Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription Luitpold Maximilian Reuter aus Regensburg, Deutschland 2015 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Frau Prof. Dr. Katja Sträßer betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, 21.09.2015 L. Maximilian Reuter Dissertation eingereicht am: 21.09.2015 1. Gutachterin / 1. Gutachter: Prof. Dr. Katja Sträßer 2. Gutachterin / 2. Gutachter: Prof. Dr. Klaus Förstemann Mündliche Prüfung am: 03.11.2015 ‘The road not taken’ TWO roads diverged in a yellow wood, And sorry I could not travel both And be one traveler, long I stood And looked down one as far as I could To where it bent in the undergrowth; THEN took the other, as just as fair, And having perhaps the better claim, Because it was grassy and wanted wear; Though as for that the passing there Had worn them really about the same, AND both that morning equally lay In leaves no step had trodden black. Oh, I kept the first for another day! Yet knowing how way leads on to way, I doubted if I should ever come back. I shall be telling this with a sigh Somewhere ages and ages hence: Two roads diverged in a wood, and I— I took the one less traveled by, And that has made all the difference. Robert Frost, 1916 Für meine Familie Summary Summary Gene expression and hence the fine-tuned and well-orchestrated transcription of genes is a fundamental process in living cells. Beside RNA polymerase II (RNAPII), which transcribes protein-coding genes and non-coding RNAs, RNA polymerase III (RNAPIII) synthesizes small RNAs, the most prominent being tRNAs. These RNAs are highly structured and have a central function in translation and cell metabolism. Although the basic mechanism of RNAPIII transcription is well understood, many molecular details of this transcription system remain elusive. In the present study, we identified - by a genome-wide approach - that Nab2, a poly(A)- binding protein important for correct poly(A) tail length of mRNAs and nuclear mRNA export, is present at all RNAPIII-transcribed genes in the model organism Saccharomyces cerevisiae. Remarkably, this occupancy is specific for RNAPIII and independent of RNAPII. Analysis of the occupancy of Nab2 at RNAPIII-transcribed genes furthermore unveiled that it is dependent on active RNAPIII transcription. After generating a novel temperature-sensitive allele of NAB2, nab2-34, we could show that Nab2 is required for the occupancy of RNAPIII at its target genes. In addition, we found that Nab2 directly interacts with RNAPIII and its precursor transcripts, suggesting a function of Nab2 in RNAPIII transcription. Importantly, impairment of Nab2 function causes an RNAPIII transcription defect in vivo and in vitro that can be rescued by the addition of recombinant Nab2. Stimulating the transcriptional activity of a minimal in vitro transcription system in a dose-dependent manner demonstrated that the function of Nab2 in RNAPIII transcription is direct. Investigation of the molecular function of Nab2 in RNAPIII transcription revealed the involvement of the essential transcription initiation factor TFIIIB. Interestingly, the TFIIIB subunit Bdp1 and thus most likely the whole TFIIIB complex is less recruited to its target genes in our mutant NAB2 strain. Consistently, Brf1, another TFIIIB subunit, interacted with Nab2 in vivo. Having found that TFIIIC was not affected by nab2-34 nor did TFIIIC interact with Nab2, we studied how Nab2 specifically influences TFIIIB on RNAPIII genes. These experiments revealed that Nab2 increased the binding of TFIIIB to promoter DNA and is thus most likely required for efficient assembly and stability of the RNAPIII transcription initiation complex in S. cerevisiae. Taken together, we discovered that Nab2, an important mRNA biogenesis factor, is a novel player required for full RNAPIII transcription by stabilizing TFIIIB and RNAPIII on promoter DNA. I Publications Publications Substantial parts of this thesis have been already published in: Reuter, L.M., Meinel, D. M., and Sträßer, K., (2015), The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription, Genes & Development, 29 (14), 1565-1575 In addition I contributed to the following publication: Meier K., Mathieu E. L., Finkernagel F., Reuter L. M., Scharfe M., Doehlemann G., Jarek M., and Brehm A., (2012), LINT, a Novel dL(3)mbt-Containing Complex, Represses Malignant Brain Tumour Signature Genes, PLOS Genetics, 8 (5), e1002676 II Abbreviations Abbreviations Amino acids are abbreviated according to the standard single or three letter code. The nucleotides Adenine, Cytosine, Guanine, Thymine, and Uracil are abbreviated A, C, G, T, and U, respectively. Standard