mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure

Chembiochem. 2004 Oct 4;5(10):1432-47. doi: 10.1002/cbic.200400219.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Surface / genetics
  • Antigens, Surface / metabolism
  • Base Pairing
  • Base Sequence
  • Binding Sites / physiology
  • ELAV Proteins
  • ELAV-Like Protein 1
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Conformation*
  • RNA Stability / genetics
  • RNA Stability / physiology*
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Structure-Activity Relationship
  • Time Factors

Substances

  • Antigens, Surface
  • ELAV Proteins
  • ELAV-Like Protein 1
  • ELAVL1 protein, human
  • RNA, Messenger
  • RNA-Binding Proteins