Fig. 6.

Reporter assays indicate that APA confers enhanced gene expression after stress. a Schematic of pRiG-based vectors for examining of PAS usage. pRiG-AD can generate two APA isoforms because of the presence of a weak PAS between RFP and IRES-EGFP regions, as indicated. pRiG does not contain a PAS and thus expresses the long isoform only. pRiG-SV-40 has a strong PAS, leading to expression of the short APA isoform only. RFP, red fluorescent protein; IRES, internal ribosome entry site; EGFP, enhance green fluorescent protein. Probe for Northern blot analysis is indicated. b Schematic showing experimental design. c Left, Northern blot analysis of two APA isoforms expressed from the pRiG-AD vector in NT or AS samples. Right, Log2(ratio) of expression level of long isoform to that of short isoform shown in the Northern blot. d Left, distribution of ratios of red fluorescence to green fluorescence in cells transfected with indicated plasmids in NT or AS cells. Higher log2(Red/Green) values indicate more expression of the short isoform encoding RFP. The difference in median log2(Red/Green) between NT and AS cells is indicated in each graph. pRiG + pRiG-SV40 is a mixture of the two plasmids with the indicated ratio in parenthesis. Right, schematic of expression of APA isoforms from indicated plasmids. e Schematic of psiCHECK2 reporters. Full 3′UTR sequences of mouse Nmt1, Dnajb1, or Timp2 were inserted downstream of the Renilla luciferase CDS. Firefly luciferase activity from the same reporter was used for normalization. f Dual luciferase assay for analysis of the effects of inserted full 3′UTR sequences on expression of Renilla luciferase in AS vs. NT cells. Error bars are standard deviation based on three replicates. g Dual luciferase assay for analysis of the effect of inserted cUTR or full 3′UTR sequence of Nmt1 on AS-induced regulation of expression of Renilla luciferase. Error bars are standard deviation based on two replicates