THE EXOSOME AND HUMAN RIBOSOME BIOGENESIS Katherine Sloan B.Sc. (Hons) Biochemistry, University of Manchester Submitted for Doctor of Philosophy Degree January 2012 Faculty of Medical Sciences University of Newcastle upon Tyne Abstract Exoribonucleases have many important functions in the cell including RNA processing, turnover and quality control. One of the key 3’-5’ exonucleases is the exosome, a multiprotein complex that has been extensively characterised in yeast. Many substrates that undergo maturation and/or degradation involving the yeast exosome have been identified and these include tRNAs, mRNAs, snRNAs, snoRNAs and rRNAs. By comparison, the human exosome is poorly understood and it is not clear whether functions of the yeast exosome are conserved in higher eukaryotes. We show that the human exosome has degradation functions including the turnover, but not the processing, of snoRNAs and the recycling of excised pre-rRNA fragments. We and others have shown that the human exosome also participates in pre-rRNA processing to form the mature 3’ end of 5.8S rRNA. Here we identify a novel role for the exosome in the processing of the pre-rRNA internal transcribed spacer 1 (ITS1). The small (18S) and large (5.8S and 28S) subunit rRNAs are co-transcribed as a single precursor. Processing of ITS1 is a key step in ribosome biogenesis as it separates 18S from the large subunit rRNAs and in higher eukaryotes it involves an additional processing step compared to yeast. We define alternative ITS1 processing pathways in human cells. In the major pathway, following an endonucleolytic cleavage to separate the small and large subunit rRNAs, the exosome, which is not involved in ITS1 processing in yeast, processes to within 25 nucleotides of the 3’ end of 18S. Our data highlight significant differences between the nucleases involved in ITS1 processing in yeast and humans. However, it appears that the roles of several yeast biogenesis factors are conserved in higher eukaryotes. Further, we have investigated mechanisms by which exonucleolytic processing of ITS1 may be regulated and suggest how this could be coordinated with the final maturation steps of the pre-40S complex. II Declaration and acknowledgements I certify that this thesis contains my own work, except where acknowledged, and that no part of this material has been previously submitted for a degree or any other qualification at this or any other University. I would like to thank my supervisor, Dr Nick Watkins, for his support, advice and encouragement throughout this project. My thanks also go to all the members of the Watkins lab, present and past, Dr Rob van Nues, Dr Kenneth McKeegan, Dr Amy Turner, Dr Andrew Knox, David Colvin, Dr Charles Debieux and Dr Hannah Richardson, for their valuable contributions. I am grateful to Dr Jeremy Brown for helpful discussions about this work. I would thank Professor David Tollervey (University of Edinburgh), Professor Ger Pruijn (Radbound University, Nijmegen) and Dr Henning Urlab (Max-Planck Institute, Gottingen) who collaborated with us on aspects of this research. I particularly wish to acknowledge the Faculty of Medical Sciences and the Institute for Cell and Molecular Biosciences for their support, which enabled me to undertake this PhD. The BBSRC and Wellcome Trust funded this work. III Table of Contents Abstract ....................................................................................................................... II Declaration and acknowledgements ........................................................................ III Index of Figures .......................................................................................................... X Index of Tables......................................................................................................... XIII Abbreviations and Acronyms ................................................................................ XIV Chapter One: Introduction 1.1 Ribosomes .............................................................................................................. 1 1.2 rRNA organisation, transcription and processing .................................................... 2 1.2.1 Pre-rRNA processing in Saccharomyces cerevisiae ..................................... 3 1.2.2 pre-rRNA processing in Xenopus laevis ........................................................ 5 1.2.3 Pre-rRNA processing in mammals ................................................................ 6 1.2.4 ITS1 processing ............................................................................................ 8 1.3 rRNA modifications and snoRNPs .......................................................................... 8 1.3.1 Box H/ACA snoRNPs .................................................................................. 10 1.3.2 Box C/D snoRNP structure ......................................................................... 10 1.3.3 snoRNA maturation ..................................................................................... 11 1.3.4 snoRNP biogenesis .................................................................................... 12 1.3.5 snoRNPs in pre-rRNA processing ............................................................... 13 1.4 Assembly of ribosomal subunits ............................................................................ 14 1.4.1 Formation of the 90S particle and the SSU processome ............................. 15 1.4.2 RRP5 .......................................................................................................... 18 1.4.3 A3-cluster proteins ....................................................................................... 18 1.4.4 RBM28 (Nop4) ............................................................................................ 21 1.5 Ribosomal proteins ............................................................................................... 22 1.6 Nuclear export and cytoplasmic maturation .......................................................... 23 1.7 Late steps in pre-40S maturation .......................................................................... 25 1.7.1 Structural reorganisation of pre-40S complexes .......................................... 26 1.7.2 ENP1 .......................................................................................................... 26 1.7.3 NOB1 and PNO1 ........................................................................................ 27 1.7.4 DIM1 ........................................................................................................... 27 1.7.5 PRP43 ........................................................................................................ 28 1.7.6 RIO2 ........................................................................................................... 29 1.8 Quality control of ribosome biogenesis.................................................................. 30 IV 1.9 Endonucleases in pre rRNA processing ................................................................ 30 1.9.1 NOB1 .......................................................................................................... 31 1.9.2 RNase MRP ................................................................................................ 32 1.9.3 RCL1 .......................................................................................................... 34 1.9.4 UTP24 and UTP23...................................................................................... 35 1.9.5 RNT1 .......................................................................................................... 35 1.10 Exonucleases ..................................................................................................... 36 1.10.1 5’-3’ exonucleases .................................................................................... 36 1.10.2 REX proteins ............................................................................................. 38 1.10.3 The exosome ............................................................................................ 39 1.10.3.1 Core exosome structure ..................................................................... 41 1.10.3.2 DIS3/DIS3L (Rrp44) ........................................................................... 42 1.10.3.3 RRP6 ................................................................................................. 44 1.10.3.4 Cofactors ............................................................................................ 45 1.11 Aims and Objectives ........................................................................................... 50 Chapter Two: Materials and methods 2.1 PCR and cloning ................................................................................................... 52 2.1.1. PCR ........................................................................................................... 52 2.1.2 Agarose gel electrophoresis ........................................................................ 52 2.1.3 DNA extraction from agarose gels and purification...................................... 53 2.1.4 Restriction digest ........................................................................................ 53 2.1.5 Ligation reactions ........................................................................................ 53 2.1.6 Transformation of chemically competent E. coli .......................................... 53 2.1.7 DNA Sequencing .......................................................................................