-2,00

-1,00

0,00

1,00

2,00

siCtrl siSRSF1 siSRSF3

Re

lati

ve e

xpre

ssio

n o

f Rac

1b

e

nd

oge

no

us

tran

scri

pt (l

og

2)

Only siRNA depletion of GSK3β and SRPK1

decreased endogenous Rac1b transcript levels A RAC1 minigene reporter

functionally reproduces the extent

of alternative splicing observed in

different colorectal cell lines

Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of

tumor-related Rac1b in colorectal cells

Vânia Gonçalves, Paulo Matos and Peter Jordan Centre for Human Genetics, Natl. Health Institute ‘Doutor Ricardo Jorge’, Lisbon, Portugal

SUMMARY The pre-messenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different

tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present

in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications such as protein phosphorylation, which are

determined by signal transduction pathways. Here we analyzed the contribution of protein kinases to the regulation of alternative splicing variant

Rac1b that is overexpressed in certain tumor types. In colorectal cells we found that depletion of AKT2, AKT3, GSK3β and SRPK1 significantly

decreased endogenous Rac1b levels. Whereas knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the

depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the

knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its

availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of

Rac1b expression in colorectal cells and provide further mechanistic insights into how the regulation of alternative splicing events by protein kinases

can contribute to sustain tumor cell survival.

The Rac1b splice variant

Jordan et al. (1999) Oncogene 18, 6835-6839

Introduction to previous work

Downstream signalling

properties

Promotes G1/S progression

and cell survival Matos & Jordan (2005) Exp Cell Res 305, 292-299;

Matos & Jordan (2008) Mol Cancer Res 6, 1178-84

Endogenous Rac1b:

little protein but highly active

Properties of Rac1b:

• Increased GDP exchange rate in vitro

• Rho-GDI cannot downregulate Rac1b in vivo

Differences in Rac1b splicing

between cell lines are not due

to genomic mutations

Conclusions:

• Depletion of AKT2, AKT3, GSK3β and SRPK1 decreased considerably the levels of endogenous Rac1b.

• AKT2 and AKT3 knockdown appears to act only post-splicing, affecting solely steady state Rac1b protein levels

in colorectal cells.

• GSK3β and SRPK1 depletion decrease Rac1b alternative splicing by regulating this event through SRSF1.

• SRPK1 knockdown or activity inhibition leads to reduced SRSF1 phosphorylation and, consequently, reduced

translocation to the nucleus limiting SRSF1 availability to enhance Rac1b alternative splicing.

Which protein kinases regulate Rac1b alternative splicing, leading to its overexpression?

Splicing factors SRSF1 and SRSF3

regulate alternative splicing of

Rac1b in an antagonistic manner

GSK3β knockdown decreases

Rac1b alternative splicing through

SRSF1

Inhibition of SRPK1 decreases SRSF1-dependent alternative splicing

of Rac1b

Overexpressed in a subgroup of

colorectal tumors (T) with B-RAFV600E

[RT-PCR, (M;L = control tissue)]

T-M T-M T-M T-M T-M T-L HT29

A1 B2 C2 C2 D meta

1.0

0.6

kb

Rac1

Rac1b

Blot:

Active Rac

SW480 DLD-1 HT29

Rac1b

Rac1RT-PCR

Rac1b

Rac1

RAC1 gene (27 kb)

2 31 4 5 63b

region sequenced

3’utr5’utr

shC

trl

sh A

KT1

shA

KT2

sh A

KT3

αtub

Rac1b

shC

LK1

sh C

LK3

sh C

LK4

sh G

SK

3β

shG

SK

3α

shSR

PK

1

shSR

PK

2

shM

SS

K1

sh C

LK2

shC

trl

shD

YR

K1

a

sh D

YR

K1

b

shD

YR

K3

sh D

YR

K4

sh P

P1α

shP

P1β

We

stern

blo

t

-1,5

-1

-0,5

0

0,5

1

1,5

shC

trl

shA

KT1

shA

KT

2

shA

KT

3

shG

SK3α

shG

SK

3β

shC

LK1

shC

LK2

shC

LK3

shC

LK4

shD

YR

K1

a

shD

YR

K1

b

shD

YR

K2

shD

YR

K3

shD

YR

K4

shP

P1α

shP

P1β

shP

P1γ

Sh

SR

PK

1

ShSR

PK

2

Sh

MS

SK

1

shC

trl

shD

YR

K2

shP

P1γ

Fold

ch

an

ge

in R

ac1

b e

xpre

ssio

n (

log

2)

AKT

αtub

Rac1b

αtub

Rac1b

SRPK1GSK3β

αtub

Rac1b

-1,2

-0,8

-0,4

0,0

0,4

0,8

1,2

si Ctrl si AKT1 si AKT2 si AKT3 si GSK3β si SRPK1

Fold

cha

nge

in R

ac1b

tra

nscr

ipt

expr

essi

on (

log 2

)

**

αtub

H2B

βcat

S NS

siCtrl siGSK3β

S NS

SRSF3

GSK3β

αtub

SRPK1

SRSF1

Rac1b

SRSF1

αtub

SRPK1

Rac1b

p-SRSF1 SRSF1

SRPK1

Rac1b

p-SRSF1

siCtrl siSRPK1

T7-SRSF1

Actin

DAPI

-1,0

-0,8

-0,6

-0,4

-0,2

0,0

0,2

0,4

0,6

0,8

1,0

si Ctrl si SRPK1

Rac1b

SRSF1

Fold

ch

ange

in

tra

nsc

rip

t e

xpre

ssio

n (

log 2

)

shRNA targeting of AKT1, AKT2 and AKT3, as well as of

GSK3β and SRPK1 led to a clear decrease in endogenous

Rac1b protein levels

-2,00

-1,00

0,00

1,00

2,00

3,00

EV SRSF1 SRSF3

Re

lati

ve e

xpre

ssio

n o

f Rac

1b

m

inig

en

etr

ansc

ript

(lo

g 2)

EV SRSF1 SRSF3

αtub

T7

We

stern

blo

t

We

stern

blo

t

αtub

Rac1b

siCtrl siSRSF1 siSRSF3

SRSF1

SRSF3