Research Collection Doctoral Thesis Tools and strategies for the unraveling of post-transcriptional gene regulatory networks Author(s): Kanitz, Alexander Publication Date: 2012 Permanent Link: https://doi.org/10.3929/ethz-a-7358945 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH Nr. 20147 TOOLS AND STRATEGIES FOR THE UNRAVELING OF POST-TRANSCRIPTIONAL GENE REGULATORY NETWORKS A dissertation submitted to ETH ZURICH for the degree of Doctor of Sciences presented by ALEXANDER KANITZ M.Sc. University of Amsterdam born November 15, 1980 citizen of Germany Accepted on the recommendation of Prof. Dr. Michael Detmar Prof. Dr. Jonathan Hall Prof. Dr. André Gerber 2012 1 SUMMARY _________________________________________________________________________________ 6 1.1 Summary _____________________________________________________________________________ 6 1.2 Zusammenfassung ______________________________________________________________________ 9 2 INTRODUCTION ________________________________________________________________________ 12 2.1 Key principles and players of post-transcriptional gene regulatory processes ____________________ 13 2.1.1 The fate of eukaryotic messenger RNAs _______________________________________________ 13 2.1.2 Cis -regulatory elements ____________________________________________________________ 15 2.1.3 Trans -acting factors _______________________________________________________________ 16 2.1.3.1 RNA-binding proteins __________________________________________________________ 16 2.1.3.2 MicroRNAs ___________________________________________________________________ 17 2.1.4 Ribonucleoprotein complexes and the RNA regulon theory _______________________________ 19 2.2 Post-transcriptional gene regulatory networks _____________________________________________ 22 2.2.1 Basic network motifs ______________________________________________________________ 23 2.2.1.1 Multiple output network motifs __________________________________________________ 23 2.2.1.2 Multiple input network motifs ___________________________________________________ 25 2.2.2 Autoregulatory, two- and multicomponent loops _______________________________________ 26 2.2.3 Composite gene regulatory networks _________________________________________________ 29 2.2.3.1 RNA-binding proteins versus transcription factors ___________________________________ 29 2.2.3.2 RNA-binding proteins versus microRNAs ___________________________________________ 30 2.3 Ribonomics methodologies for the systematic identification of basic post-transcriptional gene regulatory network motifs ___________________________________________________________________ 32 2.3.1 Top-down approaches: RIP-Chip and related methods ___________________________________ 33 2.3.2 Bottom-up: RNA affinity chromatography and related methods ____________________________ 35 2.3.2.1 Direct RNA affinity chromatography ______________________________________________ 35 2.3.2.2 Purification methods based on antisense hybridization _______________________________ 36 2.3.2.3 Aptamer-based purification methods _____________________________________________ 37 2.3.2.4 Indirect protein- and peptide-based purification methods _____________________________ 38 2.3.2.5 Bifunctional RNA tag systems ____________________________________________________ 39 2.4 Combinatorial control of cancer-related messages __________________________________________ 40 2.4.1 Combinatorial control of the angiogenesis factor vascular endothelial growth factor A _________ 40 2.4.2 Combinatorial control of the tumor suppressor CDKN1B/p27______________________________ 44 3 AIMS AND OUTLINE OF THE THESIS ________________________________________________ 48 4 IDENTIFICATION OF NEW POST-TRANSCRIPTIONAL REGULATORS OF VASCULAR ENDOTHELIAL GROWTH FACTOR A EXPRESSION ________________________ 50 4.1 Introduction __________________________________________________________________________ 50 4.2 Results ______________________________________________________________________________ 55 4.2.1 The VEGFA 3’-untranslated region contains canonical Pum consensus motifs _________________ 55 4.2.2 VEGFA is a putative target of microRNA 361-5p _________________________________________ 59 4.2.3 MicroRNA 361-5p and Pum1/2 may target other angiogenesis-related transcripts _____________ 61 4.2.4 Generation and characterization of stable Pum1/2 overexpression cell lines _________________ 64 4.2.5 Transfection of small RNAs _________________________________________________________ 67 4.2.6 The putative Pum and microRNA 361-5p recognition elements in the VEGFA 3’-UTR possess regulatory potential _______________________________________________________________ 68 