor mRNA binding motif bases 2175–2195 forms such a stem loop. That holds true for the entire AT1 receptor mRNA and for the isolated 20 base transcript used in our study as competitor and decoy. Deletion of this motif abolished the stem loop, which agrees with our finding that such a mutated mRNA no longer binds to polysomal proteins, suggesting the importance of secondary structure for protein–mRNA interaction.

Another important issue is the functional relevance of this mRNA sequences and their binding to the cytosolic mRNA binding proteins. By transfecting distinct mRNA species, we induced a decoy situation leading to competition of the corresponding binding protein from the wild-type AT1 receptor mRNA. The data suggest that the AU-rich and AUUUUA hexamer containing region base 2175–2195 of the AT1 receptor mRNA interacts with binding proteins that govern an accelerated decay of the transcripts, since competition of these proteins causes AT1 receptor mRNA up-regulation. This is consistent with our finding that angiotensin II, which induces destabilization of the AT1 receptor mRNA, causes enhanced binding activity of the identified proteins to the 3'-untranslated region of the AT1 receptor mRNA.

The data are in concert with investigations that have provided evidence that cognate sequences within the 3'-untranslated region, such as the pentamer AUUUA, and nucleotide sequences such as UUAUUUA(U/A)(U/A) and UUAUUUAUU regulate by interaction with cytosolic and nuclear-associated factor mRNA stability.

The presented study displays novel molecular mechanisms involved in post-transcriptional regulation of the AT1 receptor that will prompt further characterization of interactions between binding proteins and the AT1 receptor mRNA and will enable the structural identification of the participating binding proteins. The latter is a prerequisite for a better understanding of the complex cellular mechanisms of cytosolic mRNA turnover. In addition, the described mechanisms for AT1 receptor regulation may have relevant implications for the pathogenesis of atherosclerosis and hypertension, since pathological abnormalities of AT1 receptor regulation may drive both development and progression of these diseases.FIGURE 3

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram. Angiotensin II (angII) stimulates via AT1 receptor activation a destabilization of the AAT1 receptor mRNA that is mediated through polysomal binding proteins, which interact with the AT1 receptor mRNA bases 2175–2195. This region forms a typical stem loop. ORF = open reading frame. UTR = untranslated region.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0842fje ; to cite this article, use FASEB J. (April 18, 2001) 10.1096/fj.00-0842fje

² G.N. and F.M. contributed equally to this study.

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