Figure 2

The promoter defines the termination of transcription of hTR. (A) Analysis of the expression of the total hTR (Mature + 3′-extended) (left panel) and its 3′-extended (3′-extended) (right panel) region was performed by RT-qPCR. The mean fold-change in mature or 3′-end-extended hTR was normalized to the reference gene (GAPDH mRNA) and compared with the hTR level in HEK293T cells. Additionally, we normalized the samples to the GFP copy number determined for the cells during transduction of the reporter construct. The mean values were calculated from triplicate RT-qPCR experiments of three biological replicates with bars representing the SE. The **** indicates P < 0,0001, **P < 0,01 and *P < 0,1 by Sidak’s multiple comparisons test. (B) The ratio of the level of the 3′-extended region of hTR to the total hTR level was calculated to compare the transcription termination and processing rate independently of the different expression levels directed by various promoters in reporter constructs. The **** indicates P < 0,0001 by Sidak’s multiple comparisons test. (C) Northern blot analysis of the reporter constructs. The top panel shows hybridization to GFP specific probes to demonstrate the bicistronic construct expression. The middle panel shows hybridization hTR-specific probes to demonstrate the variations in hTR forms expressed from different promoters. The bottom panel was used as a loading control (7SL RNA) to demonstrate equal amounts of total RNA.