Supporting online material - Regulation of splicing factors by alternative splicing and NMD is conserved between kingdoms yet evolutionarily flexible

sp

ecie

sg

en

eg

en

om

e s

ou

rce

locu

srep

resen

tati

ve

gen

e m

od

el o

r m

RN

A

alt

sp

licin

g?

intr

on

rete

nti

on

?

up

str

eam

jun

cti

on

ES

Ts

do

wn

str

eam

jun

cti

on

ES

Ts

exo

n

skip

ped

ES

Ts

up

str

eam

jun

cti

on

intr

on

rete

nti

on

do

wn

str

eam

jun

cti

on

intr

on

rete

nti

on

full in

tro

n

rete

nti

on

early

po

lyA

H. sap

ien

sSR

SF5

UC

SC h

g19

chr1

4:70

2338

34-7

0238

721

NM

_006

925

yy

205

5287

685

0

M. m

uscu

lus

SRSF

5U

CSC

mm

9ch

r12:

8204

6491

-820

5149

4N

M_0

0915

9y

y2

156

4533

40

R. n

org

evic

us

SRSF

5U

CSC

rn4

chr6

:104

7282

76-1

0473

2985

NM

_019

257

yy

111

3556

271

0

C. fa

milia

ris

SRSF

5U

CSC

can

Fam

2ch

r8:4

6549

512-

4655

4334

ENSC

AFT0

0000

0262

76y

00

54

20

0

G. g

allu

sSR

SF5

UC

SC g

alG

al3

chr5

:301

6379

6-30

1684

33N

M_0

0103

1197

yy

10

514

31

0

SRSF

5aU

CSC

gal

Gal

3ch

r5:3

0595

571-

3061

2641

NM

_001

0064

76y

y1

022

37

00

X. tr

op

icalis

SRSF

5U

CSC

xen

Tro2

scaf

fold

_246

:146

3821

-146

7713

NM

_001

0047

83y

y71

4028

712

985

01

krt5

.1U

CSC

xen

Tro2

scaf

fold

_145

:676

974-

6899

26N

M_0

0100

5638

yy

10

301

20

0

F. ru

brip

es

SRSF

5bU

CSC

fr2

chrU

n:16

6973

539-

1669

7638

8EN

STR

UT0

0000

0458

080

01

00

00

SRSF

5aU

CSC

fr2

chrU

n:27

0261

895-

2702

6547

8EN

STR

UT0

0000

0272

050

00

00

00

D. rerio

SRSF

5bU

CSC

dan

Rer

5ch

r20:

3862

2161

-386

3698

8N

M_0

0100

2610

yy

51

1810

30

1

SRSF

5aU

CSC

dan

Rer

5ch

r13:

3824

6638

-382

5147

5N

M_2

0086

7y

y11

417

643

91

0

SR A

UC

SC b

raFl

o1 /

JGI v

1.0

chrU

n: 2

5461

5939

-254

6243

58ES

T cl

uste

r 260

1579

:13

y1

1?76

10

00

SR B

UC

SC b

raFl

o1 /

JGI v

1.0

chrU

n:30

9654

001-

3096

5717

4es

tExt

_fge

nesh

2_pg

.C_9

2002

7

SR C

UC

SC b

raFl

o1 /

JGI v

1.0

chrU

n: 2

5482

6591

-254

8350

12es

tExt

_fge

nesh

2_pm

.C_6

9001

4y

11?

761

00

0

S. p

urp

uratu

sSR

CU

CSC

strP

ur2

Scaf

fold

8089

2:72

4-98

31C

X551

350,

CX5

5765

1y

11

260

10

0

D. m

ela

no

gaste

rB5

2U

CSC

dm

3ch

r3R

:948

7022

-949

2613

NM

_176

488

yy

107

829

21

0

C. ele

gan

srs

p-1

UC

SC c

e6ch

rII:1

1453

896-

1145

5723

NM

_064

041

yy

20

101

30

0

rsp-

2U

CSC

ce6

chrII

:114

5136

0-11

4528

71N

M_0

6404

0y

00

151

11

0

N.v

ecte

nsis

NV

1JG

I v1.

