mRNA Openers and Closers: Modulating AU‐Rich Element‐Controlled mRNA Stability by a Molecular Switch in mRNA Secondary Structure

Abstract

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.

Overexpression of the RNA binding protein HuR impairs tumor growth in triple negative breast cancer associated with deficient angiogenesis

“…Even though HuR can theoretically bind 8% of human mRNAs which contain AREs, it does not necessarily do so. [37][38][39] Published reports from our lab and others demonstrate that HuR critically regulates myogenesis by controlling the expression of three central genes in this process. 40,41 HuR siRNA knock down prevents muscle formation, whereas HuR overexpression results in precocious differentiation.…”

hnRNP A1 regulates UV-induced NF-κB signalling through destabilization of cIAP1 mRNA

“…For example, the RNA-binding protein HuR requires both the presence of the HuR-binding site within the ARE as well as the presentation of this site in a singlestranded conformation within the 3 0 UTR structure for proper binding. 29 Although hnRNP A1 is usually considered an RNA and single-stranded DNA-binding protein, 30 accumulated evidence indicates that hnRNP A1 is capable of binding double-stranded DNA and RNA regions as well. In fact it was shown that hnRNP A1 could bind simultaneously to bipartite sequence elements with its two RRM domains, or as a dimer.…”

mRNA stability control: a clandestine force in normal and malignant hematopoiesis

“…48 RNA folds into complex structures including stems, loops and bulges. 50 Although stabilizing RNABPs display a high affinity for consensus sequences, 37,51,52 binding is also controlled by structural determinants. 53 Fialcowitz 54 has recently demonstrated that the ARE of the TNFa transcript can assume a hairpin structure to which a stabilizing (HuR) protein can bind but a destabilizing protein (AUF1p37) that recognizes the unfolded sequence can no longer bind.…”