Abstract
Association of AUF1 with A + U-rich elements (AREs) induces rapid cytoplasmic degradation of mRNAs containing these sequences, involving the recruitment or assembly of multisubunit trans-acting complexes on the mRNA. Recently, we reported that Mg(2+)-induced conformational changes in the ARE from tumor necrosis factor alpha mRNA inhibited AUF1 binding and oligomerization activities on this substrate (Wilson, G. M., Sutphen, K., Chuang, K., and Brewer, G. (2001) J. Biol. Chem. 276, 8695-8704). In this study, resonance energy transfer was employed to characterize structural changes in RNA substrates in response to cation- and AUF1-binding events. An RNA substrate containing the tumor necrosis factor alpha ARE displayed a weak conformational transition in the absence of added cations but was cooperatively stabilized by Mg(2+). Additional assays demonstrated a strong preference for small, multivalent cations, suggesting that the folded RNA structure was stabilized by counterion neutralization at discrete regions of high negative charge density. Association of AUF1 with cognate RNA substrates also induced formation of condensed RNA structures, although distinct from the folded structure stabilized by multivalent cations. Taken together, these experiments indicate that association of AUF1 with an ARE may function to remodel local RNA structures, which may be a prerequisite for subsequent recruitment of additional trans-acting factors.
Highlights
The synthetic rates of many eukaryotic gene products are regulated by the cytoplasmic stabilities of mRNAs encoding them
AUF1 exists as a family of four protein isoforms generated from a common pre-mRNA, and all associate with RNA substrates via two tandemly arranged RNA recognition motifs (RRMs) [17]
Using resonance energy transfer (RET) to monitor global changes in the solution structure of the TNF␣ A ؉ U-rich elements (AREs), we observed that this RNA substrate adopts a weak, higher order RNA structure that is cooperatively stabilized by Mg2ϩ in a sequence-specific manner
Summary
Cy-GUGAUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUUAG-Fl GUGAUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUUAG-Fl Cy-GUGAUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUUAG Fl-GUGAUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUUAG Cy-GAUCUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUA-Fl GAUCUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUA-Fl Fl-GAUCUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUA a “Fl” and “Cy” indicate the positions of the fluorescein and cyanine 3 moieties, respectively, for each RNA substrate. Using resonance energy transfer (RET) to monitor global changes in the solution structure of the TNF␣ ARE, we observed that this RNA substrate adopts a weak, higher order RNA structure that is cooperatively stabilized by Mg2ϩ in a sequence-specific manner This structure was optimally stabilized by compact, multivalent cations, indicating that ion-mediated stabilization probably occurs by counterion neutralization at localized regions of high negative charge density, resulting from the juxtaposition of negatively charged groups in the folded RNA structure. Based on calculation of intramolecular distances between the 5Ј- and 3Ј-ends of the RNA substrates, these protein-folded structures are shown to be distinct from those stabilized by cations and appear to be a direct consequence of AUF1 binding and oligomerization. These experiments define a mechanism for structural condensation of AREs that regulates their AUF1 binding activity and suggest that AUF1-induced, localized remodeling of these RNA structures may contribute to the recruitment of additional trans-acting factors to the AUF11⁄7ARE complex
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