Specific protein domains mediate cooperative assembly of HuR oligomers on AU-rich mRNA-destabilizing sequences.
The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR.ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR.ARE complexes in solution.
Fialcowitz-White
Elizabeth J
EJ
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Brewer
Brandy Y
BY
Ballin
Jeff D
JD
Willis
Chris D
CD
Toth
Eric A
EA
Wilson
Gerald M
GM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000737:chemistry*; D051959:ELAV Proteins / Q000737:chemistry*; D000067780:ELAV-Like Protein 1; D005454:Fluorescence Polarization; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D036103:Nanotechnology; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins