(A) Alignment of PMEPA1-NKILA Promoter Region Sequences of Representative Animals

Supplementary file S1

(A) Alignment of PMEPA1-NKILA promoter region sequences of representative animals.

A 1250 nucleotide human sequence, from Ensembl ( GRCh38.p2, is aligned with corresponding Ensembl database sequences for representative animals. From top to bottom these are: zebrafish (Danio rerio, dataset Zv9), coelacanth (Latimeria chalumnae, LatCha1), turtle (Chrysemys picta bellii, ChrPicBel3.0.1), opossum (Monodelphis domestica, monDom5), cattle (Bos Taurus, UMD3.1), mouse (Mus musculus, GRCm38.p3), lemur (Otolemur garnettii, OtoGar3),and chimpanzee (Pan troglodytes, CHIMP2.1.4). Letters shown below the alignment denote - in reverse orientation - the amino acids encoded by the humanPMEPA1-001 and -002 open reading frames belowthe 2nd nucleotide position of each codon. For sequences with no clear similarities to the respective human NKILA region, the 3' ends of the compared sequences were chosen somewhat arbitrarily. The sequence alignment was done using the Muscle program supplied within GENETYX Ver.12 software package (Genetyx Corp., Tokyo, Japan), and slightly modified by hand. Intron border positions are indicated by downward triangles, transcriptional start points by arrows, and orientations of transcripts by (+) or (-) symbols. Only two regions are quite similar throughout the compared species, which are the coding part of human PMEPA1-001 and parts of the intergenic promoter region that we shaded gray. Among the conserved promoter motifs there are typical cAMP-response elements (CRE) and SMAD binding element (SBE)motifs (boxed) for binding cAMP response element-binding protein (CREB)(Conkright et al., 2003, Mol. Cell 11, 1101-8) and SMAD (Hua et al., 1999, P.N.A.S. 96, 13130-5) transcription factors, respectively. The finding of SMAD binding elements is consistent with the reported upregulation of PMEPA1 by TGF- (e.g. Nakano et al., 2014, J. Biol. Chem. 289, 12680-92). The NF-B binding motif (boxed) identified by Liu et al. is well conserved among eutherian mammals, but not beyond. For the discussion on to what extentNKILAequivalents are conserved through evolution, the definition of this gene is relevant.If, for example, functional NKILA would be defined by the hairpin motifs that Liu et al. identified, then conservation would most likely be restricted to only closely related primates (in the figure below, human NKILA hairpin A and B regions are underlined). However,in mice there is an orthologous - though spliced - transcript with similar starting point as human NKILA (GenBank EST BY735079; not shown in the figure), and despite the minimal sequence similarity this can probably be considered a murine variant of an “NKILA”transcript. Although for a few more eutherian mammals “NKILA-like” transcripts can be found (not shown), database information is probably not extensive enough to determine whether they are common among most eutherian mammals. When studying database informationbeyond eutherian mammals, we could not find conservation of transcripts orthologousto NKILA.In summary, we feel that from an evolutionary point of view the sequences which are compared in this figure primarily concern an ancient PMEPA1 promoter region, and that the NF-B binding motif and NKILA transcription should be studied within that context. It appears interesting that for a paralogue of PMEPA1, C18ORF1 - alias LDLRAD4 - noncoding antisense transcripts also have been reported (see the Ensembl database).

(B) Alignment of deduced PMEPA1 amino acid sequences for the species compared in (A). This figure shows the substantial conservation of PMEPA1 through evolution. Basic residues are indicated in red, acidic ones in blue, and green residues are more hydrophilic than the orange ones (Hopp and Woods, 1981, P.N.A.S. 78, 3824-8); cysteines are in purple. The depicted sequences were aligned by hand and based on: zebrafish, GenBank AL925928 combined with Ensembl genomic sequence; coelacanth, GenBank XP_005990654; turtle, XP_005305886; opossum, XM_007475673; cattle, our prediction from Ensembl genomic sequence corrected for some nucleotide identities by comparison with GenBank EST sequences; mouse, GenBank NP_075371; lemur, our prediction from Ensembl genomic sequence; chimpanzee, GenBank XP_009435732; human, PMEPA-001 from Ensembl.

(A)

intron border PMEPA

PMEPA1(-)

Zebrafish 1:AGATACTCACAGATCGCCATACTCTGGAATGAAGTGGATCTCTGGCAGTTACATGTACAG 60

Coelacanth 1:ATTTACTTACTGATCTCCATACTCGGGAACAAA---GACCTTTTACAGTTACATGTGCAG 57

Turtle 1:AAGTACTTACTGATCTCCATAGTCTGGAACAAA---GACCTTTTGCAGTTGCATGTGCAG 57

Opossum 1:GGATACTCACTGATCTCCATGCTCTGGAACAAA---GACCTTTTGCAGTTGCATGTGCAG 57

Cattle 1:GGTCACTCACTGATCTCCATGCTCTGGAACAAA---GAGCGTTTGCAGTTGCACGTGCAG 57

Mouse 1:GCACACTCACTGATCTCCATGCTGGGGAACAAA---GAGCGCTGGCAGTTGCACGCGCAG 57

Lemur 1:GGTCACTCACTGATCTCCATGCTCTGGAACAAA---GAGCGTTTGCAGTTGCACGTGCAG 57

Chimpanzee 1:GGTCACTCACTGATCTCCATGCTCTGGAACAAA---GAGCGTTTGCAGTTGCACGTGCAG 57

Human 1:GGTCACTCACTGATCTCCATGCTCTGGAACAAA---GAGCGTTTGCAGTTGCACGTGCAG 57

Human aa (rev) I E M S Q F L S R K C N C T C S

PMEPA1(-)

