UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTÉ UPR 9002 THÈSE présentée par : Semih CETIN soutenue le : 12 septembre 2016 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline / Spécialité : Sciences du vivant / Aspects moléculaire et cellulaire de la biologie Caractérisation moléculaire du mécanisme de dégradation des microARN par un transcrit cible THÈSE dirigée par : Mr PFEFFER Sébastien Directeur de recherche CNRS, Université de Strasbourg RAPPORTEURS : Mr BRODERSEN Peter Associate Professor, Université de Copenhague Mme VANACOVA Stěpánka Associate Professor, Université de Masaryk AUTRES MEMBRES DU JURY : Mr GAGLIARDI Dominique Directeur de recherche CNRS, Université de Strasbourg Mr FILIPOWICZ Witold Professeur, Friedrich Miescher Institute for Biomedical Research Acknowledgements I would like to first of all thank Sébastien for giving me the opportunity to do my thesis in his laboratory. I am very grateful to have been a part of his team, the members of which he chooses so well to form a very diverse and interesting bunch that creates a very enjoyable learning and growing environment. Thank you for being so supportive and always accessible to talk science or any other subject. Being supervised by Sebastien was very enriching experience for which I will forever be grateful, rich in learning and experiences not only through my main work in the lab but also through the collaborations he allowed me to take part in as well as the teaching missions that he encouraged me to pursue at the faculty. I would also like to thank the members of the jury, Peter Brodersen, Stěpánka Vanacova and Dominique Gagliardi for accepting to judge my work and for their invaluable feedback through the very interesting and stressful scientific discussion during my defence. I would like to thank Joern Pütz for inspiring me to follow my scientific adventure in Strasbourg after my year as an Erasmus student, and for helping and supporting me tremendously along the way. I would like to thank all the former and present members of the Pfeffer laboratory; Gabrielle, Mélanie, Erika, Aurélie, Maud, Béatrice, Ramy, Diane, Guillaume, Lee, Ali, Ilyass, Mathieu, Antoine and Olivier, with whom we shared a great deal of time inside and outside of the laboratory. Thank you all for your support during these years and helping me grow personally and professionally. You have made this adventure very enjoyable and very memorable and I feel very lucky and grateful to have shared my time with you all. I am thankful to my family who have always been there for me and supported me in every way since the start, never failing to encourage me along the way. Finally, I would like to thank my girlfriend Nevcin, for being ever present, through our adventures from our Erasmus year abroad to our thesis studies. She has been the source of tremendous support during these times and the invaluable constant in my life. ii ii TABLE OF CONTENTS INTRODUCTION .............................................................................. 1 1. Biogenesis of miRNAs .......................................................................................................... 1 1.1 Canonical biogenesis of mammalian miRNA ..................................................................... 1 1.1.1 Generation of miRNA precursor and export into the cytoplasm ............................................... 1 1.1.2 Dicing of the pre-miRNA into miRNA/miRNA* duplex ............................................................... 5 1.2 Non canonical biogenesis of miRNA .................................................................................. 6 1.3 Formation of functional miRISC ......................................................................................... 8 2. Mode of action and Function of miRNAs ........................................................................... 10 2.1 Target recognition of miRNAs ......................................................................................... 10 2.2 Influence of target site context ....................................................................................... 11 2.3 Mechanism of action of miRNAs ..................................................................................... 12 2.4 Function of miRNAs ......................................................................................................... 15 3. Regulation of miRNA expression ....................................................................................... 16 3.1 Regulation of miRNA biogenesis ..................................................................................... 16 3.1.1 Transcriptional regulation ........................................................................................................ 16 3.1.2 Regulation of miRNA processing .............................................................................................. 17 3.1.2.1 Regulation of the Microprocessor .................................................................................. 17 3.1.2.2 Export regulation ............................................................................................................ 19 3.1.2.3 Regulation of Dicer processing ....................................................................................... 20 3.2 Regulation of RISC action ................................................................................................ 21 3.2.1 Regulation at the level of Argonaute proteins ......................................................................... 21 3.2.2 Regulation of the activity of Argonaute proteins by RNA binding proteins ............................. 22 4. RNA intrinsic regulation affecting miRNA expression and targeting ................................... 24 4.1 Modifications on miRNA precursor seQuences affecting their biogenesis and/or the targeting of miRNAs ..................................................................................................................... 25 4.1.1 Nucleobase substitutions on miRNA precursors ...................................................................... 25 4.1.2 Regulation of let-7 biogenesis by non-templated nucleotide additions .................................. 26 4.2 Sequence modifications on mature miRNA (isomiRs) ..................................................... 28 4.2.1 5’ isomiRs: shift in Drosha and/or Dicer cleavage .................................................................... 28 4.2.2 3’ IsomiRs: 3’ heterogeneity ..................................................................................................... 29 4.2.2.1 Exoribonucleases creating 3’ isomiRs ............................................................................. 29 4.2.2.2 Non-templated nucleotide additions on mature miRNA by mammalian terminal uridylyl transferases (TUTases) ....................................................................................................................... 30 4.2.2.3 Mammalian TUTase family and their function ................................................................ 30 5. Mature miRNA turnover ................................................................................................... 33 i 5.1 Trans acting factor in miRNA turnover ............................................................................ 34 5.1.1 miRNA turnover in plants ......................................................................................................... 34 5.1.2 miRNA turnover in Chlamydomonas reinhardtii ...................................................................... 35 5.1.3 miRNA turnover in Caenorhabditis elegans ............................................................................. 35 5.1.4 miRNA turnover in mammals ................................................................................................... 35 5.2 RNA-mediated miRNA turnover ...................................................................................... 37 5.2.1 Target-mediated miRNA protection (TMMP) ........................................................................... 38 5.2.2 Target RNA-mediated miRNA degradation .............................................................................. 38 5.2.3 miRNA degradation and viral infections ................................................................................... 40 RESULTS .......................................................................................... 44 1. Discovery of factors involved in Target RNA mediated miRNA degradation (TDMD) .......... 44 2. Identification of Argonaute 2 partners by proximity dependent labeling ........................... 48 2.1 Aim .................................................................................................................................. 48 2.2 Large scale protein interaction mapping techniQues ...................................................... 48 2.2.1 Affinity purification coupled to mass spectrometry ................................................................. 49 2.2.2 Tandem Affinity Purification ..................................................................................................... 49 2.2.3 Yeast two-hybrid system .......................................................................................................... 50 2.2.4 Proximity labelling strategies ................................................................................................... 50 2.2.4.1 APEX approach ...............................................................................................................