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Molecular and Cellular Biology, May 1999, p. 3561-3570, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Stem-Loop Binding Protein Facilitates 3'-End
Formation by Stabilizing U7 snRNP Binding to Histone Pre-mRNA
Zbigniew
Dominski,1,2
Lian-Xing
Zheng,1
Ricardo
Sanchez,1,2 and
William F.
Marzluff1,2,*
Department of Biochemistry and
Biophysics1 and Program in Molecular
Biology and Biotechnology,2 University of
North Carolina, Chapel Hill, North Carolina 27599
Received 21 December 1998/Returned for modification 5 February
1999/Accepted 18 February 1999
The 3' end of histone mRNA is formed by an endonucleolytic cleavage
of the primary transcript after a conserved stem-loop sequence. The
cleavage reaction requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds the stem-loop sequence, and the U7 snRNP that interacts with a sequence downstream from the cleavage site. Removal of SLBP from a nuclear extract abolishes 3'-end processing, and the addition of recombinant SLBP restores processing activity of the depleted extract. To determine
the regions of human SLBP necessary for 3' processing, various deletion
mutants of the protein were tested for their ability to complement the
SLBP-depleted extract. The entire N-terminal domain and the majority of
the C-terminal domain of human SLBP are dispensable for processing. The
minimal protein that efficiently supports cleavage of histone pre-mRNA
consists of 93 amino acids containing the 73-amino-acid RNA-binding
domain and 20 amino acids located immediately next to its C terminus.
Replacement of these 20 residues with an unrelated sequence in the
context of the full-length SLBP reduces processing >90%.
Coimmunoprecipitation experiments with the anti-SLBP antibody
demonstrated that SLBP and U7 snRNP form a stable complex only in the
presence of pre-mRNA substrates containing a properly positioned U7
snRNP binding site. One role of SLBP is to stabilize the interaction of
the histone pre-mRNA with U7 snRNP.
*
Corresponding author. Mailing address: Program in
Molecular Biology and Biotechnology, CB# 7100, University of North
Carolina, Chapel Hill, NC 27599. Phone: (919) 962-8920. Fax: (919)
966-6821. E-mail: marzluff{at}med.unc.edu.
Molecular and Cellular Biology, May 1999, p. 3561-3570, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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