FIG. 3.

hnRNP H inhibits polyadenylation. (A) To sequester endogenous hnRNP H, nuclear extract was supplemented with increasing amounts of GRS competitor RNA. In vitro polyadenylation reactions using the NRS LTR substrate were performed as described in Materials and Methods. (B) Poly(A)⁺ RNA was subsequently isolated using an oligo(dT) column. −, unpolyadenylated RNA that flowed through the column; +, poly(A)⁺ RNA eluted. (C) A control oligonucleotide containing an unrelated sequence had no effect on the polyadenylation efficiency of the NRS LTR substrate. In vitro polyadenylation reactions were performed followed by fractionation on an oligo(dT) column to separate poly(A)⁻ and poly(A)⁺ RNA. Two hundred thirty nanograms of oligonucleotide was used. (D) UV cross-linking of hnRNP H (∼53 kDa) to the NRS RNA is inhibited after incubation of the extract with increasing amounts of GRS competitor RNA but not with 230 ng of the control oligonucleotide. Protein molecular weight markers are indicated.