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Investigating the effects of MBNL1 concentration on alternative

splicing in Myotonic Dystrophy

Riti GuptaBerglund Lab

Mentor: Stacey Wagner

University of Oregon

Institute of Molecular Biology

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Myotonic Dystrophy

• Most common form of adult onset muscular dystrophy (MD)

• Autosomal dominant disorder

• 1 in 8000 individuals affected• Common symptoms include

Muscle weakness Myotonia Heart dysfunction Iridescent cataracts Mental deficiencies

http://www.nejm.org/na102/home/ACS/publisher/mms/journals/content/nejm/2009/nejm_2009.361.issue-4/nejmicm0802903/production/images/large/nejmicm0802903_f1.jpeg

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Mutations that cause Myotonic Dystrophy

Type 1 (DM1)

Unaffected (n=5-37)

Affected (n=50-4000)

Pre-mutation with no symptoms (n=38-50)

DMPK Gene CUG Repeats in the 3’ Untranslated Region

5’

3’

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Mutations that cause Myotonic Dystrophy

Type 2 (DM2)

Unaffected (n=7-24)

Affected (n=75-11000)

Pre-mutation with no symptoms (n=22-33) +/- interruptions

Znf9 Exon 1 CCUG Repeats in the 1st Intron Region5

’3’

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Mis-splicing of pre-mRNA causes disease symptoms in DM1

CU

G

G CU

C GU

CU

G

G CU

CU

G

G CU

CU

G

G CU

DMPK

MBNL

MBNL

MBNL

MBNL

MBNL

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Alternative Splicing

Exon Skipping:

Ç√

Pre-mRNA:

1 32

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What happens when MBNL1 is sequestered to CUG repeats?

TNNT2 splicing event

Fetal isoform

4 6

4 5 6

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64Adult isoform

Transcripts Mis-Spliced in DM:INSR (insulin resistance) Skeletal MuscleCLCN-1 (myotonia) Skeletal Muscle TNNT2 (cTNT) HeartATP2A1 (SERCA1) Skeletal MuscleRyR

Skeletal MuscleMTMR1Skeletal Muscle/HeartNMDAR1 BrainMAPT (Tau) Brain90+ others Tissue Specific

MBNL

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Patient splicing profiles

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What do we know?

Free level of MBNL1 available is reduced in the disease state

Level of sequestration is dependent on expression levels and number of repeats

QUESTION: Do splicing events respond differently when MBNL1 levels are varied?

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Experimental Design: Construction of inducible MBNL1 expressing cell

line

TATA TetO2 MBNL1

TetR

TetO2

TetR

+ dox ( )

TATA TetO2 MBNL1

TetR

TetO2

TetR

TATA TetO2 MBNL1TetO2

TetRTetR

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Experimental Design: Construction of inducible MBNL1 expressing cell

line

induced using doxycycline shows varying expression as dox is

titrated 0 point shows not a leaky promoter

Stacey Wagner, unpublished data

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Cell-Based Splicing Assay

PCRReverse Transcription

(RT)

% Exon Inclusion

HEK293 cells

Tet-on MBNL1 system

RNA Isolation

Add doxycycline

+ Splicing reporter minigene

http://www.pan-biotech.com/content/images/stories/serumfreiesysteme/panexinnt/nta-hek.jpg

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Effect of MBNL1 concentration in the TNNT2 splicing event

4 5 6

64

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Response variation by event

• TNNT2 clearly negatively regulated• ATP2A1 positively regulated• What’s interesting?• Difference between DMPK and 0 of TNNT2 and ATP2A1• Is there a threshold necessary

to ‘turn it on’• Endogenous is enough in TNNT2, not in ATP2A1

Stacey Wagner, unpublished data

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Conclusions/Future Directions

A stable cell line has been created in which MBNL1 expression is regulated by doxycyline

Different splicing events show different responses to varying amounts of MBNL1

In the future, determine how the binding sites in each event are important to these differing responses

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AcknowledgementsAndy Berglund (Principle Investigator)Stacey Wagner (Mentor)

Leslie CoonrodElaine DeLorimierDylan FarnsworthJulia OddoRuth SiboniRodger VoelkerDave Youngentob

SPUR – NICHD R25 Summer Research Program NIH-1R25HD070817