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Mol Cell Biol. 2001 Feb;21(4):1228-38.

hnRNP F influences binding of a 64-kilodalton subunit of cleavage stimulation factor to mRNA precursors in mouse B cells.

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1
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

Abstract

Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H' was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H', and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.

PMID:
11158309
PMCID:
PMC99576
DOI:
10.1128/MCB.21.4.1228-1238.2001
[Indexed for MEDLINE]
Free PMC Article
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