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.

The Poly(C) Binding Protein Pcbp2 and Its Retrotransposed Derivative Pcbp1 Are Independently Essential to Mouse Development

“…A number of ex vivo models support distinct biochemical functions for Pcbp1 and Pcbp2. For example, Pcbp1 is uniquely capable of regulating epithelial-mesenchymal transitions and stabilizing vascular nitric oxide synthase transcripts (37,42,70), while Pcbp2 specifically controls tumor suppressor genes in chronic myelogenous leukemia, poliovirus translation, and HIV gene expression (39)(40)(41). Thus, our in vivo analysis of embryogenesis is concordant with previous ex vivo studies in demonstrating that Pcbp1 and Pcbp2 play distinct roles in organism function.…”

IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC

“…These regulatory factors often converge on the 3’ untranslated regions (3’UTRs) of mRNAs (Jens and Rajewsky, 2015). Juxtaposition of their binding sites contributes to combinatorial mechanisms of post-transcriptional gene regulation (Glorian et al, 2011, Zhang et al, 2008, Kundu et al, 2012, Young et al, 2012, Kim et al, 2009, Jafarifar et al, 2011, Ho et al, 2013). …”

MicroRNA-765 influences arterial stiffness through modulating apelin expression

“…Second, miR-765 can also directly inhibit eNOS expression by targeting at the 3′UTR of eNOS gene (Ho et al, 2013). Ho et al found that miR-765's knockdown effect on eNOS was only evident when the RNA stabilizing protein hnRNP-E1 was depleted (Ho et al, 2013). Normally, hnRNP-E1 is abundant in HUVECs, which might explain why we did not find any change in total-eNOS amount in HUVECs transfected with miR-765 (Fig.…”

“…Plenty studies supported that APLN enhances eNOS phosphorylation through ERK1/2, Akt, and AMPK cascades (Masri et al, 2004;Shan et al, 2011). Second, miR-765 can also directly inhibit eNOS expression by targeting at the 3′UTR of eNOS gene (Ho et al, 2013). Ho et al found that miR-765's knockdown effect on eNOS was only evident when the RNA stabilizing protein hnRNP-E1 was depleted (Ho et al, 2013).…”