Zebrafish 61:GAGACATTGGCGAGGG------TGTCGGTGGTTCCGTTCGTCAGACCCATGAAG 108

Coelacanth 58:GAGACATTGGGCTGAA------TGGCTGCTGTTGTGCTGTTCAGACCCATCAAG 105

Turtle 58:GAGACATTGGGCTGTG------TGGCTGCGGCTGTGCTGTTCACACCCATCAAG 105

Opossum 58:GACACATTGGGCTGCC------CGGCGGCCGCGGTGCTGTTGAGACCCATTAAG 105

Cattle 58:GAGACATTGGGCTGCCCAGCGGCGGCGGCGGCGGCGGCGGTGCTGTTGACCCCCATCAAG 117

Mouse 58:GAGACATTGGGCTGCC------CGGCGGCGGCGGCGGCGGTGCCGTTGACCCCCATCAAG 111

Lemur 58:GAGACATTGGGCTGCC------CGGCGGCGGCGGCGGCGGTGCTGTTGAACCCCATCAAG 111

Chimpanzee 58:GAGACATTGGGCTGCC------CGGCGGCGGCGGCGGCGGTGCTGTTGACCCCCATCAAG 111

Human 58:GAGACATTGGGCTGCC------CGGCGGCGGCGGCGGCGGTGCTGTTGACCCCCATCAAG 111

Human aa (rev) S V N P Q G A A A A A T S N V G M L

PMEPA1(-)

Zebrafish 109:CTGAACATTGAGGAAATCG------ATGTGAAAAACGGACGGTTTGTCGGTTGGTTAGT 161

Coelacanth 106:TTGAACATTTAAAAAAAAA---GATATTACTAGAGATAATATGTTTAAACCAAAAAAAAA 162

Turtle 106:TGATACATTTAAAAAAAGG----GGAGAGCGAAGAACGGA------AAGTCAGC 149

Opossum 106:TTATACATTTAAAAAGGAG------GGAGAGGAAAACGGGGGACTTG-----GAGAAGAG 154

Cattle 118:CGGTGCATGGACGGCGCGGCGGAGCGGCGCGGGGCGCAGGGGGCTCG-----GGGGCGGC 172

Mouse 112:CGGT-CACGGGCGGCGCGG------GGCGCGAGGCGCGGGGCG------147

Lemur 112:CGGTGCATGGACGGCGTGGCGGCGCGGCACAGGACGCGGGGGGCGCG-----GGGACGGC 166

Chimpanzee 112:CGGTGCATGGACGGCGCGGCGGCGCGGCGCGGGGCGCGGGGGGCGCG-----GGGGCGGC 166

Human 112:CGGTGCATGGACGGCGCGGCGGCGCGGCGCGGGGCGCGGGGGGCTCG-----GGGGCGGC 166

Human aa (rev) R H M

PMEPA1(-)

Zebrafish 162:CGGTCGCCTCAGCGCGCGTTGTCGAATAAAAACATTGACGCAGGCTTTTCCTATTCTGTG 221

Coelacanth 163:CA-----CTTCTCAAGTAAGTCCAAATAAAAACAACTAAGAAGTCT------GAG 206

Turtle 150:CGAG---CTCTCCGGGCAGCACCACATAAAG------CCGGGCTCCTCCTG----GGG 194

Opossum 155:CGAGGGGGGCTCCGGGAGGCACCACATAAAGGAAGATCCTGCGGCTGCCGCTGCTTCAGC 214

Cattle 173:CGGGGGGGGCTCCGGCCGGCGCCCGGCG------CTCGGGCCCCGCATGCAG-GAG 221

Mouse 148:CGGGGGCGGCTCCGGCCGGCGC---GCGGGG------CTCGGGCCCCGCATACAGCGAG 197

Lemur 167:CGGGGGGGGCTCCGGCCGGCGCC---CGGAG------CTCGGGCCCCGCATGCAG-GAG 215

Chimpanzee 167:CGGGGGGGGCTCCGGCCGGCGCC---CGGAG------CTGGGGCCCCGCATGCAG-GAG 215

Human 167:CGGGGGGGGCTCCGGCCGGCGCC---CGGAG------CTGGGGCCCCGCATGCAG-GAG 215

PMEPA1(-)

Zebrafish 222:TC------TACCGTCCT------232

Coelacanth 207:TGAATGGAAGAAAGGAAGGA------226

Turtle 195:CGGGGGGGAGAGGGAACGGCGAACCACTCCTAAGG------229

Opossum 215:CCGGAGAGGGAAGAGAAGGCAATGCGCTTTTCTCGTTTAAAAAAAAAAATCAATTCTGGA 274

Cattle 222:GCGCGCGGCGGGGAAGGCGCGCCCCGGCTAGCCGGGCT------259

Mouse 198:CAGCGCGGCG------CGGGCT------213

Lemur 216:GCGCGCGACGGGGGAGGCGCGCCCCGGCTCTCCGGGCT------253

Chimpanzee 216:GCGCGCGGCGGGGGAGTCGCGCCCCGGCTCGCCGGGCT------253

Human 216:GCGCGCGGCGGGGGAGGCGCGCCCCGGCTCGCCGGGCT------253

PMEPA1(-)

Zebrafish 233:------CTAACCGCCGTCCCCC------248

Coelacanth 226:------226

Turtle 230:------CAGCCCCCCGAC------241

Opossum 275:GAAATTAAGTTATAAAACAAAACACCCCCCCCCCCAAAAAAAAAAACAGTGGAGGGAAGA 334

Cattle 260:------CCGGGCTCTGCC------AAGTTACCGGGG 283

Mouse 214:------CGGGGCGCCGCC------GAGGTCCC---- 233

Lemur 254:------CGGGTCGCAGCC------AAGTTCCCGAGG 277

Chimpanzee 254:------CGGGTCGCCGCC------AAGTTCCCGGGG 277

Human 254:------CGGGTCGCCGCC------AAGTTCCCGGGG 277

PMEPA1(-)

