Structural remodeling of an A + U-rich RNA element by cation or AUF1 binding.
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.
Wilson
G M
GM
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. wilsongm@umdnj.edu
Sutphen
K
K
Moutafis
M
M
Sinha
S
S
Brewer
G
G
D002412:Cations; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
D002412:Cations; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D007477:Ions; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA; D008274:Magnesium