0sc

affo

ld_1

1:47

2975

-477

003

estE

xt_f

gene

sh1_

pg.C

_110

055

y3

418

00

00

NV

2JG

I v1.

0sc

affo

ld_1

03:5

6740

5-57

0642

2667

685_

5y

11

521

00

0

NV

3JG

I v1.

0sc

affo

ld_3

18:1

4436

5-14

8353

estE

xt_f

gene

sh1_

pg.C

_318

0016

y0

011

01

00

NV

4JG

I v1.

0sc

affo

ld_1

3:13

3190

1-13

3473

3es

tExt

_fge

nesh

1_pg

.C_1

3016

9y

y3

10

02

00

NV

5JG

I v1.

0sc

affo

ld_3

79:9

4033

-945

96es

tExt

_fge

nesh

1_pg

.C_3

7900

06

NV

6JG

I v1.

0sc

affo

ld_9

3:61

4755

-619

479

estE

xt_f

gene

sh1_

pg.C

_930

062

yy

32

200

10

0

N. crassa

NC

U07

069

Entre

z G

ene

NW

_001

8497

96.1

:c26

7293

-265

451

XM_9

5524

1y

A. n

iger

An16

g038

90En

trez

Gen

eN

T_16

6531

.1:c

8741

59-8

7192

2XM

_001

3976

63y

S. p

om

be

srp2

Entre

z G

ene

NC

_003

424.

3:c4

4269

65-4

4252

50N

M_0

0101

9999

Tabl

e S1

– S

RSF

5 or

thol

ogs i

nclu

ded

in a

naly

sis. C

ount

s of E

STs a

nd m

RNA

s sho

win

g al

tern

ativ

e sp

licin

g ar

e no

ted

in th

e la

st co

lum

ns

(incl

udin

g pa

rtial

evi

denc

e of

intro

n re

tent

ion,

such

as E

STs e

xten

ding

into

the

intro

n). W

e as

signe

d te

mpo

rary

nam

es to

gen

es in

C. i

ntes

tinal

is,

B. fl

orid

ae, S

. pur

pura

tus,

and

N. v

ecte

nsis

befo

re p

hylo

gene

tic c

lass

ifica

tion

sp

ecie

sg

en

eg

en

om

e

so

urce

locu

srep

resen

tati

ve

gen

e m

od

el o

r m

RN

A

alt

sp

licin

g?

intr

on

rete

nti

on

?

sp

licin

g in

2n

d in

tro

n -

up

str

eam

jun

cti

on

ES

Ts

sp

licin

g in

2n

d in

tro

n -

do

wn

str

eam

jun

cti

on

ES

Ts

sp

licin

g in

2n

d in

tro

n -

exo

n

skip

ped

ES

Ts

sp

licin

g in

2n

d

intr

on

-

up

str

eam

jun

cti

on

in

tro

n

rete

nti

on

sp

licin

g in

2n

d

intr

on

-

do

wn

str

eam

jun

cti

on

in

tro

n

rete

nti

on

sp

licin

g in

2n

d in

tro

n -

full in

tro

n

rete

nti

on

early

po

lyA

sp

licin

g in

1st

intr

on

-

up

str

eam

jun

cti

on

ES

Ts

sp

licin

g in

1st

intr

on

-

do

wn

str

eam

jun

cti

on

ES

Ts

sp

licin

g in

1st

intr

on

-

exo

n

skip

ped

ES

Ts

sp

licin

g in

1st

intr

on

-

up

str

eam

jun

cti

on

in

tro

n

rete

nti

on

sp

licin

g in

1st

intr

on

-

do

wn

str

eam

jun

cti

on

in

tro

n

rete

nti

on

sp

licin

g in

1st

intr

on

-

full in

tro

n

rete

nti

on

early

po

lyA

H. sap

ien

sS

RS

F4

UC

SC

hg19

chr1

:29474251-2

9508637

NM

_005626

y (

i1, i2

)y (

i2)

