Analysis of Mir-30B As a Regulator of Tso45-G Anterograde Trafficking
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Analysis of miR-30b as a Regulator of tsO45-G Anterograde Trafficking DISSERTATION submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Sanchari Roy 2013 Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by MSc in Molecular Biology Sanchari Roy born in Kolkata, India Oral-examination: 30.01.2014 Analysis of miR-30b as a Regulator of tsO45-G Anterograde Trafficking Referees: Prof. Dr. Walter Nickel Dr. Vytaute Starkuviene-Erfle To my Dear Parents ACKNOWLEDGEMENT First of all, I would like to express my sincere gratitude to my supervisor Dr. Vytaute Starkuviene-Erfle for giving me the opportunity to join her research group ‘Screening of Cellular Networks’, BioQuant, University of Heidelberg and successfully carry out my PhD work. Furthermore, I would like to thank for her guidance throughout four years of my project. I would like to thank Prof. Dr. Walter Nickel for being the first supervisor of my PhD studies and useful discussion during TAC meetings. I would also like to thank Prof. Dr. Stefan Wiemann and Prof. Dr. Ursula Kummer for agreeing to be the examiners for my doctoral thesis. I would sincerely like to thank Dr. Vladimir Kuryshev for sharing his exceptional expertise in bioinformatics with me and providing me confidence and motivation during the tenure of the project. I want to thank my lab colleagues Dr. Andrius Serva and Susanne Reusing for creating a nice and pleasant atmosphere in the laboratory. I am also thankful to Jürgen Beneke, Nina Beil and Dr. Holger Erfle for their assistance and fun-filled time in the laboratory. I am indebted to my friends specially Devanjali, Sumit, Sahil and Mrinal for the wonderful time spent together having lunch and ‘chai’ as well as providing me unconditional support during ‘ups and ‘downs’ of my PhD time. I would like to take the opportunity to thank my parents for providing me the opportunity to study far away from home, giving me everything I need, providing me immense mental strength, for their continuous support and care. Last but not the least, my biggest thanks to my nephew Ron and my lovely sister with whom every time spent was joyful. 1 TABLE OF CONTENTS ABBREVIATIONS 5 FIGURE INDEX 7 TABLE INDEX 8 SUMMARY 9 ZUSAMMENFASSUNG 10 1. INTRODUCTION 12 1.1 Principles of membrane trafficking in mammalian cells 12 1.2 ER exit 13 1.3 Intra-Golgi trafficking 15 1.3.1 Post-Golgi transport 16 1.4 Endocytosis 17 1.5 Vesicular Stomatitis Virus Glycoprotein 19 1.6 Challenging the central dogma: Non-coding RNA 22 1.7 A novel class of regulators: miRNAs 22 1.7.1 miRNA biogenesis 23 1.7.2 miRNA: mRNA interaction 25 1.7.3 miR-30 family 27 1.7.4 miRNAs in membrane trafficking 27 1.8 Long non-coding RNA 28 1.9 Aims 30 2. MATERIALS AND METHODS 32 2.1 Materials 32 2.1.1 Instruments 32 2.1.2 Disposables 33 2.1.3 Chemicals and reagents 34 2.1.4 Kits 35 2.1.5 Size Markers and loading buffer 35 2.1.6 Cell lines 36 2.1.7 Enzyme 36 2.1.8 Bacteria 36 2.1.9 Media and solutions 37 2 2.1.10 Buffers 37 2.1.11 Antibodies 39 2.1.12 miRNAs 39 2.1.13 siRNAs 39 2.1.14 Oligoucleotides 41 2.1.15 Plasmids 46 2.2 Methods 46 2.2.1 Cell culture 47 2.2.2 Transfection of cells 47 2.2.3 Preparation of protein lysates 47 2.2.4 Western Blot 47 2.2.5 Blot and Development 48 2.2.6 qRT-PCR for mRNAs 48 2.2.7 Cloning of 3’UTRs for luciferase assay 49 2.2.8 Luciferase reporter assay 51 2.2.9 VSV-G (tsO45-G) trafficking assay 52 2.2.10 Golgi complex integrity assay 52 2.2.11 Transferrin assay 53 2.2.12 Computational miRNA target prediction 53 2.2.13 Pseudogene data set selection 53 2.2.14 Primer design and evaluation of transcription evidence 54 2.2.15 Microscopy 54 2.2.16 Statistical analysis 55 3. RESULTS 57 3.1 Large-scale Anti-miR screen 57 3.1.1 Additional phenotype: Golgi complex integrity 59 3.2 Choice of miR-30b 60 3.3 Selection of putative targets 62 3.4 Validation of targets 64 3.4.1 tsO45-G assay on 18 putative targets 64 3.4.2 Dual-Luciferase reporter assay of miR-30b and its targets 66 3.4.3 Over-expression of miR-30b decreases RNA and protein level of 3 targets 67 3.4.4 Mutagenesis of miR-30b binding site 69 3.4.5 Rescue effects of Anti-miR-30b 70 3.5 Post-Golgi inhibition of tsO45-G transport 71 3 3.6 Over-expression of miR-30b inhibits transferrin recycling 73 3.7 Morphology of the Golgi complex 74 3.8 Bioinformatic search of retroposed transcripts of RAB GTPases 76 3.8.1 