Terminal Intron Dinucleotide Sequences Do Not Distinguish between U2- and U12- Dependent Introns  Rosemary C Dietrich, Robert Incorvaia, Richard A Padgett  Molecular Cell  Volume 1, Issue 1, Pages 151-160 (December 1997) DOI: 10.1016/S1097-2765(00)80016-7

Figure 1 Products of In Vivo Splicing of the Wild-Type and Mutant Human P120 Intron F (A) Spliced RNA products were detected by RT-PCR analysis. The various products are identified by number and keyed to the corresponding splicing patterns shown in (B). Lane 1 is the PCR product of the transvected DNA as a marker for unspliced RNA. The other lanes are products from cells transvected with wild type (AU-AC, lane 2), the A-to-G mutation at the 5′ splice site (GU-AC, lane 3), the C-to-G mutation at the 3′ splice site (AU-AG, lane 4), the double mutation (GU-AG, lane 5), and the double mutation plus the C to G mutation at position 5 of the 5′ splice site (GU-AG+C5G, lane 6). (B) The various spliced products observed. The numbers refer to the bands shown in (A). Arrows, the locations of the normal U12-dependent splice junctions; underlining, mutant positions; and bold, terminal nucleotides of the splice junctions used in each product. Some of the products could be due to splicing in frames alternate to those shown. The sites shown preserve the /XU and AX/ dinucleotide pattern and minimize the number of sites used for the various products. Molecular Cell 1997 1, 151-160DOI: (10.1016/S1097-2765(00)80016-7)

Figure 2 In Vitro Splicing Patterns of P120 Intron Mutants (A) The P120 intron F wild type, A1G and C99G single mutant, and the A1G/C99G double mutant were transcribed, and the RNA was spliced in vitro in the presence of antisense 2′-O-methyl oligonucleotides directed against either U2, U12 snRNAs, or both. The RNAs are diagrammed on the left and from top to bottom are precursor, spliced exon product, 5′ exon intermediate, and lariat intron product. The band (*) between the spliced exons and the free 5′ exon intermediate is a degradation product, which comigrates with the lariat intron 3′ exon intermediate. Lanes 1–3, wild-type intron; lanes 4–6, A1G mutant intron; lanes 7–9, C99G mutant intron; lanes 10–12, A1G/C99G double mutant intron. Lanes 1, 3, 4, 6, 7, 9, 10, and 12 contained 2 μM anti-U12 oligo, while lanes 2, 3, 5, 6, 8, 9, 11, and 12 contained 8 μM anti-U2 oligo. Lanes 1, 4, 7, and 10 also contained 20 μM of a random sequence DNA oligo (AGCGGATAACAATTTCAC ACAGGAATATCCTT), which suppressed nonspecific degradation of the RNA. (B) Comparison of the in vivo and the U12-dependent in vitro splicing products. RNA was extracted from the spliced product region of the gel from reactions containing the anti-U2 oligonucleotide, reverse transcribed, and amplified by PCR. The in vitro spliced products of the wild type and the three mutants were run next to the analogous in vivo spliced products. The numbering of the spliced products follows that used in Figure 1. (C) Comparison of the in vivo and the U2-dependent in vitro splicing products. Analysis was as in Figure 2B but using RNA from in vitro reactions containing the anti-U12 oligonucleotide. Molecular Cell 1997 1, 151-160DOI: (10.1016/S1097-2765(00)80016-7)

Figure 3 Splice Site Sequences of Putative U12-Dependent Introns The distance listed in the right column of each entry is the number of nucleotides from the presumptive branch site adenosine residue to the 3′ splice junction. Sequences are from the following: human SCN4A (George et al. 1993); human SCN5A (Wang et al. 1996); human and mouse SCN8A (Kohrman et al. 1996); human CACNL1A4 (Ophoff et al. 1996); human CACNL1A1 (Soldatov 1994); human CACNL1A2 (Yamada et al. 1995); human CACNL1A3 (Hogan et al. 1996); human Sm E protein (Stanford et al. 1988); human ADPRP (Auer et al., 1989 and this work); human c-raf-1 (Bonner et al. 1985). Molecular Cell 1997 1, 151-160DOI: (10.1016/S1097-2765(00)80016-7)

