Abstract
In mammals, rapid mRNA turnover directed by AU-rich elements (AREs) is mediated by selective association of cellular ARE-binding proteins. These trans-acting factors display overlapping RNA substrate specificities and may act to either stabilize or destabilize targeted transcripts; however, the mechanistic features of AREs that promote preferential binding of one trans-factor over another are not well understood. Here, we describe a hairpin-like structure adopted by the ARE from tumor necrosis factor alpha (TNFalpha) mRNA that modulates its affinity for selected ARE-binding proteins. In particular, association of the mRNA-destabilizing factor p37(AUF1) was strongly inhibited by adoption of the higher order ARE structure, whereas binding of the inducible heat shock protein Hsp70 was less severely compromised. By contrast, association of the mRNA-stabilizing protein HuR was only minimally affected by changes in ARE folding. Consistent with the inverse relationship between p37(AUF1) binding affinity and the stability of ARE folding, mutations that stabilized the ARE hairpin also inhibited its ability to direct rapid mRNA turnover in transfected cells. Finally, phylogenetic analyses and structural modeling indicate that TNFalpha mRNA sequences flanking the ARE are highly conserved and may stabilize the hairpin fold in vivo. Taken together, these data suggest that local higher order structures involving AREs may function as potent regulators of mRNA turnover in mammalian cells by modulating trans-factor binding selectivity.
Highlights
The rate of mRNA turnover is highly variable among the cytoplasmic mRNA population and plays a significant role in regulating the steady-state concentrations of individual mRNA species available to program protein synthesis
A Candidate Stem-Loop Folded Structure for the TNF␣ AU-rich elements (AREs)—Previous data indicated that an RNA substrate containing the core TNF␣ ARE sequence adopts a unimolecular, condensed structure in a cation-dependent manner [33, 38]
Site-directed Mutations within the TNF␣ ARE Modulate the Stability of RNA Folding—Whereas the nuclease mapping data are largely consistent with the stem-loop ARE structure predicted by mFold, this model was further tested by creating single point mutations within the ARE[38] sequence that were predicted to alter the thermodynamic stability of its folded structure
Summary
RNA Substrates—Synthesis, deprotection of 2Ј-hydroxyl groups, and purification of RNA substrates were performed by Dharmacon Research or Integrated DNA Technologies. EFRET between donor and acceptor fluorophores may be resolved using Equation 2 [44, 46], where FDA is the fluorescence of the donor in the presence of the acceptor, whereas FD is the fluorescence measured from parallel reactions containing RNA substrates labeled with the donor fluorophore (i.e. Fl) alone. To correct for this, donor emission from (Cy3 ϩ Fl)-labeled RNA substrates (FCy-Fl) was interpreted by Equation 3, where fDA is the efficiency of acceptor labeling, in this case Cy3 (typically 75–90%) Incorporating this function into Equation 2 and substituting FD ϭ FFl yields Equation 4. RNA binding affinity was quantitatively measured by monitoring the changes in the fluorescence anisotropy of Fl-conjugated RNA substrates across a titration of protein essentially as described [33, 37]. Following normalization to ␣-globin mRNA, turnover rates of -globin and G-ARE mRNAs were calculated by nonlinear regression of the percentage of (-globin or G-ARE) mRNA remaining as a function of time following Dox treatment, yielding first-order decay constants (k)
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