HuR Stabilizes Vacuolar H+-translocating ATPase mRNA during Cellular Energy Depletion

Selvi Jeyaraj,Duaa Dakhlallah,Stephanie R Hill,Beth S Lee

doi:10.1074/jbc.m502883200

Selvi Jeyaraj, Duaa Dakhlallah + Show 2 more

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https://doi.org/10.1074/jbc.m502883200

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Abstract

V-ATPases are multisubunit membrane proteins that use ATP binding and hydrolysis to transport protons across membranes against a concentration gradient. Although some cell types express plasma membrane forms of these transporters, all eukaryotes require V-ATPases to maintain an acidic pH in membrane-bound compartments of endocytic and secretory networks to facilitate protein trafficking and processing. Mammalian cells that completely lack V-ATPases are not viable; yet, the abundance of V-ATPases can differ among cell types by an order of magnitude or more, requiring precise control of their expression. We previously showed that mRNA stability appears to play a major role in regulating overall abundance of V-ATPases. In this report, we demonstrate that the stability of V-ATPase mRNA is regulated through AU-rich elements in 3'-untranslated regions. Unlike some mRNAs that are short-lived due to the presence of these elements, V-ATPase mRNAs have half-lives of hours to days. However, during stress induced by ATP depletion, AU-rich elements are necessary to maintain stability of these transcripts and their presence in the cytoplasm. HuR, an RNA-binding protein that interacts with and stabilizes AU-rich mRNAs, shows increased binding to some V-ATPase mRNAs during ATP depletion. siRNA-mediated knockdown of HuR results in diminished V-ATPase expression. These results indicate that AU-rich elements and associated proteins can play a role in regulation of even very stable mRNAs by protecting against loss during cellular stress.

Highlights

Ance by the kidney [11], bone resorption by osteoclasts [12], and maintenance of pH within the inner ear [13] and the male reproductive tract [14, 15]
Proximal tubules cells, are susceptible to damage by ischemic injury [33], and because high V-ATPase levels are critical to kidney cell function, we investigated whether V-ATPase mRNA expression in these cells might be affected by stress
To determine relative stabilities of the resulting mRNAs, expression constructs were transfected in equal amounts into LLC-PK1 cells, both singly and pairwise, and levels of the expressed mRNAs were compared by Northern analysis

Summary

Introduction

Ance by the kidney [11], bone resorption by osteoclasts [12], and maintenance of pH within the inner ear [13] and the male reproductive tract [14, 15]. Defined in short-lived mRNAs such as those encoding lymphokines, cytokines [21, 22], and proto-oncogenes [23], they consist of U-rich runs, often containing AUUUA cores [24, 25] These sequences are bound by a multiplicity of regulatory proteins that can stabilize or destabilize the transcript, or affect its translatability [26, 27]. HuR Stabilizes V-ATPase mRNA depletion, and that the RNA-binding protein HuR mediates this stabilization These results show that even very long-lived mRNAs can possess functional AREs that act primarily not as destabilizing moieties, but as positive regulators of expression during cell stress events

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