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Molecular and Cellular Biology, July 1999, p. 4572-4581, Vol. 19, No. 7
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Assembly of the
-Globin mRNA Stability Complex Reflects Binary
Interaction between the Pyrimidine-Rich 3' Untranslated Region
Determinant and Poly(C) Binding Protein
CP
Alexander N.
Chkheidze,
Dmitry L.
Lyakhov,
Alexander V.
Makeyev,
Julia
Morales,
Jian
Kong, and
Stephen A.
Liebhaber*
Howard Hughes Medical Institute and
Departments of Genetics and Medicine, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania
Received 9 November 1998/Returned for modification 17 December
1998/Accepted 30 March 1999
Globin mRNAs accumulate to 95% of total cellular mRNA during
terminal erythroid differentiation, reflecting their
extraordinary stability. The stability of human
-globin mRNA is
paralleled by formation of a sequence-specific RNA-protein
(RNP) complex at a pyrimidine-rich site within its 3' untranslated
region (3'UTR), the
-complex. The proteins of the
-complex are
widely expressed. The
-complex or a closely related complex also
assembles at pyrimidine-rich 3'UTR segments of other stable mRNAs.
These data suggest that the
-complex may constitute a general
determinant of mRNA stability. One or more
CPs, members of a family
of hnRNP K-homology domain poly(C) binding proteins, are essential
constituents of the
-complex. The ability of
CPs to homodimerize
and their reported association with additional RNA binding proteins
such as AU-rich binding factor 1 (AUF1) and hnRNP K have suggested that
the
-complex is a multisubunit structure. In the present study, we
have addressed the composition of the
-complex. An RNA titration
recruitment assay revealed that
CPs were quantitatively incorporated
into the
-complex in the absence of associated AUF1 and hnRNP K. A
high-affinity direct interaction between each of the three major
CP
isoforms and the
-globin 3'UTR was detected, suggesting that each of
these proteins might be sufficient for
-complex assembly. This
sufficiency was further supported by the sequence-specific binding of
recombinant
CPs to a spectrum of RNA targets. Finally, density
sedimentation analysis demonstrated that the
-complex could
accommodate only a single
CP. These data established that a single
CP molecule binds directly to the
-globin 3'UTR, resulting in a
simple binary structure for the
-complex.
*
Corresponding author. Mailing address: Room 428 Clinical Research Building, 415 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 898-7834. Fax: (215) 898-1257. E-mail:
Liebhaber{at}mail.med.upenn.edu.

Present address: CNRS UPR 9042, 29680 Roscoff,
France.
Molecular and Cellular Biology, July 1999, p. 4572-4581, Vol. 19, No. 7
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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