Systematic Evaluation of the Effect of Common SNPs on Pre-mRNA Splicing Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Abdou Gomaa Abdou ElSharawy B.Sc., M.Sc. Kiel, November 2008 II Referent:…………………………………………………….. Prof. Dr. Frank Kempken Koreferent:………………………………………………….. Prof. Dr. Stefan Schreiber Tag der mündlichen Prüfung: Kiel, den 11.11.08………... Zum Druck genehmigt: Kiel, den ..………………. ................................................... Der Dekan III To my parents my wife and my sons Ahmed & Amr IV TABLE OF CONTENTS 1 INTRODUCTION .............................................................................................................. 1 1.1. Single nucleotide polymorphisms: Biology and functional relevance ....................... 1 1.2. Pre-mRNA splicing: Mechanism and challenges ....................................................... 2 1.3. Alternative splicing and biological complexity: One gene, many proteins ............... 5 1.3.1. Patterns of alternative pre-mRNA splicing ........................................................ 5 1.3.2. Splicing regulatory mechanisms at genomic dimensions .................................. 7 1.3.3. Global functions and communication of alternative splicing ............................. 8 1.3.4. Components influencing exon recognition and alternative splicing .................. 9 1.4. Pre-mRNA (mis)splicing as a primary cause of disease .......................................... 11 1.4.1. Cis-acting mutations: Possible dramatic effects upon the splicing code ......... 12 1.4.2. Trans-acting mutations: Disruption of the splicing machinery ........................ 13 1.5. Study of allele-dependent splicing: Motivations and Perspectives .......................... 14 1.5.1. Genomic and mechanistic perspectives ............................................................ 15 1.5.2. Disease relevance of allele-dependent splicing ................................................ 15 1.5.3. Recent surveys approaching allele-dependent splicing .................................... 17 1.5.4. Biomedical perspective and drug design strategies .......................................... 18 1.5.5. Predicting effects of splice-relevant SNPs ....................................................... 18 1.6. Aims of the study ..................................................................................................... 21 2 METHODS ....................................................................................................................... 22 2.1. Selection of putative splice SNPs ............................................................................. 22 2.2. General methods ....................................................................................................... 22 2.2.1. DNA extraction and quality control ................................................................. 22 2.2.2. Total RNA extraction and quality control ........................................................ 23 2.2.3. First-strand cDNA synthesis and quality control ............................................. 24 2.2.4. Gel electrophoresis ........................................................................................... 24 2.2.5. Elution of DNA fragments from agarose and PCR clean-up ........................... 25 2.2.6. Measurement of DNA/RNA concentrations .................................................... 25 2.2.6.1. PicoGreen® assay ......................................................................................... 25 2.2.6.2. NanoDrop assay ........................................................................................... 26 2.2.7. Polymerase chain reaction ................................................................................ 26 2.2.8. Digestion of DNA with restriction endonucleases ........................................... 29 2.2.9. Cloning ............................................................................................................. 29 2.2.9.1. TA Cloning for PCR products ...................................................................... 29 V 2.2.9.2. Cloning using T4-DNA ligase ...................................................................... 29 2.2.9.3. Transformation ............................................................................................. 30 2.2.10. Site-directed mutagenesis ................................................................................. 30 2.2.11. Plasmid DNA purification ................................................................................ 30 2.2.12. DNA Sequencing .............................................................................................. 31 2.2.13. Transfection of cultured HeLa cells using FuGene 6 Reagent ......................... 32 2.2.14. Protein lysate preparation and Western blotting .............................................. 33 2.2.15. Fluorescent Activated Cell Sorter (FACS)-Analysis ....................................... 34 2.3. Generation of matching DNA- cDNA pairs ............................................................. 35 2.3.1. Recruitment ...................................................................................................... 35 2.3.2. Plate layout ....................................................................................................... 36 2.3.3. Quality control checkups .................................................................................. 37 2.4. Whole-genome amplification and genotyping ......................................................... 37 2.4.1. Whole-genome amplification ........................................................................... 37 2.4.2. Genotyping of amplified DNA samples ........................................................... 39 2.4.2.1. SNPlexTM Genotyping: An advanced high-throughput technology ............. 39 2.4.2.2. TaqMan® genotyping assay: A fluorogenic 5 nuclease assay ..................... 44 2.5. Arraying of corresponding cDNA ............................................................................ 46 2.6. Transcript analysis using nested RT-PCR in genotyped cDNA samples ................. 47 2.6.1. Primer design criteria and semi-automation ..................................................... 47 2.6.2. Nested RT-PCR ................................................................................................ 48 2.7. Direct sequencing ..................................................................................................... 49 2.8. Analysis Software: SNPSplicer ................................................................................ 50 2.9. Validation of allele-dependent splicing by cloning .................................................. 51 2.10. Development of an in vitro splice reporter system................................................... 51 3 RESULTS ......................................................................................................................... 54 3.1. A high-throughput assay for the investigation of allele- dependent splicing ........... 54 3.1.1. MotifSNPs Tool: Extraction of splice SNPs from public database ................. 55 3.1.2. SpliceTool software: Arraying of cDNA ......................................................... 56 3.1.3. SkippedExonPrimer Tool: Semi-automation of designing nested primers ...... 58 3.1.4. SNPSplicer: A screening tool for allele-dependent splicing signals ................ 58 3.1.4.1. Example of the use of SNPSplicer showing a splicing-nonrelevant SNP .... 60 3.1.4.2. Simple positive example of the use of SNPSplicer ...................................... 62 3.1.4.3. Complicated example of the use of SNPSplicer .......................................... 63 VI 3.1.5. Direct sequencing approach ............................................................................. 66 3.2. First screening-round of allele-dependent splicing: Web-based tools ..................... 66 3.2.1. Candidate SNPs for canonical and NAGNAG splice sites .............................. 66 3.2.2. Putative splice SNPs at ESEs ........................................................................... 69 3.3. Second screening-round: Neural network assessment of canonical splice sites ...... 71 3.4. Combined outputs and observations from both screening rounds ........................... 73 3.4.1. Observed splice effects ..................................................................................... 73 3.4.2. Allele-dependent splicing at NAGNAG tandem acceptors .............................. 74 3.4.3. Evaluation of the performance of F-SNP tool .................................................. 74 3.5. Establishment of a novel in vitro splice reporter system ......................................... 81 3.5.1. Insertion of test genomic region and coding sequence of RFP: Optimization . 81 3.5.2. Functional validation: A fluorescence-based detection method for comprehensive analysis of splice site mutations .............................................................. 82 4 DISCUSSION .................................................................................................................. 85 4.1. Characteristics of the applied approach.................................................................... 85 4.2. Prediction rate of allele-dependent splicing ............................................................. 90 4.3. Efficiency of in silico splice SNP