Fig. 2.

Release of hPSF from the GAGE6 regulatory DNA by hPSF-binding RNAs. (A) Binding of hPSF to GAGE6 regulatory DNA. ³²P-labeled GAGE6 regulatory DNA was mixed with a nuclear extract of YU-SIT1 melanoma cells, and 20 min later an anti-PSF monoclonal, or an anti-ATCB monoclonal antibody that does not bind hPSF, was added. The samples were incubated for 20 min at room temperature and fractionated by PAGE, and the gel was autoradiographed. Lane 1: GAGE6 regulatory DNA; Lane 2: GAGE6 regulatory DNA + nuclear extract; Lane 3: GAGE6 regulatory DNA + nuclear extract + anti-PSF antibody; Lane 4: GAGE6 regulatory DNA + nuclear extract + anti-ATCB antibody; Lane 5: GAGE6 regulatory DNA + E. coli protein. The high mobility bands at the bottom of the gel indicate free GAGE6 regulatory DNA, and the low mobility bands marked by an arrow indicate the GAGE6 regulatory DNA/hPSF complex. (B) Release of hPSF from GAGE6 regulatory DNA in vitro by hPSF-binding RNAs. ³²P-labeled GAGE6 regulatory DNA was incubated with a nuclear extract of YU-SIT1 cells, and 20 min later a hPSF-binding RNA or control RNA was added. Lane 1: GAGE6 regulatory DNA + nuclear extract without added RNA; Lanes 2–14: GAGE6 regulatory DNA + nuclear extract with added RNA; Lane 2: control RNA encoded by pcDNA-3.1; Lanes 3 and 4: mouse VL30–1 RNA; Lanes 5 and 6: L1PA16 RNA fragment; Lanes 7 and 8: MALAT-1 RNA fragment; Lanes 9 and 10: HN RNA fragment; Lanes 11 and 12: unidentified RNA fragment; Lanes 13 and 14: MER11C RNA fragment. The high mobility bands at the bottom of the gel indicate the free GAGE6 regulatory DNA, and the low mobility bands marked by an arrow indicate the GAGE6 regulatory DNA/hPSF complex. The molar ratio of RNA to GAGE6 regulatory DNA was 30 in Lanes 3, 5, 7, 9, 11, and 13, and 100 in Lanes 2, 4, 6, 8, 10, 12, and 14. (C) Release of hPSF from GAGE6 regulatory DNA in vivo by human hPSF-binding RNA fragments. The parental yusac line was stably transfected with plasmid pcDNA-3.1 and plasmid pcDNA-3.1 encoding 1 of the human hPSF-binding RNA fragments, and yusac-pcDNA3.1 and yusac-RNA lines were cloned. An anti-PSF antibody was used to immunoprecipitate hPSF from the cell lines, and the amount of GAGE6 regulatory DNA co-precipitated with hPSF was assayed by semiquantitative PCR. Upper shows the immunoprecipitated GAGE6 regulatory DNA, and Lower shows the amount of total GAGE6 regulatory DNA used to normalize the amount of total DNA in the samples. Lane 1: yusac-pcDNA3.1 control; Lane 2: yusac-L1PA16; Lane 3: yusac-MALAT-1; Lane 4: yusac-HN; Lane 5: yusac-unidentified RNA; Lane 6: yusac-MER11C. (D) Activation of GAGE6 transcription in vivo by human hPSF-binding RNA fragments. The amount of GAGE6 mRNA was determined by semiquantitative RT-PCR. Upper shows the amount of GAGE6 cDNA, and Lower shows the amount of ATCB cDNA used to normalize the total RNA in the samples. The cell lines for Lanes 1–6 are the same as for C.