Zebrafish 249:------CGCCCCCGGCCCGCT---- 263

Coelacanth 227:------CTTGTTTCCCC--GT 239

Turtle 242:-----GGAAAGACAAGGAGACCCGAGCCGGGCGCTGCTGGCTTCCCTCCCCTCCCC---C 293

Opossum 335:AACAGTCAAACTGGGGGAGGACCTAACTGCTCTTTCTTCCCTCCCCTCGGTTTCCCTTCT 394

Cattle 284:CGCCGTGGGACTCAGTGCT--CGGGGCCGCGCTCCTCTGCGCTCCCCCGGCTGCCC--CT 339

Mouse 234:-----CGAGGCGAGCTGGG------TCCCCCGGCCGCCG---- 261

Lemur 278:TGCCGCGGGGCTCAGTGCG--CCGGGCCTCGCTCCTCTGCGC-CCCTCGGCCTCCC--CT 332

Chimpanzee 278:CGCCGCGGGGCTCAGTGCG--CGGGACCGCGCTCCGCTGCGCCCCCCCGGCCTCCC--CT 333

Human 278:CGCCGCGGGGCTCAGTGCG--CGGGACCGCGCTCCGCTGCGCCCCCCCGGCCTCCC--CT 333

PMEPA1(-)

Zebrafish 264:------GTCTGGC 270

Coelacanth 240:TAGCAGAAAGCCAGTGAG------GAAATGGAGTTGC 270

Turtle 294:CGCCAG---CCCGGGAAGTTTGCTCAGGGCTGG------GTGAAACAGACTTGC 338

Opossum 395:CTCCAGTCTCTCGGTAAGTTTCCCTTGCCCCAGCGCCGG------CGGAAAGCGCCTAGC 448

Cattle 340:CCGCAG---CCCCGGGGGCGTCCGCAGTGCCCGCGGGTGGCGTCCGGGAAATGGGCTGGC 396

Mouse 262:-AGCGGGAGCCCCGGGGCGGCGGGCCGTGGCCGCGGGCGGCGTGCGGGGCACGGGCGGGC 320

Lemur 333:CGGCTG---CCCCGGGGGCGTCGGCAGTGCCAATGGGTGGCGTCCGGAAAATGGGCTGGC 389

Chimpanzee 334:CGGCAG---CCCCGGGGGCGTCGGCAGTGCCCGCGGGTGGCGTCCGGAAAATGGGCTGGC 390

Human 334:CGGCAG---CCCCGGGGGCGTCGGCAGTGCCCGCGGGTGGCGTCCGGAAAATGGGCTGGC 390

PMEPA1(-)

