Figure 2.

HnRNP-K regulates AR expression at the AR 5′-untranslated region (5′-UTR). (A, left panel) LNCaP cells were transfected with hnRNP-K expression plasmid and treated with either 100 nM rapamycin and/or 1 nM R1881. (A, right panel) Determination of AR level in presence and absence of hnRNP-K siRNA and 100 nM rapamycin alone or in combination. All data were analysed by NIH Image 1.63 and represented as the ratio of AR and β-actin. (B, left panel) Effect of hnRNP-K on AR 5′-UTR luciferase activity in presence and absence of rapamycin. AR 5′-UTR luciferase plasmid (500 ng) or the control plasmid (500 ng) were cotransfected with 500 ng of hnRNP-K in LNCaP cells. Lysates were assayed for luciferase activity and protein content. (B, right panel) To verify that hnRNP-K binds to AR mRNA, LNCaP cells were immunoprecipitated with either IgG or hnRNP-K antibody and RNA was extracted and subjected to RT-PCR analysis using AR specific primers. Negative controls included: no added reverse transcriptase (-RT), beads (protein A/G) alone, and IgG immunoprecipitate. Specific PCR band in extracts of LNCaP cells was used as a positive control. (C) COS and LNCaP cells were transfected with control plasmid (pIR-Luc) plasmid or plasmid containing the AR 5′-UTR in the presence of varying amounts of hnRNP-K. (D, left panel) Schematic representation of AR 5′-UTR-Luc constructs highlighting the conserved hnRNP-K binding motifs (UCCCCA and UCCCU) in human AR mRNA. These two motifs were altered to UCAAAA (414 mutant) and UCAAU (478 mutant). (D, right panel) Either AR 5′UTR-Luc wild type, or single (M414 and M478) and double mutants (DM 414/478) were tested for activity in the presence of varying doses of hnRNP-K.