1. How does the repeat length of toxic RNA affect mis-splicing in a cell model of myotonic dystrophy? Irina Rapoport Berglund Lab 1

2. What is Myotonic Dystrophy (DM)? Most common form of adult onset muscular dystrophy 1 in 8000 individuals affected Common symptoms include – Muscle weakness – Myotonia – Heart dysfunction – Iridescent cataracts – Mental deficiencies Iridescent cataracts 2

3. How is it treated now? There is no cure, currently Symptomatic treatments are available None of the current treatments deal with the cause of the disease Understanding the molecular mechanism of could help treat it 3

4. What is the molecular mechanism of DM? DMPK ORF 5’ (CTG) n 3’ MBNL 5’ G  C U U C  G G  C U U C  G G  C U U C  G G  C U U 5’ C  G DMPK ORF 1. transcription 2. MBNL sequestration by (CUG) N stem loop 3. mis-splicing of MBNL- mediated RNA targets 4

5. What is mis-splicing and how does it cause DM symptoms? RNA is alternatively spliced  different isoforms Example: in DM1, insulin receptor mis-splicing (MBNL- dependent)  insulin insensitivity Mis-spliced RNA leads to incorrect protein formation

6. Splicing can be assayed to characterize DM in a cell model MBNL-dependent mis-splicing events  DM Assay proportion of different splice isoforms in cells Using HeLa cells as a model – Transfect with plasmids encoding for CUG repeat RNA – Assay different INSR isoforms 6

7. Insulin receptor is mis-spliced in the presence of 960 CUG repeats Leslie Coonrod 7 WTCUG 960 INSR isoforms Splicing assay

8. Pentamidine rescues mis-splicing Leslie Coonrod 8 WTCUG 960 Splicing assay

9. Does the severity of mis-splicing depend upon the length of (CUG) N ? Since MBNL is sequestered by CUG repeats, longer repeats will result in a greater degree of mis-splicing in the HeLa cell model Additionally, how does repeat length affect the ability of pentamidine to rescue mis- splicing? 9

10. General procedure HeLa cells 1a. Introduce different lengths of CUG repeats by transfection  DM 1b. Also transfect with plasmids encoding for the insulin receptor (INSR) minigene to later assay mis-splicing 2. Add pentamidine (0 uM – 60 uM) 2. Assay mis-splicing by proportion of INSR isoforms N = 0-960 CTG repeats 5’3’DMPK ORF(CTG) N 10 1211 ISNR minigene reporter

11. Raw data with 480 CUG repeats How the splicing assay works Transfect Add pentamidine Harvest cells Isolate RNA RNA  DNA PCR amplify INSR reporter Gel electrophoresis WT480 CUG INSR isoforms 11 Pentamidine 60 uM 0 ` `

12. Quantify splicing assay % inclusion = + x 100% 12 WT 0Pentamidine (uM) 60

13. 25 50 60 13 CUG 240 WT CUG 40 WT CUG 480 WT 0 Pentamidine (uM) 60 0 Pentamidine (uM) 0 60 CUG 0WT 0 Pentamidine (uM) 60 480 CUG repeats240 CUG repeats 40 CUG repeats0 CUG repeats

14. Tentative conclusions In HeLa cells, 240-960 CUG repeats cause a similar degree of mis-splicing With 40 or fewer repeats, mis-splicing is less severe 50 uM- 60 uM pentamidine rescues mis- splicing of INSR completely With fewer CUG repeats, higher [pentamidine] leads to over-rescue of mis-splicing 14

15. What next? Do more trials Test other small molecules Vary transfection conditions – Add lower [DNA] to see how much is enough to get different degrees of mis-splicing of different repeats Study the mechanism of pentamidine’s action 15

16. Thank You To: The Berglund Lab – Andy Berglund – Leslie Coonrod – Stacey Wagner – Ruth Siboni – Elaine DeLorimier – Lauryn Falcone (SPUR student) Haley Lab – Mike Haley – Micah Bodner – Aaron Docter The SPUR program – Peter O’Day – Adam Ugner – NICHD Summer Research Program (NIH- 1R25HD070817) 16