Interaction of E2F3 mRNA with hnRNP-A1 and requirement of hnRNP-A1 nucleocytoplasmic shuttling for E2F3 expression. (A) Representation of the oligoribonucleotide containing the hnRNP-A1 consensus-binding site (hnRNP-A1 rODN) used as competitor in REMSA assays. Sequence comparison of mouse and human E2F3 3′ UTRs shows presence of the hnRNP-A1 consensus-binding site (UAGGGA/U). E2F3(A1) rODN represents the probe used in REMSA. (B) Left: REMSA with the E2F3(A1) rODN and cytoplasmic lysates of 32Dcl3, untreated and imatinib-treated 32D-BCR/ABL, and K562 cells. Binding specificity was assessed by competition with 100-fold excess of unlabeled hnRNP-A1 rODN and by using hnRNP-A1 immunodepleted extracts. Right: Top shows anti–hnRNP-A1 Western blot in total, anti–hnRNP-A1–immunoprecipitated, and anti–hnRNP-A1–immunodepleted 32Dcl3, 32D-BCR/ABL, and K562 cell lysates. Bottom shows UV cross-linking of binding reactions performed with labeled E2F3(A1) rODN and cytoplasmic 32Dcl3, 32D-BCR/ABL, and K562 cell lysates. Arrow indicates the hnRNP-A1/E2F3 RNA complex. *Nonspecific complex in parental 32Dcl3 cell lysates. (C) Protein levels of E2F3 and GRB2 in 32Dcl3, 32D-BCR/ABL, and in the hnRNP-A1 dominant-negative (NLS-A1-HA)–expressing 32D-BCR/ABL cells. Inset: Northern blot shows effects of imatinib on E2F3 mRNA levels in K562 cells.