Figure 5.

Hyperadenylation can be demonstrated in nuclear extract-based polyadenylation assays when NPM1 is depleted. (A) RNAs containing the pre-cleaved SVL polyadenylation signal were incubated for the indicated times in extracts from either untreated HeLa, LKO.1 transfected control HeLa cells or HeLa cells in which NPM1 was knocked down using a specific shRNA. Ext 1 and 2 denote independent extracts made from each of the indicated cell types. Polyadenylated products were analysed on a 5% denaturing acrylamide gel. The positions of the input, normally polyadenylated and hyperadenylated RNAs are indicated on the right. (B) Same as (A) except that the entire SVL polyadenylation signal was used in the RNA substrates so that transcripts were both cleaved and polyadenylated. (C) Same as the previous panels except RNAs containing the IVA2 polyadenylation signal were used. (D) Time course of polyadenylation using nuclear extracts derived from normal or NPM1 knockdown HeLa cells and the SVL RNA substrate. (E) HeLa nuclear extracts were used untreated (no Ab lane), incubated with protein A Sepharose beads with control IgG before use (control lane), or with increasing amounts of NPM1-specific antisera before removal of antigen–antibody complexes with protein A beads (NPM1 Ab lanes). The SVL RNA substrate was incubated in these treated extracts for the time indicated and polyadenylated products were analysed on a 5% acrylamide gel. (F) Increasing amounts of partially purified NPM1 protein were added back to NPM1-depleted extracts and polyadenylation reactions were performed and assayed as described above.