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Design and Application of Circular Rnas for Protein Sponging and Modulation of Alternative Splicing

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Design and Application of Circular Rnas for Protein Sponging and Modulation of Alternative Splicing Design and application of circular RNAs for protein sponging and modulation of alternative splicing Dissertation vorgelegt von Anna Didio (Master of Science in Biology) zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) Institut für Biochemie der Justus-Liebig-Universität Gießen Gießen, November 2020 Die vorliegende Arbeit wurde am Institut für Biochemie des Fachbereichs 08 der Justus- Liebig-Universität Gießen in der Zeit von November 2017 bis November 2020 unter der Leitung von Prof. Dr. Albrecht Bindereif angefertigt. Dekan: Prof. Dr. Jürgen Janek Physikalisch-Chemisches Institut Justus-Liebig-Universität Gießen 1. Gutachter: Prof. Dr. Albrecht Bindereif Institut für Biochemie Fachbereich für Biologie und Chemie Justus-Liebig-Universität Gießen 2. Gutachter: Prof. Dr. Elena Evguenieva-Hackenberg Institut für Mikro- und Molekularbiologie Fachbereich für Biologie und Chemie Justus-Liebig-Universität Gießen Contents Contents Summary…………………………………………………………………………………………….….i Zusammenfassung……………………………………………………………………………………ii 1. Introduction ................................................................................................................. 1 1.1 Splicing of mRNA ..................................................................................................... 1 1.2 Alternative splicing ................................................................................................... 4 1.3 Alternative splicing as a therapeutic target in human diseases ................................ 6 1.4 HnRNP L, a global regulator of alternative splicing .................................................. 9 1.5 Alternative splicing of CD45 ................................................................................... 12 1.6 Circular RNA .......................................................................................................... 14 1.7 Splicing of circRNA: Sequence determinants and protein factors ........................... 16 1.8 CircRNA expression systems in human cells ......................................................... 18 1.9 Functions of endogenous circRNAs and their potential application ........................ 19 1.10 Splicing of tRNA ..................................................................................................... 21 1.11 CircRNAs in human platelets ................................................................................. 21 1.12 Specific aims of this work ....................................................................................... 23 2. Materials .................................................................................................................... 24 2.1 Chemicals and reagents ........................................................................................ 24 2.2 Kits ........................................................................................................................ 25 2.3 Enzymes ................................................................................................................ 26 2.4 Antibodies .............................................................................................................. 26 2.5 Plasmids ................................................................................................................ 26 2.6 Molecular weight markers ...................................................................................... 27 2.7 Laboratory equipment ............................................................................................ 27 2.8 Consumables ......................................................................................................... 28 2.9 Oligonucleotides .................................................................................................... 29 3. Methods ..................................................................................................................... 36 3.1 DNA cloning in E. coli ............................................................................................ 36 3.2 Design of circRNA and cloning of circRNA expression constructs .......................... 36 3.2.1 Use of platelet-derived sequence elements for circRNA expression ............... 36 3.2.2 Expression of CA-SELEX sequence in tRNA intronic circular (tric)RNA .......... 37 3.2.3 In vitro circRNA production by permuted intron-exon splicing strategy ............ 37 3.2.4 Expression of circRNA from Tornado constructs ............................................. 38 3.2.5 In vitro transcription and circularization of antisense circRNA ......................... 39 3.2.6 RNA secondary structure prediction ................................................................ 39 3.3 Cell culture methods .............................................................................................. 39 3.3.1 HeLa cells ....................................................................................................... 39 3.3.2 Transfection of circular and linear PIE RNA in HeLa cells ............................... 39 Contents 3.3.3 Transfection of circRNA-expression constructs in HeLa cells ......................... 40 3.3.4 RNAi knockdown of hnRNP L in HeLa cells .................................................... 40 3.3.5 Co-transfection of CD45 minigene and antisense circRNA in HeLa cells ........ 40 3.3.6 DG75 cells ...................................................................................................... 41 3.3.7 Electroporation of antisense circRNA in DG75 cells ....................................... 41 3.4 Working with RNA and characterization of designer circRNAs ............................... 41 3.4.1 Total RNA isolation ......................................................................................... 41 3.4.2 RT-PCR and agarose gel electrophoresis....................................................... 41 3.4.3 Northern blot ................................................................................................... 42 3.4.4 E-Gel system .................................................................................................. 43 3.4.5 RNase R treatment of total RNA samples ....................................................... 43 3.4.6 Denaturing urea polyacrylamide gel electrophoresis ....................................... 43 3.4.7 In-gel Broccoli aptamer imaging with DFHBI ................................................... 44 3.4.8 RNA immunoprecipitation and Western blot ................................................... 44 3.4.9 Quantitative real-time PCR ............................................................................. 45 3.4.10 Cell fractionation ............................................................................................. 45 3.4.11 Determination of absolute concentration of expressed circRNA ...................... 45 3.4.12 Microscopy and image processing .................................................................. 46 4. Results ...................................................................................................................... 47 4.1 Repetitive elements contribute to circRNA biogenesis ........................................... 47 4.2 A tRNA-based construct optimized for circRNA expression ................................... 49 4.3 Designer PIE circRNAs act as hnRNP L sponges .................................................. 51 4.3.1 Binding of hnRNP L by transfected PIE circRNAs ........................................... 54 4.3.2 PIE circRNA sponges affect alternative splicing.............................................. 54 4.4 Expressed Tornado circRNA with protein sponge function .................................... 58 4.4.1 Tornado-expressed circRNAs bind hnRNP L in HeLa cells ............................. 64 4.4.2 Designer Tornado circRNAs alter alternative splicing of hnRNP L target genes in HeLa cells ........................................................................................ 66 4.4.3 Sponge Tornado circRNAs shift nuclear-cytoplasmic hnRNP L distribution .... 68 4.5 Antisense circRNA for splicing modulation ............................................................. 70 4.5.1 Modulation of CD45 alternative splicing by transfected antisense circRNAs ... 73 4.5.2 CD45 alternative splicing alteration by antisense circRNA involves a post- transcriptional mechanism .............................................................................. 75 4.5.3 Antisense circRNAs reduce mRNA levels of the target gene .......................... 77 5. Discussion ................................................................................................................ 81 5.1 Development of optimal vector systems for circRNA production ............................ 81 5.2 Designer circRNAs acting on hnRNP L modulate alternative splicing networks ..... 82 5.3 Modulation of alternative splicing by antisense circRNA ........................................ 85 Contents 5.4 Perspectives .......................................................................................................... 87 6. References ................................................................................................................ 90 7. Abbreviations .......................................................................................................... 102 8. Scientific achievements ......................................................................................... 105 9. Acknowledgements ...............................................................................................
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