scholarly journals Functional role for the angiotensin II receptor (AT1A) 3′-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins

1998 ◽  
Vol 329 (2) ◽  
pp. 255-264 ◽  
Author(s):  
J. Thomas THEKKUMKARA ◽  
G. Walter THOMAS ◽  
J. Thomas MOTEL ◽  
M. Kenneth BAKER
Keyword(s):  
Angiotensin Ii ◽  
Cell Surface ◽  
Intracellular Calcium ◽  
Untranslated Region ◽  
Functional Role ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ang Ii ◽  
In Cells

We demonstrate a functional role for the 3ʹ-untranslated region (3ʹ-UTR) of the angiotensin II (Ang II) receptor subtype AT1A mRNA in Chinese hamster ovary (CHO-K1) cells by stably transfecting the coding region of the receptor gene with or without the 845 bp 3ʹ-UTR. Two cell lines expressing similar levels of cell-surface receptors (with 3ʹ-UTR, Bmax = 571 fmol/mg protein; without 3ʹ-UTR, Bmax = 663 fmol/mg protein) were used in the present study. Both cell lines expressed high-affinity receptors (with 3ʹ-UTR, Kd = 0.83 nM; without 3ʹ-UTR, Kd = 0.82 nM), and binding studies with 125I-labelled Ang II in the presence of GTP[S] demonstrated that both coupled to heterotrimeric G-proteins. Despite these similarities, significant differences were observed for receptor-mediated cell signalling pathways. In cells without the 3ʹ-UTR, Ang II stimulated an increase in cAMP accumulation (11-fold above control) and in cells with the 3ʹ-UTR no stimulation was observed, which was consistent with previous observations in most endogenous Ang II receptor (AT1)-expressing cells. Activation of cAMP by Ang II in cells without the 3ʹ-UTR correlated with an inhibition of DNA synthesis, determined by [3H]thymidine incorporation. Ang II-mediated responses were blocked by EXP3174, a selective non-peptide receptor antagonist. We also observed differences in the transient profiles of intracellular calcium between cells with and without the 3ʹ-UTR in response to Ang II. In cells with the 3ʹ-UTR, a sustained level of intracellular calcium was observed after Ang II stimulation, whereas cells without the 3ʹ-UTR displayed a full return to basal level within 50 s of Ang II treatment. Even though the expressed exogenous gene is under the control of a constitutively expressing promoter (cytomegalovirus promoter), Northern-blot analysis revealed a considerably greater accumulation of AT1A mRNA in cells without the 3ʹ-UTR compared with cells with the 3ʹ-UTR. Analysis of the decay rate of the AT1A mRNA in cells with and without the 3ʹ-UTR revealed that the normally unstable AT1A receptor mRNA became highly stable by removing its 3ʹ-UTR, identifying a role for the 3ʹ-UTR in mRNA destabilization. Interestingly, both cells express similar levels of receptors at the cell surface, suggesting that the 3ʹ-UTR is also involved in the efficient translation and/or translocation of the receptor protein to the plasma membrane. We hypothesized that these 3ʹ-UTR-mediated functions of the receptor are regulated by RNA-binding proteins. To identify possible RNA-binding proteins for the AT1A 3ʹ-UTR, cellular extracts were prepared from parental CHO-K1 cells and 3ʹ-UTR-binding assays, electrophoretic mobility-shift assays and UV crosslinking studies were performed. A major cellular protein of 55 kDa was identified, which specifically interacted with the 3ʹ-UTR. Our data suggest that the 3ʹ-UTR of the AT1A can control specific receptor functions, perhaps via selective recognition of the 3ʹ-UTR by RNA-binding proteins.

Posttranscriptional mechanisms contribute to osmotic regulation of ANG type 1 receptors in cultured rat renomedullary interstitial cells

2006 ◽  
Vol 290 (1) ◽  
pp. R44-R49 ◽  
Author(s):  
Sunghou Lee ◽  
Zheng Wu ◽  
Kathryn Sandberg ◽  
S-E. Yoo ◽  
Christine Maric
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Renal Medulla ◽  
Interstitial Cells ◽  
Receptor Internalization ◽  
Osmotic Regulation ◽  
Ang Ii ◽  
Rnase Protection ◽  
Competition Analysis