Zebrafish 271:CGCTGTGC---TCCAGTATTCCCGTT------ATTCCCAGACGGTTTGTACTACTTTCC 320

Coelacanth 271:TACT------TGTTTCTCTGCCTGGG------CTCTTAGCGGCTTT---- 304

Turtle 339:TACTTTGTTTCTCTCTCGCTCCTGCCCAGG------CAGGCTCCTAGCGGCTCCCCCC 390

Opossum 449:TACTACTCTGTTTCTGCG---CTGGCCGGACGGAGCGACGGACTCCCGGCGGCGGCTTTC 505

Cattle 397:AGCCGGG----GCGCGCGCTGCCGGCGGGGCTGAGCCTCTG-CTGCTAGCTTCCCCCAGC 451

Mouse 321:GGCCGGG------GCACGGGGCCGCC-GGGCTCAGCCTGCG-CGCCGGCCGATCCGCAGC 372

Lemur 390:AGCGGGG-----CGCGCGCTGCCGCC-GGGCTGAGCCTCCG-CCGCTAGC-TTCCCTAGC 441

Chimpanzee 391:AGCGGGG-----CGCGCGCTGCCGCCGGGGCTGAGCCTCTG-CCGCTAGCTTTCCCCAGC 444

Human 391:AGCGGGG-----CGCGCGCTGCCGCCGGGGCTGAGCCTCTG-CCGCTAGCTTTCCCCAGC 444

human PMEPA1-001 transcript start

conserved motif with unknown function

Zebrafish 321:--AGCGTCTGCGC------G------CACCTCATTCAAGTCCAAGTTGG 355

Coelacanth 305:------CCCCTCATTCAAGTCCAAGGAGA 327

Turtle 391:------TCCCCGCCCCCTTCG------CTCCTCCTCATTCAAGTCCAAGGAGA 431

Opossum 506:CCAGCCACTCCGCCTCCTCCG------CCTCCTCCGCCTCCTCCTTCAAGTCCAAGGAGA 559

Cattle 452:CGAGCGCCTCCGCCGCCGCCG------CCTCCTCCTCCTCATTCAAGTCCAAGGAGA 502

Mouse 373:TGAGCGCCGCCGCCCG------CTCCTCATTCAAGTCCAAGGAGA 411

Lemur 442:CGAGCGCCTCCGCCGCCGCCG------CCTCCTCCTCATTCAAGTCCAAGGAGA 489

Chimpanzee 445:CGAGCGCCTCCGCCGCCGCCGCCGCCGCCGCCTCCTCCTCCTCATTCAAGTCCAAGGAGA 504

Human 445:CGAGCGCCTCCGCCGCCGCCGCCGCCGCCGCCTCCTCCTCCTCATTCAAGTCCAAGGAGA 504

SBE

Zebrafish 356:CTAGG------AAGCGGCAGGGGGCGGAGCCAACATTGAGGGGCGGGATCTCGAGGCAG 408

Coelacanth 328:TTCAGTTTCATGTGGATAC--TAGTCTGAAAC------AGGCAG 363

Turtle 432:TTCAGTTTCCTATGGAAAGGGGCGTCTGAGGC------AGACAG 469

Opossum 560:TTGGGTTTCATATGGAGATACGGTACTGAGGC------AGGCAG 597

Cattle 503:TCGGGTTTCGCTCCGAGACCGCGGTCGGAGGC------AGGCAG 540

Mouse 412:TCGGGTTGCGCCGCGCGCCCGCGGTCGGAGGC------AGGCAG 449

Lemur 490:TCGGGTTTCGCTCCGAGACCGCGGTCGGAGGC------AGGCAG 527

Chimpanzee 505:TCGGGTTTCGCTCCGAGACCGCGGTCGGAGGC------AGGCAG 542

Human 505:TCGGGTTTCGCTCCGAGACCGCGGTCGGAGGC------AGGCAG 542

CRE SBE

Zebrafish 409:ACAGCG-TGACGTCATTGCCAGACTTGGCC-----TTTCG--CAGAGCGT---GCGAGCC 457

Coelacanth 364:ACAGTCATGACGTCACTGCCAGACTGGGCCAGCCCCTCCCA-GGCACTGCAAAGTGGGC- 421

Turtle 470:ACAGGG-TGACGTCAGCGCTAGACTGGGACTGCCCTTTCCAGCGCCCCGCCAGGCGGGGC 528

Opossum 598:ACGGCC-TGACGTCAGCGCTAGACGGAACTGCCCTTTTCCACCGCACTGGCGGAGGGGGG 656

Cattle 541:ACGGTC-TGACGTCAGCGCTAGAC-GGGGCTGCCGGTTCC--CACCGCGC---GGGGGCC 593

Mouse 450:ACGGTC-TGACGTCAGCGCTAGAC-GGGGCTGCCGGTTCC--CACCGCGC---GGGGGCC 502

Lemur 528:ACGGTC-TGACGTCAGCGCTAGACGGGGTCC------CACCGCGC---GGGGGCC 572

Chimpanzee 543:ACGGTC-TGACGTCAGCGCTAGAC-GGGGCTGCCGGTTCC--CACCGCGC---GGGGGCC 595

Human 543:ACGGTC-TGACGTCAGCGCTAGAC-GGGGCTGCCGGTTCC--CACCGCGC---GGGGGCC 595

NF-B

Zebrafish 458:AATCGGAGTCACACGGGGATCAAACAACACACACACACACACACACACACACACACACAC 517

Coelacanth 422:------CACCC 426

Turtle 529:GGGGAGGGGAAGAGAGAGAAGGGGGCGGGTGAGT------GACGGCG 569

Opossum 657:GAGCGGGGGGAGTCTAGGAAGGAGGAGAGGAGCCCCCCTCTAGCCTTGGCGGCTGCACCA 716

Cattle 594:GGGGAGGGG------GCGCGCGC------TGCGCCA 617

Mouse 503:GCGGAGGGG------GCGCGCGC------TGCGCCA 526

Lemur 573:GGGGAGGGG------GCGCGCGC------TGCGCCA 596

Chimpanzee 596:GGGGAGGGG------GCGCGCGC------TGCGCCA 619

Human 596:GGGGAGGGG------GCGCGCGC------TGCGCCA 619

Zebrafish 518:ACACACACACACACACACACACACACACACACACACACACACACACACACACACACACAC 577

Coelacanth 427:GCTTTGACTGA------CTCCTCCCGCCACCAATCACAATTTTACTTGGAAAT 473

Turtle 570:CTCCTAGCCAA----TCCCAGCTCCGCCGCCCGGC------GGGAGG 606

Opossum 717:ATCCCTACCGAGGTTCCCCCGCACCCCCCCCTTTCTCCCTCCCAGCGCTAGGAAGGGTGG 776

Cattle 618:ATCCCCGCCGAGGTTCTCCCGCACCCCCTCCCCGGGCC------GCGGGG 661

Mouse 527:ATCCCCGCCGACGGCCCC---CGCACCCTCCTCGGCCC------GGGGGG 567

Lemur 597:ATCCCCGCCGAGGTTCCCCCGCACCCCCTCCCGGTCT------GGGAGG 639

Chimpanzee 620:ATCCCCGCCGAGGTTCCCCCGCACCCCCTCCCCGAGCC------GGGGGG 663

Human 620:ATCCCCGCCGAGGTTCCCCCGCACCCCCTCCCCGAGCC------GGGGGG 663

Zebrafish 578:TGTATTGATCGATTTCACTG------GGACAGTCAAATAAAGTGTGTG------619

Coelacanth 474:TTAAAGGGCTGTTTTTAAAG--GTACATTGCCCTTCTAAAGGCTTGTGCC------521

Turtle 607:CGGGGCGCCCGA------GCCCCTCCAGGCGCAGCTGCGGTGACCCGCA 649

Opossum 777:GGGGGGGAATGAATGAGGGGGGTGAAACTGACTCTTAAAGGGACAGAGTG------826

Cattle 662:CGGGGTGACGGGGAAGGGGG------CGGGCTCTTAAAGGGCCAGAGCA------704

Mouse 568:CGGGCCCGTCGCTCGCGGGG------CGGGCTCTTAAAGGGCCCGAGCC------610

Lemur 640:CGGGGTGACGGTAAACGGGG------CGGGCTCTTAAAGGGCCAGAGCC------682

Chimpanzee 664:CGGGGTGACGGGAAACGGGG------CGGGCTCTTAAAGGGCCAGAGCT------706

Human 664:CGGGGTGACGGGAAACGGGG------CGGGCTCTTAAAGGGCCAGAGCT------706

human NKILA (+)

Zebrafish 620:------TGTGTGTGGACGGAAAGACATCACAGAGAGCACAGACGGAATATTTTACG 669

Coelacanth 522:------AGTAAGTACAT------TTCCCCAAAATCAACCAAGAGGCAAATCT-- 561

Turtle 650:CCCGCAGTGTGGGGTGAGCGCGCGGCGAGCTCGGTCGGGCCGCGCGTGCGGCTCGCAGCT 709

Opossum 827:------CATTCGCGAATGCAG------CAAAGCCAC---CGCCACACGCCATC 864

Cattle 705:------GGGCGGTCCGCGTAGACCCGGGACCCGCGAAGCGAAAGAAGGGTGCAGCG 754

Mouse 611:------TAGC-GTCCATGTAGACCGGTCGCCGGCGCTGCGTAG-----ACCCAGCG 654

Lemur 683:------AAGCGGCCCGCGTAGACCCGAGACCAGCGCGACGGAGGAGGGGCGCTGTG 732

Chimpanzee 707:------AGGCGGCCCACGTAGACCCGGCACCCGCGCAACGGAGGAGGGGCGCTGTG 756

Human 707:------AGGCGGCCCACGTAGACCCGGCACCCGCGCAACGGAGGAGGGGCGCTGTG 756

human NKILA transcript start

human NKILA (+)

