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

Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavag e-polyadenylation reaction operating differentially during B-cell developme ntal stages. Using four complementary approaches, we demonstrate that a cha nge in the level of hnRNP F is an important determinant in the regulated us e of alternative polyadenylation sites between memory and plasma stage B ce lls. 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. I n memory B cells the activity of CstF-64 binding to pre-mRNA, but not its a mount, was reduced, Second, examination of the complexes formed on input pr e-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 plas ma cells. Formation of these large complexes is dependent on the region dow nstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs c ompete for a similar region. Third, using a recombinant protein we showed t hat hnRNP F could bind to the region downstream of a poly(A) site, block Cs tF-64 association with RNA and inhibit the cleavage reaction. Fourth, overe xpression 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 mem ory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.