<p>(<b>A</b>) U1-70K minigene constructs used for <i>in vivo</i> splicing analysis (see panel B), including exons 7, 7a, and 8; the arrows indicate the primers used for RT-PCR analysis. The enlargement below represents the region covered by the biotinylated transcripts used for <i>in vitro</i> binding studies (see panel C). The exact positions of the stop codon (dashed vertical line) and the three cryptic 5′ splice sites (bold vertical lines labeled with A, B, and C) are shown together with the 5′ splice site sequences, including all point mutations analyzed. The two solid lines above the enlarged exon mark the positions of the splice site blocking antisense morpholinos (see panel D); their labeling (AB and BC AMO) refers to the 5′ splice site they block. (<b>B</b>) Mutational analysis of U1-70K alternative splicing. Splicing patterns of U1-70K minigenes (as indicated) in control- (ctr) and U1C-knockdown (ΔC) HeLa cells were analyzed by RT-PCR, detecting alternative 3′ splice site activation (primers 7-7a; top panel), exon 7a inclusion and skipping (primers 7–8; middle panel), and as a loading control, exon 7 alone (bottom panel); splicing products are depicted on the right. Percentages of exon 7a inclusion are given below with standard deviations calculated from three individual experiments [n = 3]; the labels within the second panel indicate, which cryptic 5′ splice sites were used in each case for 7a inclusion, with letters in parentheses marking the less frequently used splice sites (see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#pgen.1003856.s003" target="_blank">Figure S3B</a></b>). (<b>C</b>) U1 snRNP binds to cryptic 5′ splice sites of U1-70K exon 7a. 3′-biotinylated RNAs spanning “exon 7a” including flanking intronic sequences (as shown enlarged in the middle of the schematic in panel A) were incubated with HeLa nuclear extracts (2% input). Bound proteins (2/3 of selected material) were analyzed by Western blotting, using antibodies against U1-70K, U1A, and U1C; bound U1 snRNA was detected by Northern blot hybridization. (<b>D</b>) Antisense morpholino (AMO) transfection in HeLa cells. In two separate assays HeLa cells were transfected with AMOs either against the cryptic 5′ splice sites of U1-70K exon 7a (AB, BC; left panel) or the 5′ end of U1 snRNA (U1; right panel), or with an unspecific control morpholino (ctr). Cells were either left untreated (−CHX) or were treated with cycloheximide (+CHX) before RT-PCR analysis. The label within the middle panel indicates which cryptic 5′ splice sites are used for exon7a inclusion, with letters in parantheses marking the less frequently used splice sites. M, DNA size markers (in bp).</p
