1. Supplementary MaterialsB A 2 9 3 1 7 10 4 5 6 8 11 12 13 14
Color key for aligment scores
2500
500
1000
1500
2000
<40
40-50
50-80
80-200
>=200
Query: Sus Scrofa ankrd1 intron 8 20 40 60
120
Query: Sus Scrofa ankrd1 intron 7 80
100
DD36
DD159
Supplementary Fig. 1. Results of BLAST searches for the intron 7 and 8 of the pig ankrd1 gene against the NCBI EST database. Pig ankrd1 intron 8 (A) and intron 7 (B) sequences were used as queries. For each query, the alignment score graph (left) and summary table (right) are shown. A - From 14 ESTs with similarity to the pig ankrd1 intron 8 sequence, eight are expressed in heart and skeletal muscle-derived libraries. White boxes - DD159 and DD36 bands (see Fig. 1). B – All ESTs with similarity to the pig ankrd1 intron 7 sequence are expressed in heart and skeletal muscle tissues. The pig ankrd1 intron 6 sequence did not reveal a significant similarity with a minimum alignment score value of 50.

2. 305
ANK1
ANK2
ANK3 e5 e6 e7 i7 e8
150
200
250
300
350
319
ANK4 e9
309 i8
339 i6
ANKRD1
ANKRD1-i8
ANKRD1-i7,8
ANKRD1-i6,7,8 FS A
1 MMVLKVEELV TGKKNGGGDA GEFLPEDFRD GEYEAAVTLE KQEDLKTLPA HFVSLGEQQW
61 KIEKEREAEL KKKKLEQRSK LENLEDLEII IQLKKRKKYR KTKVPVVKEP EPEVITEPVD
121 VPRFLKAALE NKLPVVEKFL SDKNNPDVCD EYKRTALHRA CLEGHLAIVE KLIEAGAQIE
181 FRDMLESTAI HWASRGGNLD VLKLLLNKGA KISARDKLLS TALHVAVRTG HYECAEHLIA
241 CEADLNAKDR EGDTPLHDAV RLNRYKMIRL LITYGADLNV KNCVSAQARG PSYRLSNEQA
301 WLTYLKIVF
ANKRD kDa
241 CEADLNAKDR EGDTPLHDAV RLNRYKMIRL LITYGADLNV KNCAGKTPMD LVLNWQNGTK
301 AIFDSLKENS YKASRIATF
ANKRD1-i kDa
241 CEADLNAKDR VSSLVSLGLL KLHSPFMWNP CPSRAPLQVP LAGLRPTLAP SLCLSRKEIP
301 PCTTP
ANKRD1-i7, kDa
i7-e8
e7-e8-e9
181 FRDMLESTAI HWASRGGNLD VLKLLLNKGA KISARDKVFL LLPPSFSSLP LRTCLGVWAG
241 LWGSAGSSTS QLLSTALHVA VRTGHYECAE HLIACEADLN AKDRVSSLVS LGLLKLHSPF
301 MWNPCPSRAP LQVPLAGLRP TLAPSLCLSR KEIPPCTTP
ANKRD1-i6,7, kDa i6
e7-e8-i8
i6-e7-i7 e8 e7 e9 i8 B
Supplementary Fig. 2. A - Predicted C-terminal structure of intronless and intron-retaining ANKRD1 protein isoforms. Exon/exon (e/e) and exon/intron (e/i) arrangements (vertical dotted lines, green) are shown. Regions coding by the exon- and intron-derived sequences are marked in grey and red, respectively. Ankyrin repeat (ANK) domains were mapped using the ScanProsite algorithm. Titin-binding (horizontal white rectangle) and calsequestrin-2-binding (horizontal black rectangles) sequences are shown (Mikhailov, Torrado, 2008). FS – frame shift position. In the ANKRD1-i7,8 and ANKRD1-i6,7,8, the retention of intron 7 would produce a frameshift reading (blue) of exon 8. In both ANKRD1-i7,8 and ANKRD1-i6,7,8 isoforms, a retention, respectively, of the intron 7 or intron 6 and 7 would result in modification of the C-terminus through removal of the ankyrin repeat domain 4 that, in turn, might affect these isoform’s interactions with cardiac calsequestrin-2. B - Predicted amino acid sequences of pig intron-retained ANKRD1 isoforms. Intron-derived sequences are shown in red, while adjacent exon 7, 8 and 9 sequences are shown in blue. Deduced MW values (kDa) are also indicated.

3. Supplementary Table 1: Primers used in this study
P – pig; H – human; e – exon; i – intron; F/R – forward/reverse primers. Each anchor primer has T7 sequence on the 5´end, while each arbitrary primer has M13 sequence on the 5´end (boldface).