Zebrafish 670:CACTT------TTAGGTCTGATT------AAGTTTGA 694

Coelacanth 562:------GAAAGTGTAATTTTTTTTTAAATAATTTTAAAAAAATCAGCATTTC- 607

Turtle 710:CGCCG------GGGAGTCGCGTTGTTCCCTGGTGGGACGCTGCCGTGTCCGGGCCCG- 760

Opossum 865:CGC------AGACTTCAGACA----CACACAGAGACACAGACGTGCACAC------904

Cattle 755:TCCCCTCCCAAACGGCGGTCAGTTT------GGAAGCTCTGACCCTCTCAGGCCAG- 804

Mouse 655:CCCTC------GAGACTC------CGCAGGCCCG- 676

Lemur 733:TCCCCTCCCCAGCGAGGGTCAAGTT------AGTCCCGCACAAGCTTG- 774

Chimpanzee 757:CCCTCTCCCCAACGGCGGTCAGCTTG------GAACGCCTGCCCGGCGCACGCCCG- 806

Human 757:CCCTCTCCCCAACGGCGGTCAGCTTG------GAACGCCTGCCCGGCGCACGCCCG- 806

human NKILA (+)

Zebrafish 695:TGTGCTTGAAAGATGTAATT-----AATGTAAATAT---TATGTATTTTATATCATAATT 746

Coelacanth 608:-ATGTAAGAATGATGGAATCAAAAAAAGTTCCATACATCTATTGTAGGAGTATAAGAACA 666

Turtle 761:-ATCCAAAGCCCCAAGGCTCCCGCTGGCCCCGGCGG------GCTTAGGATCGGGCCC 811

Opossum 905:-AGACTCGGAAGGGAAAATACATACGGTGGCGGTGGTGGTGACTCCATTAACTTATATAG 963

Cattle 805:-GCCCGGGAGAGCAGGATTG-----GGTCCCAGCCTTTGTGG------840

Mouse 677:-GTCCGCGGGAGCCCC------CGTGCCTGCGGCGGTCACAACACAGAACAAGAATC 726

Lemur 775:-GGCCGCGGGAGGGGGGCTC-----GGTCTCAGCCCC------ACC 808

Chimpanzee 807:-GGGCCAAGGAGCCGAACTC-----GGTGCCAGCCGC------ACC 840

Human 807:-GGGCCGGGGAGCCGAACTC-----GGTGCCAGCCGC------ACC 840

human NKILA (+)

Zebrafish 747:TGAGTGAATAA----AGC------GCTCTCCT------768

Coelacanth 667:AAAGAGAAAGAGAATGGGGATAAAAGCTCACCCATAGTGACCTGGTTTCCGAATTTCACC 726

Turtle 812:CGGCCGGGCAGCGG------GCTCGGCTCTCCGGTGGCTGGCCGCGG------C 853

Opossum 964:TAGTTGCTTAATG--AAT------GTCATTCTGAGTTATGGAATTCCCAAG------C 1007

Cattle 841:------GTGCGTTCCCCACAACCCGGACCTCGG------G 868

Mouse 727:AAGACGAAAAGTTAAGGT------GTTCTCCTCTCCAACTCCTGCCCCTGG------G 772

Lemur 809:CTGGCGTGTTGCC--GGT------GCTCCCAACCTCTGC-CCGCGCCCCGG------G 851

Chimpanzee 841:CGGGCGGGTTGCT--GGT------GCGCCCTCCCCTCGCCCCCGTCCCTGG------G 884

Human 841:CGGGCGGGTTGCT--GGT------GCGCCCTCCCCTCGCCCCCGTCCCTGG------G 884

human NKILA (+)

Zebrafish 769:---CTCGTTTTG------GGATTTTAAAGTTTGTCT 795

Coelacanth 727:AACTCTGACCAGTTCCACTGTCCCTCAATAATCAATACCAGCGTCTCTTAAAAGTATTTT 786

Turtle 854:AGCCCCGAGCGG------TGGCGGTGC--- 874

Opossum 1008:GTCCCTATTCCT------GCCTTTTTGAAG------1031

Cattle 869:GTCCTTGACCGA------GGCTTTGGGGGGAAGCTT 898

Mouse 773:GCCCTCCACCCA------GGCTTTTGGGGGCA---- 798

Lemur 852:GTCCTTGACCCA------AGCTTTGGGGGTTAGCCT 881

Chimpanzee 885:GTCCTTGACCCA------GGCTCTTGGGGCTAGCCT 914

Human 885:GTCCTTGACCCA------GGCTCTTGGGGCTAGCCT 914

human NKILA (+)

Zebrafish 796:AAAACGACATCAAAACAATAAAAGTGTAATGG----AGATTTAGA------ATGC 840

Coelacanth 787:TTGACCATACTAAATTTT----AATGTTTACA----GCATTGAGAAATATTTCATTCTGC 838

Turtle 875:------AGGTGCAGGTGGTTCCCGGC----GAAGTGGGTAATAATGAACCGTCC 918

Opossum 1032:------GGGGTGGCGA----AGATGGTGG------1050

Cattle 899:GTCCCCCTTCAGAGGAGCACGAGGTCCCTCGGACTCAGGTCAAGGGAAATT------949

Mouse 799:------CCCGAAGACTATAAGGTTTCTGGG----GCGCCTTGGAAGAGTTTA--ATGC 844

Lemur 882:AT------CTAAAGCGCAGGAGGGTCTCGGG----GGCTCAGGGAAAGGAAAT------924

Chimpanzee 915:AT----CTTCTGAGGAGCACAAGGTCCCTGGG----GGCTCAGGGAAGAGAAAT------960

Human 915:AT----CTTCTGAGGAGCACAAGGTCCCTGGG----GGCTCAGGGAAGAGAAAT------960

human NKILA (+)

