Table 1. HIPK3-T mutations used in this study Name Mutation 50-splice site mutations A1 Weak native 50-splice site (0.16) strengthened to 4.36 A2 Weak native 50-splice site (0.16) strengthened to 7.15 A3 Deletion of the weak native 50- splice site A4 Strong downstream 50-splice site in weak native 50-splice site position. ESE Mutations B1 DESE1 B2 DESE1 B3 DESE2 B4 DESE2 B5 DESE3-4 B5x B5y B5z B6 DESE3 DESE4, peak a DESE4, peak b DESE 2-3-4 Combinations of ESE and 50-splice site mutations C1 DESE2 in the intermediate strength (4.36) native 50-splice site background C2 DESE2 in the strong (7.31) native 50-splice site background C3 DESE2 in the intermediate strength (4.36) native 50-splice site background C4 DESE2 in the strong (7.31) native 50-splice site background Mutations silencers downstream exon HIPK3-T D1 Exonic silencer neutralized D2 Exonic silencer neutralized D3 Both silencer troughs in down- stream intron neutralized D4 Second silencer trough in down- stream intron neutralized Systematic name e+5,6 AA-GG e+5,6 AA-GT e.108 A-C, e+2 T-C del e.107-109, del e+(1-34) e.5 G-A e.5 G-T e.86 A-T, 88 A-T e.84 G-T, 86 A-G, 88 A-T B5x+y+z e.95 a-t e.102 g-a e.108 g-c B3+B5 B3+A1 B3+A2 B4+A1 B4+A2 e.23 T-C, e.26 A-C e.22 T-C, e.27 T-C e+47 G-C +53 T-C e+57 C insertion Nucleic Acids Research, 2011, Vol. 39, No. 18 8095 Sequence GAGgttaaaaaa to GAGgttaggaaa GAGgttaaaaaa to GAGgttagtaaa GAGgttaaaaaa to GCGgctaaaaaa deleted: GAGgttaaaaaaacatctttgcacttattctcaaata GATAaAAGATATTGAA GATAtAAGATATTGAA GCTCAGAGGGAGGAtGtAT GGGAGtAgGtAATAGAAGA GGGAGGAAGAAATAGAtGATGCAaAA GAGcATGGACT As shown in B5 As shown in B5 As shown in B5 As shown in B3 and B5 – – – – TATTGAACATTTTcGTcTTTGGT TATTGAACATTTcTGTAcTTGGT CAGGTTAGGGGAcTCAAGcTTTAGTAA CAGGTTAGGGGAGTCAAGTTTcAGTAA RNA sequences used for Pull Down and Gel Shifts (vector sequence in grey) WT HIPK-3 exon aattgggtaccGATAGAAGATATTGAACATTTTTGTATTTGGTGGGGAGAGAGCTC AGAGGGAGGAAGAAATAGAAGATGCAGAAGAGGATGGACTAATTGATGGA GCAGAGTCTTTGAG DS Downstream sequence aattgggtaccgttaaaaaaacatctttgcacttattctcaaatacaggttagg B4 DESE2 aattgggtaccGATAGAAGATATTGAACATTTTTGTATTTGGTGGGGAGAGAGCTC AGAGGGAGtAgGtAATAGAAGATGCAGAAGAGGATGGACTAATTGATGGA GCAGAGTCTTTGAG B5 DESE3-4 aattgggtaccGATAGAAGATATTGAACATTTTTGTATTTGGTGGGGAGAGAGCTC AGAGGGAGGAAGAAATAGATGAtGCAaAAGAGcATGGACTAATTGATGGA GCAGAGTCTTTGAG B6 DESE2-3-4 aattgggtaccGATAGAAGATATTGAACATTTTTGTATTTGGTGGGGAGAGAGCTC AGAGGGAGGAtGtAATAGATGATGCAaAAGAGcATGGACTAATTGATGGAG CAGAGTCTTTGAG The mutant name, systematic name, sequence effect of mutant are shown. Underneath the sequence of the HIPK3-T exon (uppercase) and downstream intron (lower case, downstream 50-splice site is italicized). The ESEs are shown in coloured text consistent with Figure 1. Systematic name: e. denotes the exonic position of the mutation, e+ denotes downstream intron.