Effects of Microprocessor target sequences (MTSs) on EDA alternative splicing and pre-mRNA processing. (A) Analysis of mature spliced transcripts. The indicated minigenes derived from pEDAL (Fig. 1A) contain different Microprocessor target sequences inserted between the EDA and the DRE. Minigenes were transfected in Hep3B cells and total RNA analyzed by RT-PCR to detect mature transcripts. (M) The 1-kb molecular weight marker. The identity of the corresponding amplified bands is indicated. The histogram shows the percentage of EDA exon inclusion. Data represent the mean ± SD of three independent experiments performed in duplicate. (B) Analysis of pre-mRNA transcripts. The indicated minigenes (0.45 μg) were cotransfected with pEDAL (0.15 μg) in Hep3B cells, and the nascent b fragment was amplified with the specific pair of primers indicated in Figure 1A. The identity of the b bands originating from pEDAL (n1) or other minigenes (n2) is indicated. (Lower panel) A control PCR, without reverse transcriptase (−RT). (C) Quantification of n1 and n2 ratio expressed as the mean ± SD of two independent experiments done in duplicate.
