The Regulation of Incorrect Splicing of ISCU in Hereditary Myopathy with Lactic Acidosis Denise F. R. Rawcliffe Department of Medical Biosciences Umeå 2018 This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN: 978-91-7601-954-2 ISSN: 0346-6612 New series number: 1978 Cover design: Denise Rawcliffe Electronic version available at: http://umu.diva-portal.org/ Printed by: UmU Print Service, Umeå University Umeå, Sweden 2018 To Bethie Ancora Imparo Table of Contents Abstract ............................................................................................ iii Original Papers ................................................................................ iv Abbreviations .................................................................................... v Enkel sammanfattning på svenska .................................................. vii Splitsning ........................................................................................................................vii Linderholms myopati .....................................................................................................vii Studie I .......................................................................................................................... viii Studie II ......................................................................................................................... viii Studie III .......................................................................................................................... ix Slutsatser ........................................................................................................................... x Introduction ....................................................................................... 1 Splicing .............................................................................................................................. 1 Constitutive and Alternative Splicing ....................................................................... 1 The Spliceosome .........................................................................................................3 Cis-acting Elements ....................................................................................................3 Exon Definition ........................................................................................................... 5 Trans-acting Factors ................................................................................................ 6 Secondary Structure ................................................................................................. 6 RNA-Binding Proteins ...................................................................................................... 7 Splicing Factors .......................................................................................................... 7 Nonsense-Mediated Decay .............................................................................................. 8 Diseases Caused by Aberrant Splicing ............................................................................ 9 Pseudoexons .............................................................................................................. 10 Splicing Prediction ................................................................................................... 10 Muscle-specific Splicing ........................................................................................... 11 Muscles ............................................................................................................................ 11 Myopathies ............................................................................................................... 13 Hereditary Myopathy with Lactic Acidosis.................................................................... 13 Symptoms and Inheritance ..................................................................................... 13 Incorrect Splicing of ISCU ....................................................................................... 15 Muscle-specific Incorrect Splicing .......................................................................... 16 The ISCU Protein and FeS Cluster Assembly ......................................................... 17 ISCU and Oxidative Phosphorylation in Cancer .................................................... 18 Muscle-specific Loss of ISCU ................................................................................... 18 PTBP1 ........................................................................................................................ 19 IGF2BP1 ................................................................................................................... 20 RBM39 ...................................................................................................................... 21 MATR3 ..................................................................................................................... 22 Other ISCU Mutations ................................................................................................... 23 Aims of Thesis ................................................................................. 25 Materials and Methods ................................................................... 26 i Human Materials ........................................................................................................... 26 Mice ................................................................................................................................ 26 Cell Culture .................................................................................................................... 26 Lentiviral Transduction .......................................................................................... 26 Magnetofection ......................................................................................................... 27 Cycloheximide Treatment ........................................................................................ 27 RNA Extraction and cDNA Synthesis ............................................................................ 27 Semi-qRTPCR and qRTPCR .......................................................................................... 27 Protein Samples and Nuclear Extracts .......................................................................... 27 Pull-down Assay ............................................................................................................. 27 Western Blot Analysis.................................................................................................... 28 Statistics ......................................................................................................................... 28 Results and Discussion ................................................................... 29 Paper I ............................................................................................................................ 29 Muscle-specific Splicing .......................................................................................... 29 SRSF3 as a Muscle-specific Activator of the Incorrect Splicing ........................... 29 Paper II........................................................................................................................... 30 Tissue-specificity of PTBP1 ..................................................................................... 30 PTBP1 inhibition of the Incorrect Splicing .............................................................. 31 Tissue Specificity ...................................................................................................... 31 Paper III ......................................................................................................................... 32 K-homology Domain Candidates ........................................................................... 32 Other Splicing Factor Candidates .......................................................................... 33 U2AF ........................................................................................................................ 33 MBNL1 ..................................................................................................................... 34 MBNL1 and RBM39 Activates the Incorrect Splicing of ISCU ..............................35 The Incorrect ISCU RNA Transcripts are Targets for NMD ................................ 36 Conclusions ..................................................................................... 38 Paper I ............................................................................................................................ 38 Paper II........................................................................................................................... 38 Paper III ......................................................................................................................... 38 Overall Discussion .......................................................................... 39 The Exercise Sensitivity of HML Patients .................................................................... 39 Effect of HML Mutation on Splicing Elements ............................................................ 40 Splicing Factor Binding Prediction ............................................................................... 42 Tissue-Specific Regulation of ISCU Splicing