Zebrafish 841:ACAT------AAAATAATG 853

Coelacanth 839:ACATCTATTACATTGTGATTTCCATCTCCTGGTCTCTAAATCCGTTCTCCCGAAGTGGTG 898

Turtle 919:CTGG------GGAGCGGCG 931

Opossum 1051:------AGAATGGGG 1059

Cattle 950:------AGGGAAAGG 958

Mouse 845:ATGG------AGATCAGAG 857

Lemur 925:-TGG------AGAAAGGGG 936

Chimpanzee 961:-TGG------AGAAAGGGG 972

Human 961:-TGG------AGAAAGGGG 972

intron border of human PMEPA1-002

PMEPA1 (-) human NKILA (+)

Zebrafish 854:------ACTATTATCAATTTTTCTCACGGTTTCCAGAAGA------887

Coelacanth 899:AAAAAAGTCTGCCCCTGTGTAGATACTCTTATCTGCCTCAGTATT------943

Turtle 932:GTGAGCACCTTGC------CCTCCCATGCACCCGAA--TTGAAGGGGGG------972

Opossum 1060:AAAGGAG------AGTTGGTTTTGT------1078

Cattle 959:CGGGGAGCCCTTCAGA------TCATCCGT-TACCTGAT--TTCGCAGTCAACTCCCC 1007

Mouse 858:CATGGAGGGT-CTGGAGTAAAGACCCATCCAT-AGCTGTAT--TTCTCAGGAGA------907

Lemur 937:AGGGAAGCCC-CCAAGATCAA---TCACCCAT-TGCCTGAT--TTCAATGGAGACTCTCC 989

Chimpanzee 973:GAGGAAGCCC-CCAAGATGGA---TCACCCAT-TGCCTGAT--TTCGCAGGAGACTGTCC 1025

Human 973:GAGGAAGCCC-CCAAGATGGA---TCACCCAT-TGCCTGGT--TTCGCAGGAGACTGTCC 1025

Human aa (rev) M

PMEPA1 (-) human NKILA (+)

Zebrafish 888:--TCCTAAAATTTCACTGACATTGAGTCTAA---TAATTGAGTCTCCAACAGTTGTGTTC 942

Coelacanth 944:---TTTAGTACTCAAATTACCCCTCCTCACAATCTCCTTGTCTTTGGGAAGAAAATACTA 1000

Turtle 973:------AGCTAGCCACCTGCACACGGGGACACCCCCAGCAGGCCCCCTGAGGAGGCAACT 1026

Opossum 1079:------CAATGGCAAGCTTTTTCATATCTGGCACATTCCCCTCAGACTTATCC 1125

Cattle 1008:GATTCCAGATCTGCAGCTCCAAGAGGCCCCAAGCCCACTGCGCCCCCCGC----GCAATT 1063

Mouse 908:------CTGAGAATTAGTCTGAGCCCCTCCGCAACCCCCAACCCCGCGCGG 952

Lemur 990:GCCCTCGGTTCTGCTGCAGCACGCGG------CCACCGAGCCCTG----GGTGCACTT 1037

Chimpanzee 1026:GCCTTCAGTTCTCCAGCAGCTCGGGGATCATGGCCCACTGAACCCCC-----AAGCGCTT 1080

Human 1026:GCCTTCAGTTCTCCAGCAGCTCGGGGATCATGGCCCACTGAACCCCC-----AAGCGCTT 1080

human NKILA hairpin A region

PMEPA1 (-) human NKILA (+)

Zebrafish 943:CCTTTTATTCACCAGGGGGTCACCACAACGGAATGAACCACCAACTATTTCAGCATATGT 1002

Coelacanth 1001:TTTTTTTTTCGTTATAACAACAATTTAGATCTTGGAATCATTCTTTATGCTTTTTTGCGT 1060

Turtle 1027:CCTGCCAGGCCTGGGGCTGCAGGCTAGCGAGGGGTGGTGACACATCGCCCCCCTTCCCCT 1086

Opossum 1126:TATTT------1130

Cattle 1064:TATCTCGACCTAGAGGAGGTCGACTGGGGAAAA------C 1097

Mouse 953:CTCTTTAACT------GGGCCTTTTGGATAGA------C 979

Lemur 1038:TAACCCGAATCCAAGGAGGTCGACTTGGCAAGA------C 1071

Chimpanzee 1081:TCACCCGAACCCAAGGAGGACGACCAGGAAAGA------C 1114

Human 1081:TCACCCGAACCCAAGGAGGACGACCAGGAAAGA------C 1114

PMEPA1 (-) human NKILA (+)

Zebrafish 1003:TGTACGCAGCGCATACCCTTCCAGCTGCAACCCAGTACTGGGAAACACCCAAACACACAC 1062

Coelacanth 1061:GCTTTTTTGCGTACTTTTTCCGGTGTATCAATGCTTTTCTTGAAACAAGACGACCAAAAC 1120

Turtle 1087:GGGGTCTAGCGCAGATTCTCC------CCCCCCCCCCAAAAAAAAAGCGAGACTGCAGCC 1140

Opossum 1131:------GGAAACTTCCTCC------GCAGTGGGGTACAGCC 1159

Cattle 1098:TAGAACTCCTGTAGACACGCC------CCGGACTGCTCCGAGAAACGCC------C 1141

Mouse 980:TGGAATTTCTGTAGACACGCC------CCGAGAAGCGCC------C 1013

Lemur 1072:TGGAACTCGCATAGACACGCC------C 1093

Chimpanzee 1115:GGGAACTCGCGTAGACACGCC------C 1136

Human 1115:GGGAACTCGCGTAGACACGCC------C 1136

human NKILA hairpin B region

human PMEPA1-002 transcript start

intron border of human NKILA transcript DA866558

human NKILA (+)