43

157

47

10

68

159

00

01

SR

SF

6U

CS

C h

g19

chr2

0:4

2086504-4

2092242

NM

_006275

yy

58

6132

10

90

0

M. m

uscu

lus

SR

SF

4U

CS

C m

m9

chr4

:131429554-1

31457640

NM

_020587

y (

i1)

y (

i1)

00

00

00

03

835

00

00

SR

SF

6U

CS

C m

m9

chr2

:162757244-1

62762854

NM

_026499

yy

219

94

319

10

R. n

org

evic

us

SR

SF

4U

CS

C r

n4

chr5

:150731169-1

50758938

NM

_001108685

y (

i1)

00

00

00

01

13

00

00

SR

SF

6U

CS

C r

n4

chr3

:153795474-1

53800796

NM

_001014185

yy

13

23

014

00

C. fa

milia

ris

SR

SF

4U

CS

C c

anF

am

2chr2

:74298034-7

4323762

BU

751463, D

N425514 (

gap in 1

st in

tron)

00

00

00

0

SR

SF

6U

CS

C c

anF

am

2chr2

4:3

4189662-3

4192538

EN

SC

AF

T00000014759, D

N355700

yy

11

20

10

0

G. g

allu

sS

RS

F4

UC

SC

galG

al3

chr2

3:3

043852-3

048526

BX

936077, B

X931065

00

00

00

0

SR

SF

6U

CS

C g

alG

al3

chr2

0:6

9285-7

4296

NM

_001030843 (

gap in intr

on)

yy

03

13

03

00

X. tr

op

icalis

SR

SF

4U

CS

C x

enT

ro2

scaffold

_492:4

34541-4

41727

CX

803859

y (

i2)

y (

i2)

01

93

10

10

SR

SF

6U

CS

C x

enT

ro2

scaffold

_38:8

05274-8

14512

NM

_001001248

yy

613

72

30

70

0

F. ru

brip

es

SR

SF

4U

CS

C fr2

chrU

n:1

39894565-1

39897692

EN

ST

RU

T00000030178

SR

SF

6U

CS

C fr2

chrU

n:3

55227627-3

55231373

EN

ST

RU

T00000007652

y0

10

00

00

D. rerio

SR

SF

4U

CS

C d

anR

er5

chr1

9:2

3988960-2

3998507

NM

_199574

y (

i2)

00

84

11

30

1

SR

SF

6a

UC

SC

danR

er5

chr2

3:3

858771-3

870268

NM

_213466

00

70

30

0

SR

SF

6b

UC

SC

danR

er5

chr1

1:3

91396-4

00063

NM

_001008732

yy

12

11

43

00

C. in

testi

nalis

SR

AU

CS

C c

i2chr0

1q:6

846290-6

849708

AK

116209

y*

SR

BU

CS

C c

i2chr0

1q:6

888465-6

893171

BW

305692, B

W324472

y*

SR

CU

CS

C c

i2chr0

2q:6

965692-6

967083

AK

114189

SR

DU

CS

C c

i2chr0

5q:4

307321-4

308319

ES

Ts (

unspliced in O

RF

)

SR

EU

CS

C c

i2chr0

5q:4

319181-4

321175

AK

112385

Tabl

e S2

– S

RSF

4 an

d SR

SF6

orth

olog

s inc

lude

d in

ana

lysi

s. A

s in

tabl

e S1

. “i1

” in

dica

tes a

ltern

ativ

e sp

licin

g or

intro

n re

tent

ion

in th

e fir

st in

tron

of S

RSF

4 or

thol

ogs,

and

“i2”

indi

cate

s the

seco

nd in

tron.

All

alte

rnat

ive

splic

ing

even

ts in

SR

SF6

are

in th

e se

cond

intro

n, a

nd

first

-intro

n al

tern

ativ

e sp

licin

g in

SR

SF4

was

onl

y ob

serv

ed in

hum

an, m

ouse

, and

rat,

so m

any

cells

are

not

app

licab

le a

nd a

re le

ft em

pty.