Previously, we showed that ANG II receptors in cultured rat renomedullary interstitial cells (RMICs) are osmotically regulated ( 19 ). The current study examined the mechanisms underlying this osmotic regulation in RMICs cultured in isoosmotic (300 mosmol/kgH2O) and hyperosmotic (600 mosmol/kgH2O) conditions. Radioligand competition analysis coupled with RNase protection assays (RPA) and ligand-mediated receptor internalization studies revealed that RMICs primarily express the type 1a angiotensin receptor (AT1aR). When cultured under hyperosmotic conditions, the density (Bmax) of AT1R in RMIC membranes decreased by 31% [Bmax (pmol/mg protein): 300 mosmol/kgH2O, 6.44 ± 0.46 vs. 600 mosmol/kgH2O, 4.42 ± 0.37, n = 8, P < 0.01], under conditions in which no detectable changes in AT1aR mRNA expression or in the kinetics of ligand-mediated AT1R internalization were observed. RNA electromobility shift assays showed that RNA protein complex (RPC) formation between RMIC cytosolic RNA binding proteins and the 5′ leader sequence (5′LS) of the AT1aR was increased 1.5-fold under hyperosmotic conditions [5′LS RPC (arbitrary units): 300 mosmol/kgH2O, 0.79 ± 0.08 vs. 600 mosmol/kgH2O, 1.17 ± 0.07, n = 4, P < 0.01]. These results suggest that the downregulation of AT1aR expression in RMICs cultured under hyperosmotic conditions is regulated at the posttranscriptional level by RNA binding proteins that interact within the 5′LS of the AT1aR mRNA. The downregulation of AT1aR expression under hyperosmotic conditions may be an important mechanism by which the activity of ANG II is regulated in the hyperosmotic renal medulla.

scholarly journals Current techniques for visualizing RNA in cells

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 775 ◽  
Author(s):  
Lilith V.J.C. Mannack ◽  
Sebastian Eising ◽  
Andrea Rentmeister
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
State Of The Art ◽  
Living Cells ◽  
Enzymatic Modification ◽  
Current State ◽  
Rna Imaging ◽  
In Cells ◽  
Art Techniques

Labeling RNA is of utmost interest, particularly in living cells, and thus RNA imaging is an emerging field. There are numerous methods relying on different concepts ranging from hybridization-based probes, over RNA-binding proteins to chemo-enzymatic modification of RNA. These methods have different benefits and limitations. This review aims to outline the current state-of-the-art techniques and point out their benefits and limitations.

scholarly journals RNA-Binding Proteins Tia-1 and Tiar Link the Phosphorylation of Eif-2α to the Assembly of Mammalian Stress Granules

1999 ◽  
Vol 147 (7) ◽  
pp. 1431-1442 ◽  
Author(s):  
Nancy L. Kedersha ◽  
Mita Gupta ◽  
Wei Li ◽  
Ira Miller ◽  
Paul Anderson
Keyword(s):  
Environmental Stress ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Binding Protein ◽  
Plant Cells ◽  
Stress Granules ◽  
Binding Domains ◽  
Translational Arrest ◽  
In Cells

In response to environmental stress, the related RNA-binding proteins TIA-1 and TIAR colocalize with poly(A)+ RNA at cytoplasmic foci that resemble the stress granules (SGs) that harbor untranslated mRNAs in heat shocked plant cells (Nover et al. 1989; Nover et al. 1983; Scharf et al. 1998). The accumulation of untranslated mRNA at SGs is reversible in cells that recover from a sublethal stress, but irreversible in cells subjected to a lethal stress. We have found that the assembly of TIA-1/R+ SGs is initiated by the phosphorylation of eIF-2α. A phosphomimetic eIF-2α mutant (S51D) induces the assembly of SGs, whereas a nonphosphorylatable eIF-2α mutant (S51A) prevents the assembly of SGs. The ability of a TIA-1 mutant lacking its RNA-binding domains to function as a transdominant inhibitor of SG formation suggests that this RNA-binding protein acts downstream of the phosphorylation of eIF-2α to promote the sequestration of untranslated mRNAs at SGs. The assembly and disassembly of SGs could regulate the duration of stress- induced translational arrest in cells recovering from environmental stress.

scholarly journals Microtubules as platforms for probing liquid–liquid phase separation in cells – application to RNA-binding proteins

2018 ◽  
Vol 131 (11) ◽  
pp. jcs214692 ◽  
Author(s):  
Alexandre Maucuer ◽  
Bénédicte Desforges ◽  
Vandana Joshi ◽  
Mirela Boca ◽  
Dmitry A. Kretov ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation ◽  
In Cells

scholarly journals Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

1994 ◽  
Vol 14 (9) ◽  
pp. 5898-5909 ◽  
Author(s):  
R Stripecke ◽  
C C Oliveira ◽  
J E McCarthy ◽  
M W Hentze
Keyword(s):  
Binding Sites ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Yeast Cells ◽  
General Mechanism ◽  
Translational Repressor

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

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