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Mol Cell Biol, February 1998, p. 685-693, Vol. 18, No. 2
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Specificity of RNA Binding by CPEB: Requirement for
RNA Recognition Motifs and a Novel Zinc Finger
Laura E.
Hake,
Raul
Mendez,
and
Joel D.
Richter*
Worcester Foundation for Biomedical Research,
Shrewsbury, Massachusetts 01545
Received 20 June 1997/Returned for modification 4 September
1997/Accepted 6 November 1997
CPEB is an RNA binding protein that interacts with the
maturation-type cytoplasmic polyadenylation element (CPE) (consensus UUUUUAU) to promote polyadenylation and translational activation of
maternal mRNAs in Xenopus laevis. CPEB, which is conserved from mammals to invertebrates, is composed of three regions: an amino-terminal portion with no obvious functional motif, two RNA recognition motifs (RRMs), and a cysteine-histidine region that is
reminiscent of a zinc finger. In this study, we investigated the
physical properties of CPEB required for RNA binding. CPEB can interact
with RNA as a monomer, and phosphorylation, which modifies the protein
during oocyte maturation, has little effect on RNA binding.
Deletion mutations of CPEB have been overexpressed in
Escherichia coli and used in a series of RNA gel shift
experiments. Although a full-length and a truncated CPEB that lacks 139 amino-terminal amino acids bind CPE-containing RNA avidly, proteins
that have had either RRM deleted bind RNA much less efficiently. CPEB
that has had the cysteine-histidine region deleted has no detectable capacity to bind RNA. Single alanine substitutions of specific cysteine or histidine residues within this region also abolish RNA
binding, pointing to the importance of this highly conserved domain of
the protein. Chelation of metal ions by 1,10-phenanthroline inhibits
the ability of CPEB to bind RNA; however, RNA binding is restored if
the reaction is supplemented with zinc. CPEB also binds other metals
such as cobalt and cadmium, but these destroy RNA binding. These data
indicate that the RRMs and a zinc finger region of CPEB are essential
for RNA binding.
*
Corresponding author. Present address: Department of
Molecular Genetics and Microbiology, University of Massachusetts
Medical School, 222 Maple Ave., Shrewsbury, MA 01545. Phone: (508)
842-8921, ext. 340. Fax: (508) 842-3915. E-mail:
joel.richter{at}banyan.ummed.edu.

Present address: Department of Biology, Boston College, Chestnut
Hill, MA 02167-3811.

Present address: Department of Molecular Genetics and
Microbiology, University of Massachusetts Medical School, Shrewsbury,
MA 01545.
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