Figure 4 Effect of 2′-O-Methyl Antisense Oligonucleotides against U snRNAs on In Vitro Splicing of U2- and U12-Dependent Introns (A) In vitro splicing of the human ADPRP intron 22. ATP and creatine phosphate were omitted from the reaction in lane 1 and included in all the others. Splicing reactions were preincubated with no oligonucleotide (lanes 1 and 2), 20 μM anti-U1 (lane 3), 8 μM anti-U2 (lane 4), 8 μM anti-U2 plus 10 μM anti-U6 (lane 5), 8 μM anti-U2 plus 5 μM anti-U6atac (lane 6), or 8 μM anti-U2 plus 2 μM anti-U12 (lane 7) oligonucleotides for 10 min, followed by addition of 32P-labeled RNA substrate for 3 hr at 30°C. The size markers in lane 8 are φ174 replication form (RF) DNA digested with HaeIII. The RNAs are diagrammed on the left, and from top to bottom are lariat intron product, precursor, spliced exon product, and 5′ exon intermediate. The multiple spliced exon bands are due to 3′ heterogeneity since only a single product was obtained after reverse transcription and PCR amplification (data not shown). (B) In vitro splicing of the U2-dependent adenovirus substrate RNA. Splicing reactions were preincubated with either no oligonucleotide (lane 2), 20 μM anti-U1 (lane 3), 8 μM anti-U2 (lane 4), 10 μM anti-U6 (lane 5), 5 μM anti-U6atac (lane 6), or 2μM anti-U12 (lane 7) oligonucleotides for 10 min followed by addition of 32P-labeled RNA substrate for 1 hr at 30°C. The positions of the various RNAs are indicated as in (A). Lane 1, RNA markers in nucleotides (Ambion). (C) In vitro splicing of the U12-dependent P120 intron F substrate RNA. Splicing reactions were preincubated with no oligonucleotide (lane 1), 0.5 μM (lane 2), 2 μM (lane 3), or 8 μM (lane 4) anti-U2 oligonucleotide, or with 20 μM anti-U1 oligonucleotide (lane 5). Lanes 6–10 all contained anti-U2 oligonucleotide at a concentration of 8 μM as well as 10 μM anti-U6 oligonucleotide (lane 6), 0.5 μM (lane 7), 2 μM (lane 8), or 5 μM (lane 9) anti-U6atac oligonucleotide, or 2 μM anti-U12 oligonucleotide (lane 10). Oligonucleotides were preincubated with extract and ATP for 10 min at 30°C followed by addition of 32P-labeled RNA substrate for 3 hr at 30°C. The positions of the various RNAs are indicated as in (A). Molecular Cell 1997 1, 151-160DOI: (10.1016/S1097-2765(00)80016-7)

Figure 5 Splice Site Sequences of Introns Homologous to Human SCN4A Intron 21 Genomic clones containing the introns shown were generated and sequenced as described in Experimental Procedures. Consensus sequences of U2-dependent (Senapathy et al. 1990) and U12-dependent (Hall and Padgett 1994) introns are shown (Y, pyrimidine). Molecular Cell 1997 1, 151-160DOI: (10.1016/S1097-2765(00)80016-7)

Figure 6 Effect of 2′-O-Methyl Antisense Oligonucleotides on In Vitro Splicing of the SCN4A Intron 21 Substrate RNA Splicing reactions were preincubated with no oligonucleotide (lane 1), 20 μM anti-U1 (lane 2), 8 μM anti-U2 (lane 3), 10 μM anti-U6 (lane 4), 5 μM anti-U6atac (lane 5), or with 2 μM anti-U12 (lane 6) oligonucleotides for 10 min, followed by addition of 32P-labeled RNA substrate for 3 hr at 30°C. The positions of the various RNAs are indicated as in Figure 4. Molecular Cell 1997 1, 151-160DOI: (10.1016/S1097-2765(00)80016-7)