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. 2013 Jan;87(1):243–256. doi: 10.1128/JVI.01693-12

Fig 8.

Fig 8

PABPC1 binds ORF57 RNA in vivo and inhibits translation of ORF57 protein but not transcription and export of ORF57 RNA. (A) PABPC1 binds ORF57 RNA in vivo. HEK293 cells cotransfected with ORF57-FLAG and HA-PABPC1 expression vectors at 24 h posttransfection were UV cross-linked for CLIP assays with an anti-HA antibody. The RNA-PABPC1 complexes present in the pulldown assay mixtures treated with proteinase K were extracted and digested with DNase. Input RNA was extracted from 100 μl of the cell extracts before anti-HA immunoprecipitation. ORF57 RNA was detected by RT-PCR using a primer pair of oVM1 and oVM11. +RT, reverse transcription before PCR; −RT, no reverse transcription before PCR. (B) PABPC1 inhibits PAN expression without affecting export of ORF57 RNA. HEK293 cells (5 × 105 cell/well in a 6-well plate, 2 wells per group) were cotransfected with 1 μg of ORF57-FLAG (pVM7) and 1 μg of PAN (pJM1) in the absence (FLAG) or presence of 600 ng PABPC1-myc expression vector. At 24 h after transfection, total (T) RNA (4 μg/lane) extracted from the cells in one well and fractionated nuclear (N) and cytoplasmic (C) RNA (4 μg/lane) extracted from the cells in another well were analyzed by Northern blotting for PAN, ORF57, and GAPDH RNA, as described in the legend to Fig. 7. U6 RNA served as a fractionation efficiency control. PAN-Neo chimeric RNA was derived from the usage of an alternative SV40 poly(A) site downstream of the neomycin-coding region (see Fig. S5 in the supplemental material). (C) PABPC1 inhibits steady-state expression or translation of ORF57 protein but not transcription of ORF57 RNA. HEK293 cells were cotransfected with 1 μg of PAN (pJM1) and 0.2 or 1 μg of ORF57-FLAG (pVM7) in the presence of 600 ng of PABPC1-myc or FLAG control vector (PABPC1 −). Total protein and RNA from transfected cells (24 h after transfection) were detected by Western blotting (WB) for ORF57, PABPC1, and β-tubulin protein by using anti-ORF57, anti-myc (for PABPC1), or anti-β-tubulin antibody or by Northern blotting (NB) for ORF57, PAN, and GAPDH RNA, as described in the legend to Fig. 7. The relative level of ORF57 or PAN (including PAN-Neo) RNA, normalized to the level of the corresponding GAPDH for sample loading, in each sample is shown at the bottom of the Northern blots.