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Molecular and Cellular Biology, July 2000, p. 4765-4772, Vol. 20, No. 13
Norris Cancer Center, University of Southern
California, Keck School of Medicine, Los Angeles, California
90089-9176,1 and Huntsman Cancer
Institute, University of Utah, School of Medicine, Salt Lake City,
Utah 841322
Received 24 November 1999/Returned for modification 10 January
2000/Accepted 4 April 2000
Human neuron-specific RNA-binding protein HuD belongs to the family
of Hu proteins and consists of two N-terminal RNA recognition motifs
(RRM1 and -2), a hinge region, and a C-terminal RRM (RRM3). Hu proteins
can bind to AU-rich elements in the 3' untranslated regions of unstable
mRNAs, causing the stabilization of certain transcripts. We have
studied the interaction between HuD and prototype mRNA instability
elements of the sequence UU(AUUU)nAUU using equilibrium methods and real-time kinetics (surface plasmon resonance using a
BIACORE). We show that a single molecule of HuD requires at least three
AUUU repeats to bind tightly to the RNA. Deletion of RRM1 reduced the
Kd by 2 orders of magnitude and caused a
decrease in the association rate and a strong increase in the
dissociation rate of the RNA-protein complex, as expected when a
critical RNA-binding domain is removed. In contrast, deletion of either
RRM2 or -3, which only moderately reduced the affinity, caused marked
increases in the association and dissociation rates. The slower binding and stabilization of the complex observed in the presence of all three
RRMs suggest that a change in the tertiary structure occurs during
binding. The individual RRMs bind poorly to the RNA (RRM1 binds with
micromolar affinity, while the affinities of RRM2 and -3 are in the
millimolar range). However, the combination of RRM1 and either RRM2 or
RRM3 in the context of the protein allows binding with a nanomolar
affinity. Thus, the three RRMs appear to cooperate not only to increase
the affinity of the interaction but also to stabilize the formed
complex. Kinetic effects, similar to those described here, could play a
role in RNA binding by many multi-RRM proteins and may influence the
competition between proteins for RNA-binding sites and the ability of
RNA-bound proteins to be transported intracellularly.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
HuD RNA Recognition Motifs Play Distinct Roles in
the Formation of a Stable Complex with AU-Rich RNA
*
Corresponding author. Mailing address: USC/Norris
Cancer Center, Room NOR 6420, 1441 Eastlake Ave., Los Angeles, CA
90089-9176. Phone: (323) 865-0655. Fax: (323) 865-0158. E-mail:
ilaird{at}hsc.usc.edu.
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