Transcriptional and experimental evidence 76 3.8.2 Initial characterization of RAB42p 77 4. DISCUSSION 80 4.1 Large-scale assay to analyze membrane trafficking 80 4.2 miR-30b and membrane trafficking 81 4.3 Retroposed transcript: a new complexity in transcriptome 84 5. REFERENCES 86 6. APPENDIX 103 Appendix Table 6.1 103 Appendix Table 6.2 111 Appendix Table 6.3 112 Appendix Table 6.4 121 Appendix Table 6.5 122 Appendix Table 6.6 124 4 ABBREVIATIONS Ago Argonaute ANC Anti-miRNA negative control Anti-miR Synthetic single-stranded RNA molecule to inhibit miRNA AllStars siRNA negative control AP Adaptor protein AP3S1 Adaptor protein 3 sigma subbunit ARF ADP-ribosylation factor ARL ARF-like CDS Coding sequence CFP Cyan fluorescent protein CI-M6PR Cation-independent Mannose-6 phosphate receptor ConA Concanavalin A COPI Coat protein complex I COPII Coat protein complex II EE Early endosome EEA1 Early endosome antigen 1 ER Endoplasmic Reticulum ERC Endosomal recycling compartment ERES ER exit site ERGIC ER-Golgi intermediate compartment GOLGA1 Golgin-97 GTP Guanosine triphosphate LE Late endosome lncRNA Long non-coding RNA miRNA MicroRNAs miRISC MiRNA-induced silencing complex miRLC MiRISC loading complex ncRNA Non-coding RNA PGC Post-Golgi carriers PM Plasma membrane PNC Pre-miR negative control Pre-miRNA Precursor miRNA Pri-miRNA Primary miRNA transcript 5 qRT-PCR Quantitative Real-time PCR RAB Ras-related GTP binding protein RNAi RNA interference rER Rough ER SCAMP1 Secretory carrier membrane protein 1 SD Standard deviation SEM Standard error mean SNARE Soluble NSF attachment protein receptor Tf Transferrin TfR Transferrin receptor TGN trans -Golgi network tsO45-G Temperature-sensitive VSV (Orsay strain) glycoprotein UTR Untranslated region VSV-G Vesicular Stomatitis virus glycoprotein YFP Yellow fluorescent protein 6 FIGURE INDEX Figure 1.1 Overview of the biosynthetic and endocytic pathway Figure 1.2 Bidirectional transport between ER and Golgi cisternae Figure 1.3 Localization of Golgins across the Golgi complex Figure 1.4 Endocytic recycling pathways Figure 1.5 Primary structure of VSV-G protein Figure 1.6 Detection of fluorescently labelled tsO45-G anterograde transport Figure 1.7 MicroRNA biogenesis pathway Figure 1.8 Origin of processed pseudogene and retrogenes Figure 2.1 psiCHECK TM -2 vector map Figure 3.1 Microscopy-based tsO45-G screen on miRNAs Figure 3.2 Effectors of tsO45-G transport with Anti-miR hits Figure 3.3 cis -Golgi phenotype with down-regulation of miRNA Figure 3.4 Test of Pre-miRs and Anti-miRs Figure 3.5 Effect of miR-30b on the transport of tsO45-G Figure 3.6 Venn diagram showing common predicted targets of miR-30b Figure 3.7 Effect on tsO45-G secretion with down-regulation of predicted targets of miR-30b Figure 3.8 Four genes are targeted by miR-30b Figure 3.9 Expression of four targets on RNA level Figure 3.10 Expression of four targets on protein level Figure 3.11 Effect on protein amount with varying concentrations of miR-30b Figure 3.12 AP3S1 and GOLGA1 are directly targeted by miR-30b Figure 3.13 Rescue of tsO45-G transport inhibition of miR-30b by combinatorial knockdown of 3 targets Figure 3.14 Time-course transport of tsO45-G Figure 3.15 Post-Golgi transport of tsO45-G Figure 3.16 Over-expression of miR-30b inhibits transferrin recycling Figure 3.17 Effect on Golgi morphology with down-regulation of AP3S1, GOLGA1 and SCAMP1 Figure 3.18 Expression of retroposed transcripts RAB42p and RAB5Cp Figure 3.19 Effect on tsO45-G secretion with down-regulation of RAB42p, RAB5Cp and RAB28p Figure 3.20 qRT-PCR of RAB42 and RAB42p Figure 4.1 Network analysis between AP3S1, GOLGA1 and SCAMP1 7 TABLE INDEX Table 2.1 List of siRNAs with respective sequences Table 2.2 List of siRNAs for pseudogenes and parent genes Table 2.3 List of PCR primers for the cloning miRNA binding site and 3’UTRs in luciferase vector Table 2.4 List of primers of validated targets for qRT-PCR Table 2.5 List of PCR primers for pseudogenes Table 2.6 Protein trafficking related pseudogenes and their classes Table 2.7 List of excitation and emission filters Table 3.1 List of Golgi hits transfected with Anti-miRs Table 3.2 Overlap of the targets with other prediction programs Table 3.3 Expression of parent and retroposed transcript of RAB GTPases in different cell lines 8 SUMMARY MicroRNAs (miRNAs), small-sized ~22 nucleotide noncoding RNA (ncRNA) regulate diverse biological processes, mainly developmental timing and tumorigenesis.