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Matije Lucic Thesis Research Collection Doctoral Thesis The Good, the Bad and the Ugly: a three-way duel in microRNA targeting Author(s): Lucic, Matije Publication Date: 2019 Permanent Link: https://doi.org/10.3929/ethz-b-000335030 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 NO. 25693 The Good, the Bad and the Ugly: a three-way duel in microRNA targeting A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH Zurich (Dr. sc. ETH Zurich) presented by MATIJE LUCIC M.Sc. Pharm. Sciences, ETH Zurich born on 09.01.1986 citizen of Muralto, Switzerland accepted on the recommendation of Prof. Dr. Jonathan Hall, examiner Prof. Dr. Constance Ciaudo, co-examiner 2019 To my loved ones. For ‘Science & Cigars’, may this be the start. ETH Zurich Matije Lucic 2 Table of contents Acknowledgements ............................................................................................................... 6 Abstract ................................................................................................................................ 7 Riassunto .............................................................................................................................. 8 1 Introduction ................................................................................................................... 9 1.1 Introduction to microRNAs (miRNAs) ..................................................................... 9 1.1.1 miRNA gene families ................................................................................... 10 1.1.2 miRNA nomenclature ................................................................................... 10 1.1.3 miRNA guide and passenger strands ........................................................... 11 1.1.4 miRBase: the miRNA sequence repository .................................................. 12 1.2 miRNA biogenesis ............................................................................................... 14 1.3 Strand selection and RISC assembly ................................................................... 15 1.4 miRNA-mediated gene silencing .......................................................................... 16 1.4.1 Argonaute-catalyzed slicing mechanism ...................................................... 17 1.4.2 Slicing-independent translational repression and mRNA decay ................... 18 1.5 miRNA target recognition ..................................................................................... 19 1.5.1 5′ end of the miRNA .................................................................................... 19 1.5.2 Central region of the miRNA ........................................................................ 20 1.5.3 3′ end of the miRNA .................................................................................... 21 1.5.4 Non-canonical miRNA targeting ................................................................... 22 1.5.5 Model for miRNA target recognition ............................................................. 23 1.5.6 Identifying the miRNA targetome ................................................................. 24 1.5.7 miRNA target prediction strategies ............................................................... 25 1.6 miRNA targets fight back on miRNAs .................................................................. 26 1.7 Complexity and redundancy of miRNA function ................................................... 26 1.8 Oncomirs and tumor suppressors ........................................................................ 27 1.8.1 miR-17~92 cluster ........................................................................................ 28 1.8.2 let-7 family ................................................................................................... 30 1.9 Aim of the project................................................................................................. 31 2 Results ........................................................................................................................ 32 2.1 Sequence homology between miR-17 and let-7 families ...................................... 32 2.2 Model for competitive non-canonical binding at let-7 target sites ......................... 33 2.3 Investigation of the let-7 transcriptome in HEK293T cells .................................... 34 2.3.1 Canonical repression by let-7a ..................................................................... 37 2.3.2 Canonical repression by miR-106a and miR-106b ....................................... 39 2.3.3 miR-106a seed-mutation abolishes canonical repression............................. 41 2.3.4 3′ end-mutated miR-106a retains canonical repressive activity .................... 43 2.3.5 miR-106a and miR-106b are unable to repress let-7 targets ........................ 45 2.3.6 Co-transfection of let-7a with either miR-106a or miR-106b ......................... 47 2.3.7 Co-transfection of let-7a with seed-mutated miR-106a ................................. 49 2.3.8 Co-transfection of let-7a with 3′ end-mutated miR-106a .............................. 51 2.4 Follow-up on putative let-7 targets: HMGA2 and LIN28B ..................................... 55 ETH Zurich Matije Lucic 3 2.5 let-7 competition by miR-106a-5p strand ............................................................. 58 2.6 Seed-homology sequences in human miRNAs .................................................... 61 3 Discussion ................................................................................................................... 64 4 Outlook........................................................................................................................ 67 5 Contributions ............................................................................................................... 68 6 Side projects ............................................................................................................... 69 6.1 RNAi activity of hybrid duplexes with parallel orientation ..................................... 69 6.2 Targeting miR-122 in RISC with conjugated antimiRs.......................................... 70 6.3 Antagonizing Lin28-pre-let-7 interaction with ‘looptomirs’ .................................... 71 6.4 Mono- and bis-labeling of pre-miRNAs ................................................................ 72 7 Materials and methods ................................................................................................ 73 7.1 Materials .............................................................................................................. 73 7.1.1 miRNAs and siRNAs .................................................................................... 73 7.1.2 Plasmids ...................................................................................................... 74 7.1.3 RT-qPCR primers ........................................................................................ 74 7.1.4 Antibodies .................................................................................................... 74 7.2 Methods .............................................................................................................. 75 7.2.1 Cultivation and maintenance of mammalian cell lines .................................. 75 7.2.2 Seeding of the cells...................................................................................... 75 7.2.3 Transient transfections of miRNAs and siRNAs ........................................... 75 7.2.4 RT-qPCR ..................................................................................................... 75 7.2.5 Western blot................................................................................................. 75 7.2.6 Cloning and transfections of luciferase reporter plasmids ............................ 76 7.2.7 Luciferase assay .......................................................................................... 76 7.2.8 RNA integrity and quantification ................................................................... 76 7.2.9 Library preparation ....................................................................................... 77 7.2.10 Clustering and sequencing........................................................................... 77 7.2.11 Analysis of sequencing data sets ................................................................. 77 7.2.12 Library ID ..................................................................................................... 78 7.2.13 miRNA target predictions ............................................................................. 79 7.2.14 Statistical analysis........................................................................................ 80 References ......................................................................................................................... 81 Supplementary information ................................................................................................. 90 Posters ............................................................................................................................. 104 Curriculum vitae ................................................................................................................ 108 ETH Zurich Matije Lucic 4 Table of figures and
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