Zebrafish 1063:TCACACTCATACACTACG---GCCAG------TGTAGTTGATCAGTTCCCCTATAGC 1110

Coelacanth 1121:TATATCCAATAT----TGTAAATGAGGCCTAAACAAGTAATTGTTCCATGCTAAT----- 1171

Turtle 1141:CCAAACTCACGCAGCACGTCGGTGAC------AGTCAGGAATCGAACCCTGGGGCCA 1191

Opossum 1160:TAGGATCCAGGC----TTTGGGGGGGAGGTGTGTGTGTGGTGTATTTGTGTTT------1208

Cattle 1142:GGGAACCCTTGT--CATGTAAATACG------TGTTGGGGACTTGCAC------1181

Mouse 1014:CCTGGTCCTTGC--CTTGTAAATTAG------TGCCCGGGACTGGGGGTGAGAGTGT 1062

Lemur 1094:GGGAGCCGTTGT--CATGTAAATAGA------GGTCCTTGACTA------1129

Chimpanzee 1137:GGAAGCCCTTGT--CATGTAAATAGC------TGTCGGGGACTG------1172

Human 1137:GGAAGCCCTTGT--CATGTAAATAGC------TGTCGGGGACTG------1172

human NKILA (+)

Zebrafish 1111:GCATGTGTTTG---GACTGTGGGGGAAACC------GGAGCACCCGGA-----GGAAAC 1155

Coelacanth 1172:------GTAAATTCTCTTGATTCAAATTTGATATTCCTGGC----TATAATT 1213

Turtle 1192:------AATTCTGATCTAAACGCTGCCAGTGTAAATCCAGA-----GTAACT 1232

Opossum 1209:------GTCTGTTTGTGTCTCTCTGCCTGTCTGTGTCTGTGTGTCTGTGTCT 1254

Cattle 1182:------GGTCCCGGCCAGCGAGGCCCGGG-----GCGAAT 1210

Mouse 1063:GTGTGTGTGTGTGTGTGTGTGTGTGTGTTACTATTATGGGACACCCAAGCAGTTACACAC 1122

Lemur 1130:------GTGTATTGTGGCCGCCCCACCCGGCGGGGCCCGGG-----GCGAAT 1170

Chimpanzee 1173:------GTGTATTGTCGCCGCCCCAGCCGGCGGGACCTGGG-----GCGAAT 1213

Human 1173:------GTGTATTGTCGCCGCCCCAGCCGGCGGGACCTGGG-----GCGAAT 1213

human NKILA (+)

Zebrafish 1156:CCACACCAACACGGGGAGAACATGCAAACTC-CAC------AC------AGAAACA 1198

Coelacanth 1214:TCAAGTAGTC------TGTTTGCTCATTCAACTGTGCAGAGTGTTTCAGTTGTA 1261

Turtle 1233:CCACAGTGGC------TACTCCAGCTC-CACAGTGGTGTAATGGAAATTAGGTGTG 1281

Opossum 1255:C------TGTGTGTTT-TGC------AGGAGGGGTGTGGGGCG 1284

Cattle 1211:CCACACCCTA------CGTCTGCTG-CCC------AA------AGGGCCA 1241

Mouse 1123:CCTCACCCTC------TATCTGCCG-CCC------CA------AGGGCCA 1153

Lemur 1171:CCACACCCTT------TGTCTGCTG-CCC------GA------GGGGCTA 1201

Chimpanzee 1214:CCACACCCAT------TGTCTGCTG-CCC------GA------GGGGCCA 1244

Human 1214:CCACACCCAT------TGTCTGCTG-CCC------AA------GGGGCCT 1244

human NKILA (+)

Zebrafish 1199:CCAACTGACCCAGCCGAGACTCAAACCTTCTTACTGCGCCACCGTGCAGCCCTTATTTTA 1258

Coelacanth 1262:TTATTT------AGAATGCGATAGTCCTTTTCTTTAATCTACCTT------1300

Turtle 1282:CTGGCTCCCAGCACCAGGT------1300

Opossum 1285:CTGCGTAACTCTGGGA------1300

Cattle 1242:CTGGCC------1247

Mouse 1154:CCGACT------1159

Lemur 1202:CCGGCT------1207

Chimpanzee 1245:CCGGCT------1250

Human 1245:CCGGCT------1250

(B)