4.2.7 Pum1, Pum2 and microRNA 361-5p repress the expression of VEGFA 3’-UTR reporters _________ 71 4.2.8 The repressive effects of Pum proteins and microRNA 361-5p on VEGFA 3’-UTR reporter activity are additive ______________________________________________________________________ 73 4.2.9 Endogenous VEGFA expression is regulated by microRNA 361-5p __________________________ 76 4.2.10 MicroRNA 361-5p is down-regulated in cutaneous squamous cell carcinoma _________________ 78 4.3 Discussion ___________________________________________________________________________ 83 4.3.1 The putative Pum and microRNA recognition elements exhibit regulatory potential ___________ 83 4.3.2 Combinatorial control of VEGFA expression by microRNA 361-5p and the Pum proteins ________ 85 4.3.3 The regulation of VEGFA expression by microRNA 361-5p and the Pum proteins may be dependent on each other __________________________________________________________ 87 4.3.4 The influence of microRNA 361-5p and Pum1 on VEGFA secretion rates _____________________ 89 4.3.5 A potential role of microRNA 361-5p and Pum proteins in cancer development and progression _ 90 4.3.6 Bioinformatics analyses suggest common functions of microRNA 361-5p and Pum proteins beyond the regulation of VEGFA expression ____________________________________________ 92 4.3.7 Conclusion ______________________________________________________________________ 93 4.4 Materials and Methods_________________________________________________________________ 93 4.4.1 Ethics statement__________________________________________________________________ 93 4.4.2 Plasmids ________________________________________________________________________ 93 4.4.3 Cell culture and tissue samples ______________________________________________________ 95 4.4.4 MicroRNA target gene prediction and pathway analysis __________________________________ 97 4.4.5 Immunoblot analysis ______________________________________________________________ 98 4.4.6 Flow cytometry___________________________________________________________________ 99 4.4.7 Quantitative reverse transcription PCR ________________________________________________ 99 4.4.8 Immunocytochemistry ____________________________________________________________ 100 4.4.9 Luciferase reporter assays _________________________________________________________ 101 4.4.10 Enzyme-linked immunosorbent assay ________________________________________________ 102 4.5 Contributions ________________________________________________________________________ 102 5 A NOVEL RNA TANDEM AFFINITY TAG FOR THE PURIFICATION OF RIBONUCLEOPROTEIN PARTICLES ______________________________________________________ 104 5.1 Introduction _________________________________________________________________________ 104 5.2 Results _____________________________________________________________________________ 106 5.2.1 Aptamer selection _______________________________________________________________ 106 5.2.2 Oligonucleotide selection _________________________________________________________ 108 5.2.3 Arrangement of the HAMMER tandem affinity tag system _______________________________ 110 5.2.4 Purification strategy ______________________________________________________________ 112 5.2.5 Plasmid generation ______________________________________________________________ 114 5.2.6 Secondary structures of HAMMER-tagged RNAs are largely unaffected _____________________ 116 5.2.7 HAMMER-tagged RNAs are expressed in transiently transfected cells ______________________ 119 5.2.8 Purification of HAMMER-tagged in vitro transcripts via hybridization to antisense oligonucleotides _________________________________________________________________ 122 5.2.9 Purification of HAMMER-tagged in vitro transcripts via the S1 aptamer_____________________ 125 5.3 Discussion __________________________________________________________________________ 127 5.3.1 Expression of tagged transcripts ____________________________________________________ 127 5.3.2 Capturing of tagged transcripts by antisense hybridization _______________________________ 128 5.3.3 Elution of transcripts immobilized by hybridization _____________________________________ 129 5.3.4 Aptamer-mediated purification of tagged transcripts ___________________________________ 130 5.3.5 Reflections on tag folding and insertion ______________________________________________ 131 5.3.6 Limitations of RNA secondary structure prediction algorithms ____________________________ 132 5.3.7 Conclusion _____________________________________________________________________ 133 5.4 Materials and Methods________________________________________________________________ 134 5.4.1 Tag design and