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1
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
2
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’
4
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’
5
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
5
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