Characterization of the Regulation of CD46 Alternative Splicing
Total Page：16
File Type：pdf, Size：1020Kb
This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Characterization of the regulation of CD46 alternative splicing Tang, Sze Jing 2016 Tang, S. J. (2016). Characterization of the regulation of CD46 alternative splicing. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/65984 https://doi.org/10.32657/10356/65984 Downloaded on 29 Sep 2021 15:37:19 SGT CHARACTERIZATION OF THE REGULATION OF CD46 ALTERNATIVE SPLICING TANG SZE JING SCHOOL OF BIOLOGICAL SCIENCES 2016 CHARACTERIZATION OF THE REGULATION OF CD46 ALTERNATIVE SPLICING TANG SZE JING School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2016 ACKNOWLEDGEMENTS First of all, I would like to express my sincere gratitude to my supervisor, Dr. Xavier Roca, for his constant guidance and patience throughout the course of my project. I am thankful to School of Biological Sciences, Nanyang Technological University for the advance core facilities and financial support. I am also sincerely appreciated my Thesis Advisory Committee, Professor Alex Law and Dr. Eugene Makeyev, for their valuable suggestions on my project. I would like to thank Dr. Karen Yap Lin Choo and Fursham Hamid for their help in one of the experiments. I would also like to thank Heidi Berger for her help in maintaining Jurkat T cells. I am indebted to all my present and past lab members especially Luo Shufang and Dr. Liu Jun for their help and guidance throughout these four years. I am grateful to my friends for their support and accompanying. Last but not least, I would like to dedicate this thesis to my family who always support me. P a g e | 2 TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................. 1 LIST OF FIGURES ............................................................................................. 7 LIST OF TABLES .............................................................................................. 9 ABBREVIATIONS ............................................................................................10 ABSTRACT .......................................................................................................16 1. INTRODUCTION ..........................................................................................17 1.1 Splicing .....................................................................................................17 1.1.1 Exon definition ...................................................................................19 1.2 Alternative splicing ...................................................................................21 1.2.1 Types of alternative splicing ...............................................................21 1.2.2 Regulation of alternative splicing ........................................................22 1.2.2.1 Splice-site strength ................................................................ 22 1.2.2.2 Cis-acting elements and trans-acting factors .......................... 25 1.2.2.2.1 SR proteins ................................................................ 30 1.2.2.2.2 SRSF1 ....................................................................... 30 1.2.2.3 Transcription and chromatin .................................................. 32 1.2.2.4 RNA secondary structure ....................................................... 33 1.2.3 Alternative splicing and NMD ............................................................33 1.2.4 Alternative splicing and diseases .........................................................35 1.3 The complement system ............................................................................38 1.4 Adaptive immune system ..........................................................................41 1.4.1 B cells.................................................................................................41 P a g e | 3 1.4.2 T cells .................................................................................................43 1.5 CD46 ........................................................................................................47 1.5.1 Gene structure .....................................................................................47 1.5.2 CD46 Function ...................................................................................49 1.5.2.1 Role in complement regulation .............................................. 49 1.5.2.2 CD46 and fertilization ........................................................... 49 1.5.2.3 Role of CD46 in epithelial cells ............................................. 49 1.5.2.4 Role of CD46 on B cells ........................................................ 50 1.5.2.5 CD46 in T-cell regulation ...................................................... 51 1.5.3 Implications in diseases ......................................................................54 1.5.4 Alternative splicing of CD46 ..............................................................57 1.6 Alternative splicing in immune responses ..................................................58 1.7 Objective ...................................................................................................60 2. MATERIALS AND METHODS ....................................................................61 2.1 Cell culture................................................................................................61 2.2 Plasmids ....................................................................................................61 2.2.1 Minigene construction ........................................................................61 2.2.2 Generation of mutant minigenes .........................................................63 2.2.3 Heterologous minigene .......................................................................70 2.2.3.1 pSXN plasmid ....................................................................... 70 2.2.3.2 CD46 exon 13 in Universal Minigene Vector (UMV) ............ 71 2.2.4 Overexpression plasmid ......................................................................72 P a g e | 4 2.3 Bacterial transformation and plasmid extraction ........................................73 2.4 Transfection ..............................................................................................74 2.4.1 Adherent cells .....................................................................................74 2.4.1.1 Transfection with minigenes .................................................. 74 2.4.1.2 siRNA and antisense oligonucleotides (ASOs) transfection ... 74 2.4.2 Suspension cells ..................................................................................77 2.4.2.1 Transfection with minigenes .................................................. 77 2.4.2.2 siRNA and antisense oligonucleotides (ASO) transfection ..... 77 2.5 Jurkat E6.1 cell activation .........................................................................77 2.6 RNA extraction and DNase I treatment .....................................................78 2.7 Reverse transcription (RT)-PCR and gel electrophoresis ...........................78 2.8 Semi-quantitative (Semi-Q) PCR...............................................................80 2.9 Analysis of CD46 alternative splicing patterns in human tissues ................80 2.10 Western blot ............................................................................................80 2.10.1 Sample preparation ...........................................................................80 2.10.2 SDS-PAGE and transfer ....................................................................81 2.10.3 Detection ..........................................................................................81 2.11 RNA pull-down .......................................................................................82 2.12 Silver staining .........................................................................................83 2.13 Flow cytometry .......................................................................................83 2.13.1 Cell sorting .......................................................................................83 2.13.2 Cell analysis .....................................................................................83 P a g e | 5 2.14 Inhibition of NMD ..................................................................................83 2.15 Preparation of HeLa nuclear extract .........................................................84 3. RESULTS ......................................................................................................85 3.1 Alternative splicing of CD46 is tissue selective .........................................85 3.2 The 5'ss flanking exons 7 and 8 are recognized by U1 using asymmetric loop register ............................................................................................................88 3.3 Multiple cis-acting elements regulate the alternative splicing pattern of exon 13....................................................................................................................96 3.3.1 Exon 13 is rich in ESEs and ESSs .......................................................96 3.3.2 The intronic sequences near exon 13 have less regulatory effect ....... 102 3.3.3 Validation of cis-acting elements by point mutations