A

ltern

ativ

e sp

licin

g in

C. i

ntes

tinal

is S

R A

and

B w

as n

ot h

omol

ogou

s to

alte

rnat

ive

splic

ing

in o

ther

spec

ies.

anemone 5

S. pombe srp2

N. crassa srp2

A. niger srp2

anemone 6

anemone 4

fly b52

lancelet b

sea squirt c

worm rsp1

worm rsp2

lancelet c

lancelet a

urchin c

fugu srsf4

zebrafish srsf4

frog srsf6

dog srsf6

rat srsf6

mouse srsf6

human srsf6

chicken srsf6

zebrafish srsf6a

fugu srsf6

zebrafish srsf6b

frog srsf4

chicken srsf4

rat srsf4

dog srsf4

human srsf4

mouse srsf4

sea squirt b

sea squirt a

sea squirt d

sea squirt e

chicken srsf5a

frog krt5.1

zebrafish srsf5a

fugu srsf5a

zebrafish srsf5b

fugu srsf5b

dog srsf5

rat srsf5

human srsf5

mouse srsf5

frog srsf5

chicken srsf5

anemone 3

anemone 2

anemone 1

At 31

81

4

1

332

3

1

47

39 73

22

32

63

69

68

95

4

1

0

0

0

100

100

96

88

34

98

23

95

95

92

31

5442

7184

87

87

70

7163

28

anem

one

5

S. p

ombe

srp

2

N. c

rass

a sr

p2A

. nig

er s

rp2

anem

one

6an

emon

e 4

fly b

52

lanc

elet

b

sea

squi

rt c

wor

m rs

p1w

orm

rsp2

lanc

elet

cla

ncel

et a

urch

in c

fugu

srs

f4ze

braf

ish

srsf

4

frog

srs

f4

dog

srsf

4ra

t srs

f4m

ouse

srs

f4hu

man

srs

f4

chic

ken

srsf

4

zebr

afis

h sr

sf4a

fugu

srs

f4ze

braf

ish

srsf

4b

frog

srs

f4ch

icke

n sr

sf4

rat s

rsf4

dog

srsf

4

hum

an s

rsf4

mou

se s

rsf4

sea

squi

rt b

sea

squi

rt a

sea

squi

rt d

sea

squi

rt e

chic

ken

srsf

5afr

og k

rt5.

1ze

braf

ish

srsf

5afu

gu s

rsf5

a

zebr

afis

h sr

sf5b

fugu

srs

f5b

dog

srsf

5ra

t srs

f5

hum

an s

rsf5

mou

se s

rsf5

frog

srs

f5ch

icke

n sr

sf5

anem

one

3an

emon

e 2

anem

one

1

A B

Figure S1 – A. Maximum likelihood tree of all animal and fungi SRSF4/5/6 genes, with bootstrap values. Vertebrate SRSF4/6 (blue) and SRSF5 (green) orthologs are grouped. Red indicates genes with no introns. B. Alignment of all SRSF4/5/6 protein sequences indicating exon positions. Colors denote exons. Sequences were trimmed after the second RRM domain before alignment. Black bars indicate the positions of alternative exons in human SRSF4, SRSF5, and SRSF6.

human

mouse

rat

chicken(SRSF5)

chicken(SRSF5)

frog(SRSF5)

zebrafish(SRSF5b)

zebrafish(SRSF5a)

lancelet(A)

urchin(C)

fly

worm(rsp-1)

anemone(1)

3 4

18

20 5

52

87 68

2 1

56

45 33

11 1

35

56 27

1

5

14 3

1

22

3 7

11 4

176

43 9

1 1

76

1

1 1

26

1

10 7

83

9 2

2

10

1

AAA

5 1

18

10 31

71 40

287

85AAA1129

Figure S2 – Transcript evidence for alternative splicing of SRSF5. Alternative regions of each gene are shown with the upstream and downstream constitutive exons in blue and alternative exon in orange (to scale). Counts of ESTs and mRNAs showing alternative splicing are noted (including partial evidence of intron retention, such as ESTs extending into the intron) and splice junction lines are scaled to this count.