Zebrafish 1:MFSFMGLTNGTTD----TLANVSCTCNCQRSTSFQSMAISQLEFVQILVIVVVMMMMVLV 56

Coelacanth 1:MFNLMGLNSTTAA----IQPNVSCTCNCKRSL-FPSMEITDLEFVQIIIIVVVMMVMVVV 55

Turtle 1:MYHLMGVNSTAAA----TQPNVSCTCNCKRSL-FQTMEITELEFVQIIIIVVVMMVMVVV 55

Opossum 1:MYNLMGLNSTAAA----GQPNVSCTCNCKRSL-FQSMEITELEFVQIIIIVVVMMVMVVV 55

Cattle 1:MHRLMGVNSTAAAAAAAGQPNVSCTCNCKRSL-FQSMEITELEFVEITIIVVVVMVMVVV 59

Mouse 1: MGVNGTAAAAA--GQPNVSCACNCQRSL-FPSMEITELEFVQIVVIVVVMMVMVVM 53

Lemur 1:MHRLMGFNSTAAAAA--GQPNVSCTCNCKRSL-FQSMEITELEFVQIIIIVVVMMVMVVV 57

Chimpanzee 1:MHRLMGVNSTAAAAA--GQPNVSCTCNCKRSL-FQSMEITELEFVQIIIIVVVMMVMVVV 57

Human 1:MHRLMGVNSTAAAAA--GQPNVSCTCNCKRSL-FQSMEITELEFVQIIIIVVVMMVMVVV 57

Zebrafish 57:ITCLLNHYRLSARSLMSRHTHERRRHLPLPSEGSLWSSDGPGSSSAMSE--VYTP-RAPD 113

Coelacanth 56:ITCLLNHYKLSARSFINRHSQGRRREENLSPEGSLWPSDSTVSGSGMTE-QIYTP-RSTE 113

Turtle 56:ITCLLNHYKLSARSFINRHSQGRRRDENLSSEGSLWPSESTVSGNGVTEQQIYTP-RPTD 114

Opossum 56:ITCLLNHYKLSARSFIHRHSQGRRREENLSSEGSLWPSESTVSGNGLTE-QIYAPSRSAD 114

Cattle 60:ITCLLSHYKLSARSFIGRHSQSRRREDALSSEGCLWPSESTVSGNGIPEPQVYAPPRPTD 119

Mouse 54:ITCLLSHYKLSARSFISRHSQARRRDDGLSSEGCLWPSESTVSG-GMPEPQVYAPPRPTD 112

Lemur 58:ITCLLSHYKLSARSFISRHGQGRRREDALSSEGCLWPSESTVSGSGIPEPQVYTPPRPAD 117

Chimpanzee 58:ITCLLSHYKLSARSFISRHSQGRRREDALSSEGCLWPSESTVSGNGIPEPQVYAPPRPTD 117

Human 58:ITCLLSHYKLSARSFISRHSQGRRREDALSSEGCLWPSESTVSGNGIPEPQVYAPPRPTD 117

Zebrafish 114:R--VPSFLQRERVSRFQPTFPFLPPVIELPPTIALSDGEEPPPYQGPCTLQLRDREQQLE 171

Coelacanth 114:RLTVPSFLQRDRFNRFQPTYPYLQHEIDLPPTISLSDGEEPPPYQGPCTLQLRDPEQQME 173

Turtle 115:RLSVPSFLQRDRFNRFQPTYPYMQHEIDLPPTISLSDGEEPPPYQGPCTLQLRDPEQQME 174

Opossum 115:RLTVPSFLQRDRFNRFQPTYPYLQHEIDLPPTISLSDGEEPPPYQGPCTLQLRDPEQQME 174

Cattle 120:RLAVPAFAQRDRFHRFQPTYPYLQHEIDLPPTISLSDGEEPPPYQGPCTLQLRDPEQQLE 179

Mouse 113:RLAVPPFIQR---SRFQPTYPYLQHEIALPPTISLSDGEEPPPYQGPCTLQLRDPEQQLE 169

Lemur 118:RLAVPAFAQRDRLHRFQPTYPYLQHEIDLPPTISLSDGEEPPPYQGPCTLQLRDPEQQLE 177

Chimpanzee 118:RLAVPPFAQRDRFHRFQPTYPYLQHEIDLPPTISLSDGEEPPPYQGPCTLQLRDPEQQLE 177

Human 118:RLAVPPFAQRERFHRFQPTYPYLQHEIDLPPTISLSDGEEPPPYQGPCTLQLRDPEQQLE 177

Zebrafish 172:LNRESVRPPPNRTVYDSAL------THTGIS------GGRLQGPPPAYSEVIG 212

Coelacanth 174:LNRESVRAPPNRTIFDSDLIDTSMYGGPCPPSSNSGISATCYSSNGRMEGPPPTYNEVIG 233

Turtle 175:LNRESVRAPPNRTIFDSDLIDNSVFGGPCPPSSNSGISATCYGSNGRMEGPPPTYSEVIG 234

Opossum 175:LNRESVRAPPNRTIFDSDLIDNSMYGGPCPPSSNSGISATCYGSNGRMEGPPPTYNEVIG 234

Cattle 180:LNRESVRAPPNRTIFDSDLMESAMLGGPCPPSSNSGISATCYGGGGRMEGPPPTYSEVIG 239

Mouse 170:LNRESVRAPPNRTIFDSDLIDSTMLGGPCPPSSNSGISATCYSSGGRMEGPPPTYSEVIG 229

Lemur 178:LNRESVRAPPNRTIFDSDLMDSARLGGPCPPSSNSGISATCYGSGGRMEGPPPTYSEVIG 237

Chimpanzee 178:LNRESVRAPPNRTIFDSDLMDSARLGGPCPPSSNSGISATCYGSGGRMEGPPPTYSEVIG 237

Human 178:LNRESVRAPPNRTIFDSDLMDSARLGGPCPPSSNSGISATCYGSGGRMEGPPPTYSEVIG 237

Zebrafish 213:HYYH------PTTLPQAAALVHGFI---QLSRADTRSKTQPKAQPV 249

Coelacanth 234:HYPG-STFYQHQQ-NNGMPSILEGTRL-HSQINGLESTIARNKDKEKSKGHPF 283

Turtle 235:HYPG-STFFQHQQNNNGMPSILEGSRLHHSQINGLESTTAWNKDKEKQKGQPLLKKNKKN 293

Opossum 235:HYPG-STFYQHQQ-NNGTPSILENNRLHQSQISSLESTIAWNKEKEKQKGHPF 285

Cattle 240:HYPGAST-FQHQQ-SSGPASLLEGTRLHPAHIAPLESTAAWSKEKDKQKGHPL 290

Mouse 230:HYPG-SS-FQHQQ-SNGPSSLLEGTRLHHSHIAPLE-----NKEKEKQKGHPL 274

Lemur 238:HYPG-SS-FQHQQ-SNGPPSLLEGTRLQHTHMAPLESAAIWSKEKDKQKGHPL 287

Chimpanzee 238:HYPG-SS-FQHQQ-SSGPPSLLEGTRLHHTHIAPLDSAAIWSKEKDKQKGHPL 287

Human 238:HYPG-SS-FQHQQ-SSGPPSLLEGTRLHHTHIAPLESAAIWSKEKDKQKGHPL 287

Turtle 294:QTRK 297

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