Emanuele Buratti

RNA splicing mutations and human disease: Pompe disease.

Author

INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY

One CENTRE made of three Components, two Outstations and a Network of

38 Affiliated Centres in 63 Member States and 83 Signatory Countries

Trieste Component

Regulation of mRNA processingMammalian DNA replication,Chromosomal stability in yeastMechanisms of DSB repair

Gene and cell therapy of cardiovascular disorders

Genetics of ant ibodies

Molecular biology of viral infect ions

Protein structure and bioinformatics

Quorum sensing in bacteria

Production of recombinant proteins for human therapy

Inherited genetic disorders

Basic molecular biology

ICGEB

Pompe disease, Glycogenosis type II or acid maltase deficiency

Autosomal recessive lysosomal storage disease (1:40.000 live births)

Due to the deficiency of a-glucosidase (GAA) or acid maltase

Impaired glycogen degradation and accumulation within the lysosomes leads to enlargement of cardiac and skeletal muscle.

Phenotypic continuum:

•Manifests soon after birth•Rapidly progressive disease course

•Progressive muscle weakness •Cardiomegaly and cardiomyopathy

•Hypotonia •Respiratory insufficiency

•Feeding difficulties •Moderate hepatomegaly

•Markedly elevated CK•no GAA activity

•Manifests in children or adults•Progressive muscular

weakness •No cardiac involvement

•Respiratory insufficiency•Exercise intolerance•Swallowing difficulty

•Moderate hepatomegaly•Elevated CK

•Residual GAA activity

Infantile onset

Late onset

http://www.unitedpompe.com/aboutpomhttp://www.unitedpompe.com/aboutpompe.cfmpe.cfm

3’ 5’ 3’ 5’ 3’ 5’3’ 5’ 3’ 5’

AAAAAAAAAA

aug uag

5’UTRORF

3’UTR

AAAAAAAAAA

pre-mRNA splicing:

Poly-A5’cap

PolII PolII PolII

PolII

Splicing

Export and Translation

DNA

pre-mRNA

mRNA

protein

aug uag

Disease-causing mutations can occur Disease-causing mutations can occur in all these types splicing controlling in all these types splicing controlling

elements:elements:

What happens when one of the basic elements is altered?

Splicing mutations can be found in virtually any intron-containing gene. The frequency depends on overall

length and individual susceptibilities

Baralle D. et al. EMBO Rep 2009; 10:810-816.

Focus on the splicing mutations:

ATG TAG

Molecular analysis

Close to 200 mutations have been described; most of them are private.

Some mutations are common in different ethnic groups (p.R854X among African-Americans; p.D645E among Asians and del525T in Dutchs)

The mutation profile is very heterogeneous. In late onset patients the leaky c.-32-13T>G is present in about 40-70% of the alleles.

Infantile GSDII

mutation profile highly heterogeneous all mutations described are considered to be severe

Late onset GSDII

c.-32-13T>G is the most frequent GAA mutation (allelic frequency: 42.3%).

Combination of severe with mild mutations correlates with late onset of the disease

c.525delT(11.8%)

c.1064T>C(7.9%)

c.1655T>C(10.5%)

c.2237G>A (10.3%)

c.-32-13T>G (42.3%)

Each horizontal bar represents the disease duration of an individual patient. *Patients have died. van der Ploeg and Reuser, Pompe's disease The Lancet, Volume 372, Issue 9646, 2008, 1342 - 1353

Age at onset of symptoms and the current age of a cohort of 36 patients with GSDII disease

Despite the common genotype, patients present with a great variability in residual enzyme activity, age of appearance of clinical

signs and rate of disease progression

GSDII in Italy:

exon 1

exon 2 (578 bp)

exon 3

cgggtgaga

35 bp

gcg/gtaaca

tcttctcccgcaggc….

60 bp

….acggtgggc catctcttctagat

g

-13T>Gtcttccccaag/ga

5’ss(c1) 3’ss(c2)

Why is the -13T>G mutation so harmful?:

Exon 2 is very long with respect to the majority of normal human exons

Sakharkar et al., 2005

The 5’splice site is poorly defined according to consensus

CAGGURAGU 3’5’

cauu u u

cc

g

U53’

gagaca

U63’

guccauucauapppGU1

3’m3

ACGGUGGGC 3’5’

cauu u u

cc

g

U53’

gagaca

U63’

guccauucauapppGU1

3’m3

Normal interactions GAA exon 2

exon 1

exon 2 (578 bp)

exon 3

cgggtgaga

35 bp

gcg/gtaaca

tcttctcccgcaggc….

60 bp

….acggtgggc catctcttctagat

g

-13T>Gtcttccccaag/ga

N

SV1

SV2

SV3

5’ss(c1) 3’ss(c2)

c1

c2

What was already known regarding the effects of this mutation:

Huie ML et al.,, HMG, 1994

Analysis of two patients that express ONLY the allele carrying the -13T/G mutation:

-13u gccucccugcugagcccgcuuucuucucccgcagGCCUGUAugugugug-13g gccucccugcugagcccgcuugcuucucccgcagGCCUGUAugugugug

-13u-13g Beads

U2AF65

TDP-43

-13 3‘ssug-tail

BP(yncuray)

-13u-13g BeadsMW

83

58

47.5

32.5

kDa

P. red

Western

Pulldown analysis showed that weakening of the splice site was associated to loss of binding of one

of the basic splicing factors, U2AF65:

T7

cccgcuuucuucucccgcagGCCUGUAGGAGCUGUCCAGGcccgcuugcuucucccgcagGCCUGUAGGAGCUGUCCAGG

-13u -13g

pre-mRNA

mRNA

minutes1 30 90 1 30 90

3‘ss GAA exon 285 nt.

