1. Riti Gupta Berglund Lab Mentor: Stacey Wagner University of Oregon Institute of Molecular Biology 1

2. 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 2 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

3. 3 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. 4 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 1 st Intron Region 5’ 3’

5. C U G GC U CG U C U G GC U C U G GC U C U G GC U DMPK MBNL 5

6. 6 Exon Skipping: Ç√ Pre-mRNA: 13 2

7. 7 TNNT2 splicing event Fetal isoform 4 6 456 5 64 Adult isoform Transcripts Mis-Spliced in DM: INSR (insulin resistance)Skeletal Muscle CLCN-1 (myotonia)Skeletal Muscle TNNT2 (cTNT) Heart ATP2A1 (SERCA1)Skeletal Muscle RyRSkeletal Muscle MTMR1Skeletal Muscle/Heart NMDAR1Brain MAPT (Tau)Brain 90+ othersTissue Specific MBNL

8. 8

9.  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? 9

10. 10 TATA TetO 2 MBNL1 TetR TetO 2 TetR + dox ( ) TATA TetO 2 MBNL1 TetR TetO 2 TetR TATA TetO 2 MBNL1 TetO 2 TetR

11. 11  induced using doxycycline  shows varying expression as dox is titrated  0 point shows not a leaky promoter Stacey Wagner, unpublished data

12. PCR Reverse Transcription (RT) % Exon Inclusion HEK293 cells Tet-on MBNL1 system RNA Isolation Add doxycycline 12 + Splicing reporter minigene http://www.pan-biotech.com/content/images/stories/serumfreiesysteme/panexinnt/nta-hek.jpg

13. 13 456 64

14. 14 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and ATP2A1 Is there a threshold necessary to ‘turn it on’ Endogenous is enough in TNNT2, not in ATP2A1 Stacey Wagner, unpublished data

15.  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 15

16. Andy Berglund (Principle Investigator) Stacey Wagner (Mentor) Leslie Coonrod Elaine DeLorimier Dylan Farnsworth Julia Oddo Ruth Siboni Rodger Voelker Dave Youngentob SPUR – NICHD R25 Summer Research Program NIH-1R25HD070817 16