Active Stabilization of Human Endothelial Nitric Oxide Synthase mRNA by hnRNP E1 Protects against Antisense RNA and MicroRNAs

Abstract

Human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Posttranscriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially under hypoxic conditions. Here, we show that the human eNOS 3′ untranslated region (3′ UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and RNA electrophoretic mobility shift assays (EMSAs) revealed the formation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1)-containing RNP complexes at these 3′-UTR elements. Knockdown of hnRNP E1 decreased eNOS mRNA half-life, mRNA levels, and protein expression. Significantly, these stabilizing RNP complexes protect eNOS mRNA from the inhibitory effects of its antisense transcript sONE and 3′-UTR-targeting small interfering RNAs (siRNAs), as well as microRNAs, specifically, hsa-miR-765, which targets eNOS mRNA stability determinants. Hypoxia disrupts hnRNP E1/eNOS 3′-UTR interactions via increased Akt-mediated serine phosphorylation (including serine 43) and increased nuclear localization of hnRNP E1. These mechanisms account, at least in part, for the decrease in eNOS mRNA stability under hypoxic conditions. Thus, the stabilization of human eNOS mRNA by hnRNP E1-containing RNP complexes serves as a key protective mechanism against the posttranscriptional inhibitory effects of antisense RNA and microRNAs under basal conditions but is disrupted under hypoxic conditions.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01257-12.

ACKNOWLEDGMENTS

We declare that there are no financial or other conflicts of interest related to this work.

J.J.D.H. is the recipient of an Ontario Graduate Scholarship award. G.B.R. is the recipient of a Canadian Institute of Health Research/Heart and Stroke Foundation of Canada Doctoral Research Award. P.A.M. is a Heart and Stroke Foundation of Canada Career Investigator and is supported by a grant from the Heart and Stroke Foundation of Canada (grant T-6777).

We thank Stephen A. Liebhaber, Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, for kindly providing anti-hnRNP antibodies. We thank Philip H. Howe, Hollings Cancer Center, Medical University of South Carolina, for kindly providing FLAG-tagged wild-type and S43A mutant hnRNP E1 plasmids.