SP6/T7promoter

Labelled RNA is transcribedin vitro and incubated in nuclear

extract for 2-4 hours.

Splicing productsare separated

in a denaturing gelor amplified by

RT-PCR

In vitro splicing

In vitro splicing assay confirmed that 3’ss was indeed weakened:

WT Mut

N

SV3SV2

WT

Mut

N

SV3SV2

N

SV3SV2

- SRSF1 (A

SF/SF2

)

SRSF3 (S

Rp20)

SRSF9 (S

Rp30c

)

SRSF2 (S

C-35)

SRSF5 (S

Rp40)

SRSF6 (S

Rp55)

SRSF4 (S

Rp75)

SV3SV2

- hnRNP A

1hn

RNP A

2hn

RNP C2

YB-1DAZA

PTD

P-43

TIA-1

hnRNP H

hn

RNP F

N

N

SV3SV2WT

Mut

Nd

e1

50nt 50nt

GAAExon 2SV40

Nd

e1

pAa2-3 Bra2

Looking for the factors that can influence exon 2 inclusion both in the wild-type and mutant version

Making a minigene to mimic the effects of the -13T>G mutation

SRSF4

Tubulin

scra

mbl

esi

RN

A

Relative normal spliced mRNA expression (% of scramble)

Relative normal spliced mRNA expression (% of mock transfected cells)

*

*

*

Overexpression of SRSF4 in patient fibroblasts:

Knockdown of SRSF4 in patient fibroblasts:

Relative normal spliced mRNA expression (% of WT)

Resveratrol can improve mRNA and enzyme production in the fibroblasts of patient cells:

No other HDAC inhibitors except for SAHA can act like Resveratrol to improve normal splicing levels:

Highthroughput drug screening for compounds capable of increasing GAA exon

2 inclusion:

Future directions 1: setting up an HTS assay to test for compounds/factors capable of increasing exon 2

inclusion:

Nd

e1

50nt 50nt

GAAExon 2

SV40pA

Nd

e1

CMV

SV40 pACMVpEGFP-N1

pEGFP-N1 WTand MUT (-13T>G)50nt50nt

5’ 3’

EGFP

5’ 3’

Transfection in HeLa cellsExon skippingExon inclusion or

cryptic 3’ss activation

WT

MU

T (-

13u/

g)

Con

t

83

62

47.5

32.5

25

62

47.5

α-EGFP

α-Tubulin

kDa

EGFP

578nt

WT MUT (-13u/g)

HeLa HeLa

exon 1

exon 2 (578 bp)

exon 3

cgggtgaga

35 bp

gcg/gtaaca

tcttctcccgcaggc….

60 bp

….acggtgggc catctcttctagat

g

-13T>Gtcttccccaag/ga

5’ss(c1) 3’ss(c2)

Approaches to find new therapeutic tragets and strategies:

1) Search for silencer elements:

exon 2 (578 bp)tcttctcccgcaggc….

60 bp

….acggtgggc

g

-13T>Gtcttccccaag/ga

3’ss(c2)

Set of overlapping deletions

Once identified, use of ASO technology to improve inclusion

3) Use of U7snRNA to block cryptic site usage:

Sense1 TTCTTCCCCAAGGACATCCTGA 

Antisense TCAGGATGTCCTTGGGGAAGAA 

Sense2 CCCCACCTTCTTCCCCAAGGAC 

Antisense GTCCTTGGGGAAGAAGGTGGGG 

Sense3 CCAAGGACATCCTGACCCTGCG 

Antisense CGCAGGGTCAGGATGTCCTTGG

U7snRNA1

U7snRNA2

U7snRNA3

2) Improvement of 5’ss recognition: making a mutant U1snRNP that can recognize the poorly defined 5’splice site:

Previous examples have worked very well:

ACGGUGGGC 3’5’

cauu u u

cc

g

U53’

gagaca

U63’

guccauucauapppGU1

3’m3

ACGGUGGGC 3’5’

cauu u u

cc

g

U53’

gagaca

U63’

ugccacucguapppGU1

3’m3

GAA exon 2 GAA exon 2+mutant U1snRNP

mutant

Normal exon

Future directions 2: test these compounds on skeletal muscle cells obtained through

differentiation of hSKIN-Multipotent Adult Stem Cells (MASC) isolated from LO patients bearing the c.-32-

13T>G mutation

Skeletal muscle differentiation:

Acknowledgements:

Elisa GoinaCristiana StuaniMaurizio RomanoFrancisco E. Baralle

Andrea DardisIrene ZaninStefania ZampieriBruno Bembi

Project detail:TTNumber: GGP14192Durata: 3 yearsData inizio: 30/10/2014

Conclusions:

a)The -13T>G is a common splicing mutation in late onset GSDII disease.

b)The functional effects of this mutation are to lower recognition of the 3’ss of exon 2.

c)This exon is very long and poorly defined even in its normal status, and this is the reason why this mutation has a huge effect on its recognition.

d)Several RNA-based strategies can be made available to rescue exon 2 recognition in the presence of -13T>G.

e)These strategies may involve the use of antisense nucleotides to inhibit splicing regulatory regions, modified U1snRNPs to favour 5’ss recognition, or the discovery of small molecules capable of increasing the expression of positive factors.

f)At the same, however, it is important to develop suitable cellular models to study the efficacy of these strategies.