unit prefixes are used when needed. The abbreviations used throughout this study are listed below in an alphabetical order: α Anti EMSA Electromobility shift assay aa Amino acid ER Endoplasmic reticulum °C Celsius FACT Facilitates chromatin AID Auxin-inducible degron transcription APS Ammonium persulfate 6-FAM 6-carboxyfluorescein ARE AU-rich element Fig. Figure bp Base pair gDNA Genomic DNA C- Carboxy-terminal GFP Green fluorescent protein CBP Calmodulin binding GST Glutathione-S-transferase peptide h Hour cDNA CopyDNA HEK293 A human embryonic ChIP Chromatin kidney cell line Immunoprecipitation HeLa A human cervical cancer CPF Cleavage and derived cell line (Henrietta Polyadenylation factor Lacks) CRAC Crosslinking and analysis HEPES 4-(2-hydroxyethyl)-1- of cDNA piperazineethanesulfonic Da Dalton acid DAPI 4′,6-Diamidin-2- HMG High mobility group phenylindole hnRNP Heterogenous nuclear ddH2O Double-distilled water ribonucleoprotein DEPC Diethylpyrocarbonate Ig Immunoglobuline DMSO Dimethyl sulfoxide IPTG Isopropyl β-D-1- DNA Desoxyribonucleic acid thiogalactopyranoside dNTP Desoxynucleoside M Molar triphosphate MCS Multiple Cloning Site ds Double stranded min Minute DTT Dithiothreitol mRNA Messenger RNA EDTA Ethylene diamine tetra mRNP Messenger acetic acid ribonucleoprotein EM Electron microscopy ncRNA Non-coding RNA III Abbreviations N- Amino-terminal SANT Transcription regulation NLS Nuclear localization domain (Swi3, Ada2, N- sequence Cor and TFIIIB) nt Nucleotides SCF Skp1, Cullin, and F-box NTP Nucleoside triphosphate SD Standard devitation OD Optical density SRP Signal recognition particle ORF Open reading frame ss Single stranded PABP Poly(A)-binding protein SUMO Small ubiquitin-like PAF Polymerase associating modifier factor Tab. Table PAGE Polyacrylamide gel TAE Tris-acetate-EDTA electrophoresis TAP Tandem affinity PAR-CLIP Photoactivatable- purification Ribonucleoside-Enhanced TBE Tris-borate EDTA Crosslinking and TEMED Tetramethyl- Immunoprecipitation ethylenediamine PBS Phosphate buffered saline TEV Tobacco etch virus PCG Protein coding gene cleavage site PCR Polymerase chain reaction TF Transcription factor PDB Protein Data Bank tgm tRNA gene-mediated PEG Polyethylene glycol silencing pH Potentia hydrogenii THSC Thp1-Sac3-Sus1-Cdc31 poly(A) Poly adenosine TPR Tetratricopeptide repeat RBP RNA binding protein TREX Transcription and Export RNA Ribonucleic acid TRIS Tris(hydroxyl- RNAP DNA dependent RNA methyl)aminomethane polymerase tRNA Transfer RNA RNase Ribonuclease ts Temperature-sensitive RNP Ribonucleoprotein qPCR Quantitative PCR rpm Rounds per minute U Unit of enzyme activity, RGG Arginine-glycine-glycine conversion of 1 µmol repeats substrate min-1 RRM RNA recognition motif v/v Volume per volume RSC Remodel the Structure of w/v Weight per volume Chromatin wt Wild-type RT Room temperature Zn Zinc s Seconds Znf Zinc finger IV Table of contents Table of contents Summary……………………………………………………………………………………………….I Publications…………………………………………………………………………………………..II Abbreviations……………………………………………………………….……………………….III Table of contents…………………………………………………………………………………....V 1. Introduction ..................................................................................................................... 1 1.1 Gene expression ...................................................................................................... 1 1.2 mRNA biogenesis and export ................................................................................. 1 1.2.1 Three platforms provide the recruitment of mRNA binding proteins ................... 1 1.2.2 mRNA processing and mRNP formation ............................................................ 3 1.2.3 Nuclear export of mRNPs ................................................................................... 6 1.3 The poly(A)-binding protein Nab2 .......................................................................... 9 1.3.1 Regulation of poly(A) tails ................................................................................. 13 1.3.2 Nab2 mediates export of mature mRNPs ......................................................... 14 1.3.3 Nab2 is conserved from yeast to humans ........................................................ 14 1.4 RNA polymerase III transcribes tRNA and other ncRNA genes ........................ 15 1.4.1 The RNA polymerase III transcription apparatus .............................................. 15 1.4.2 RNAPIII promoter elements .............................................................................. 16 1.4.3 The RNAPIII transcription factor TFIIIA ...........................................................