Supplementary Material(Carriero & Damha)

1 10 20 30 40 50 60 5’-GGGCGAATTCGAGCTCACTCTCTTCCGCATCGCTGTCTGCGAGGTACCCTACCAGGTGAG3’-CCCGCTTAATCTCGAGTGAGAGAAGGCGTAGCGACAGACGCTCCATGGGATGGTCCACTC

61 70 80 90 100 110 120 TATGGATCCCTCTAAAAGCGGGCATGACTTCTAGAGTAGTCCAGGGTTTCCGAGGGTTTCATACCTAGGGAGATTTTCGCCCGTACTGAAGATCTCATCAGGTCCCAAAGGCTCCCAAAG

121 130 140 150 160 170 180 CGTCGACGATGTCAGCTCGTCTCGAGGGTGCTGACTGGCTTCTTCTCTCTTTTTCCCTCAGCAGCTGCTACAGTCGAGCAGAGCTCCCACGACTGACCGAAGAAGAGAGAAAAAGGGAGT

181 190 200 210 220 230GGTCCTACACAACATACTGCAGGACAAACTCTTCGCGGTCTCTGCATGCAAGCTT-3’CCAGGATGTGTTGTATGACGTCCTGTTTGAGAAGCGCCAGAGACGTACGTTCGAA-5’

5’-SS

3’-SS

branchpointpolypyrimidine tract

CQ27 PBS

S1: Sequence of the PIP85.B splicing substrate gene. The 235-nt sequence is inserted between the T7 promoter region and Hind III restriction site of pBS- (Stratagene). When PCR amplified, the T7 promoter region (upstream of +1) and the 234-nt splicing sequence are produced. The 5’- and 3’-splice sites (5’-SS and 3’-SS), branch point adenosine and polypyrimidine tract are shown above. The primer binding sites (PBS) used for PCR amplification are the CQ27 primer (shown) and the universal M13F primer (not shown; located at position -47). CQ27: 5’-AGC TTG CAT GCA GAG ACC-3’; M13F: 5’-CGC CAG GGT TTT CCC AGT CAC GAC-3’

S2: PCR amplification of the PIP85.B splicing substrate gene. Panel A: Simplified schematic representation of PCR amplification of a double-stranded DNA substrate using two diverse primers (e.g. M13F & CQ27) and the thermophilic Taq DNA polymerase. Amplification over a number of number of cycles (N=30-35 cycles) produces an exponential amount of amplified DNA material (2N). Panel B: Analysis of the PIP85.B PCR product on a 2% agarose gel containing 20 ng/mL of ethidium bromide. The expected length of the PCR product is 281-nt.

506

396344298

1 Kbladder

PIP85.B

PCR

1 2

M13FCQ27

1. Heat denature (95°C)2. Anneal primers (45°C)

3. Taq DNA Polymerase

Repeat steps 1-3N cycles (N=30-35)

22NN copies of DNA copies of DNA

A. B.

0 10 20 30 60 90 min

1 2 3 4 5 6

E2E1

E2

E2E1

179-nt125-nt

234-nt

109-nt

S3: In vitro pre-mRNA splicing reaction of the PIP85.B substrate gene at various time intervals. Reaction mixtures were partitioned on a 15% (19:1 crosslink) denaturing gel (8 M urea) at constant power (75 Watts). As the reaction time progresses, the amount of mature RNA (mRNA) and lariat intron accumulate, whereas the amount of pre-mRNA and lariat-3’-exon decrease. E1: exon 1; E2: exon 2.

E2

E2E1

(-)v

e(+

)ve

5 10 20 0.1 0.5 2 5 10 20 M

14 15

ara-A ara-A Y-RNAY-RNA

(no L-dC)

ara-Aara-AY-RNAY-RNA

(with L-dC)

1 2 3 4 5 6 7 8 9 10 11

S4: Inhibition of pre-mRNA splicing in HeLa nuclear extract with variable concentrations of cold Y-RNAs containing an arabino-adenosine branchpoint (14 and 15). Splicing reactions were stopped after 30 minutes. Intermediates and products were partitioned on a 15% (19:1 crosslink) denaturing gel (8 M urea) and visualized by autoradiography. The negative control [(-)ve] represents the pre-mRNA alone. The positive control [(+)ve] is the spliced RNA in the absence of any inhibitor.

S5: Inhibition of pre-mRNA splicing in HeLa nuclear extract with small branched oligonucleotides. Cold linear, V-shaped and Y-shaped RNAs were incubated with the pre-mRNA transcript under splicing conditions. Splicing reactions were stopped after 30 minutes. Intermediates and products were partitioned on a 15% (19:1 crosslink) denaturing gel (8 M urea) and visualized by autoradiography. Oligomer 22 is a dodecamer Y-RNA, i.e., cUAA2’,5’(GUc)

3’,5’GUc. The negative control [(-)ve] represents the pre-mRNA alone. The positive control [(+)ve] is the spliced RNA in the absence of any inhibitor. Bracketed residues indicates nucleotides linked by a 2’,5’-phosphodiester bond.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

E2

E2E1

(-)v

e

(+)v

e5 10 20 5 10 20 5 10 20 5 10 20 5 10 20 5 10 20 5 10 20 M

AUC A(U)U A(U)G A(G)U UA(U)U AA(G)G 22