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The 3′-untranslated region length and AU-rich RNA location modulate RNA–protein interaction and translational control of β2-adrenergic receptor mRNA

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Abstract

Posttranscriptional controls play a major role in β2-adrenergic receptor (β2-AR) expression. We recently reported that β2-AR mRNA translation is suppressed by elements in its 3′-untranslated region (UTR). We also identified T-cell-restricted intracellular antigen-related protein (TIAR) and HuR as prominent AU-rich (ARE) RNA-binding proteins that associate with β2-AR mRNA 3′-UTR. In this study, we identified a poly(U) region at the distal end of the 3′-UTR as critical for TIAR binding to β2-AR mRNA and for translational suppression. Here, we also report that the locations of the poly(U) and ARE sequences within the 3′-UTR are important determinants that control the translation of β2-AR mRNA. Consistent with this finding, a 20-nucleotide ARE RNA from the proximal 3′-UTR that did not inhibit mRNA translation in its native position was able to suppress translation when re-located to the distal 3′-UTR of the receptor mRNA. Immunoprecipitation and polysome profile analysis demonstrated the importance of 3′-UTR length and the ARE RNA location within the 3′-UTR, as key determinants of RNA/protein interactions and translational control of β2-AR mRNA. Further, the importance of 3′-UTR length and ARE location in TIAR and HuR association with mRNA and translational suppression was demonstrated using a chimeric luciferase reporter gene.

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Abbreviations

β2-AR:

β2-adrenergic receptor

AREs:

A + U-rich element(s)

TIA-1:

T-cell-restricted intercellular antigen-1

TIAR:

T-cell-restricted intracellular antigen-related protein

hnRNP:

Heterogeneous nuclear ribonucleoprotein

REMSA:

RNA electrophoretic mobility shift assay

TTP:

Tristetraprolin

UTR:

Untranslated region

ORF:

Open reading frame

CR:

Coding region

IP:

Immunoprecipitation

nt:

Nucleotide(s)

CHO:

Chinese hamster ovary

GST:

Glutathione S-transferase

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Acknowledgments

This work was supported in part by grants from United States Public Health Services (RO1 GM 58740), from the Mid-Atlantic Affiliate of the American Heart Association (0555470U) and a URC institutional award from the Medical University of South Carolina to BGT.

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Author notes

  1. Kothandharaman Subramaniam

    Present address: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Singapore

  2. Karthikeyan Kandasamy

    Present address: Cell and Tissue Engineering, Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, ‘The Nanos’, Singapore, 138669, Singapore

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA

    Kothandharaman Subramaniam, Karthikeyan Kandasamy, Kusumam Joseph, Eleanor K. Spicer & Baby G. Tholanikunnel

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  1. Kothandharaman Subramaniam
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Correspondence to Baby G. Tholanikunnel.

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The authors wish it to be known that the first two authors should be regarded as joint first authors: Kothandharaman Subramaniam, Karthikeyan Kandasamy.

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Subramaniam, K., Kandasamy, K., Joseph, K. et al. The 3′-untranslated region length and AU-rich RNA location modulate RNA–protein interaction and translational control of β2-adrenergic receptor mRNA. Mol Cell Biochem 352, 125–141 (2011). https://doi.org/10.1007/s11010-011-0747-z

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