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Molecular Genetic Studies of the Prp8 Splicing Factor Andrew James

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Molecular Genetic Studies of the Prp8 Splicing Factor Andrew James Molecular Genetic Studies of the Prp8 Splicing Factor Andrew James Brown A Thesis Presented for the Degree of Doctor of Philosophy University of Edinburgh. September 1995 p.. - Declaration I declare that I alone have composed this thesis, and that the work I present is my own, except where stated otherwise. September 1995 11 Abstract Introns which interrupt transcripts from eukaryotic genes are removed in a splicing reaction. Introns are recognised by particles called snRNPs, which assemble into a spliceosome complex in which the reactions of splicing occur. RNA components of the snRNPs are present at the spliceosome active site, and splicing is believed to be catalysed, at least in part, by RNA. A factor which seems to have roles throughout spliceosome assembly and catalysis is the Prp8 protein of the budding yeast Saccharomyces cerevisiae. Prp8p is a component of the U5 snRNP important for entry of U5 and other snRNPs into the spliceosome, and is present in the active site(s) at the times when the splicing reactions occur. Genes encoding Prp8p have been isolated from yeast and other eukaryotes and are extraordinarily well conserved, consistent with the multiple roles of this factor. This thesis focuses on the only region of yeast Prp8p not known to be common to other eukaryotes: a repetitive acidic and proline-rich domain at the N-terminus. Removal of part or all of this domain inhibits function, but cells lacking this domain are viable if the truncated protein is overproduced. A reconstruction approach suggests that proline is the most important feature of this domain, and thus function may be analogous to proline-rich regions of other proteins which in general promote complex assembly. The phenotype of truncation mutants is consistent with this. Spliceosome components are present in the yeast nucleus at lower concentration than in other eukaryotes, suggesting a reason as to why this otherwise strongly conserved protein possesses the extra domain. This thesis also describes the analysis of several mutants of yeast PRP8 which have the highly unexpected phenotype of a block to cell cycle progression. The data indicate that these mutants also affect splicing. The growth defect of one of them (dbf3- 1) is suppressed by a cDNA copy of the TUB] gene. TUB] contains an intron and encodes a microtubule subunit (a-tubulin) functional in M-phase. Suppression separates the cell cycle and splicing defects, as the splicing defect is unaffected by suppression. These data strongly support the hypothesis that the cell cycle defect is a secondary consequence of a splicing defect, the link being a gene (TUB]) which functions in the cell cycle and which contains an intron. The cell cycle block is II' observed because the splicing defect is mild: except for the TUB1 intron, splicing in a dbJ3-1 cell is sufficient to maintain growth. iv Abbreviations Amp Ampicillin APS Ammonium persuiphate ARS Autonomous Replication Sequence ATP Adenosine 5-triphosphate A260 Absorbance at 260nm BCIP 5-Bromo-4-chloro-3-indolyl Phosphate bisacrylamide NN'-methylene-bisacrylamide bp Base pair BPS Branch Point Sequence BSA Bovine Serum Albumin Degrees celsius CEN Centromere cDNA Complementary DNA Ci Curie (2.2x1012 disintegrations/min) CTP Cytidine 5-triphosphate dATP Deoxyadenosine 5-triphosphate dCTP Deoxycytidine 5'-triphosphate DEPC Diethylpyrocarbonate dH20 Distilled water DMSO Dimethyl Suiphoxide DNA Deoxyribonucleic acid dNTP Deoxynucleoside triphosphate ds Double-stranded D'fl' Dithiothreitol EDTA Ethylenediaminetetraacetic acid EMBL European Molecular Biology Laboratory g Gram or Grammes Galactose D-galactose Glucose D-glucose hr Hour(s) HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid IPTG Isopropyl-f3-D-thiogalactopyranoside k Kilo- (1O) kb Kilobase kDa KiloDalton Litre(s) LB Luria-Bertani broth (medium) M Molar m Milli- (10-1 ) mg Milligram(mes) m3G 2,2,7-trimethyl guanosine mM Millimolar ruin Minute(s) ml Millilitre(s) MOPS 3-(N-morpholino)propanesulphonic acid mRNA Messenger RNA N Purine or pyrimidine NBT Nitroblue tetrazolium NP-40 Nonidet P-40 detergent NTP Nucleoside 5-triphosphate 0D600 Optical Density at 600nm OLB Oligo Labelling Buffer ORF Open Reading Frame SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis PAS Protein A-Sepharose PCR Polymerase Chain Reaction PEG Polyethylene glycol Pol Polymerase R Purine nucleotide RNA Ribonucleic acid RNase Ribonuclease RNasin Ribonuclease inhibitor rpm, Revolutions per minute rRNA Ribosomal RNA SDS Sodium dodecyl sulphate sec Second(s) snRNA SmailNuclear RNA snRNP Small Nuclear Ribonucleoprotein Particle SSC Saline sodium citrate buffer TAE Tris-acetate buffer TBE Tris-borate buffer TBS Tris-buffered saline TE Tris/EDTA TEMED N,N,N',N-tetramethyl ethylenediamine tRNA Transfer RNA Tris Tris(hydroxymethyl)aminomethane (I U6) p. Micro 99 Microgram(mes) PI Microlitre(s) jilvI Micromolar UV Ultraviolet light v/v Volume per unit volume wlv Weight per unit volume X-Gal 5-Bromo-4-chloro-3-indolyl-3-D-ga1actoside Y Pyrimidine nucleotide YCp Yeast Centromeric Plasmid YEp Yeast Episomal Plasmid vi Amino Acids (single letter code) A Alanine M Methionine C Cysteine N Asparagine D Aspartate P Proline E Glutamate Q Glutamine F Phenylalanine R Arginine G Glycine S Serine H Histidine T Threonine I Isoleucine V Valine K Lysine W Tryptophan L Leucine Y Tyrosine A Note on Nomenclature Almost all yeast genes are designated with three letters and a number. In this thesis yeast genes are described in italics according to convention, with upper-case for dominant alleles (mostly the wild-type) and lower-case for recessive alleles. Alleles are numbered (prp8-1, prp8-2, etc). Names of deletion mutations incorporate the character A. Names for gene products are not italicised, and where there is no common name (eg tubulin, U5 snRNA) the gene product is named after the gene, thus PRPS encodes the Prp8p (p for protein). The adopted system of numbering within the PRP8 gene assigns A of the ATG start codon as position 1. Vii CONTENTS Declaration ii Abstract iii Abbreviations v Note on Nomenclature vii Contents viii CHAPTER 1: INTRODUCTION 1.1 The Discovery of Introns 1 1.2 The Mechanism of Splicing 1 1.3 The Origin of Introns 3 1.4 The Study of Splicing 5 1.5 The Distribution of Introns 5 1.6 SnRNP5 and the Spliceosome: An Overview 7 1.7 The Cis-acting Intron Determinants 7 1.7.1 The 5' Splice Site 8 1.7.2 The Branchpoint 8 1.7.3 The 3' Splice site and Polypyrimidine Tract 9 1.8 SnRNAs and SnRNP5 10 1.9 Spliceosome Assembly 11 1.9.1 Association of the Ui snRNP 12 1.9.2 Commitment and Alternative Splicing 14 1.9.3 The Association of the U2 snRNP 16 1.9.4 Association of the U4/U6.U5 tri-snRNP Particle 18 1.10 Conformational Change in the Spliceosome 21 1.10.1 Dissociation of U4 and U6 snRNAs 21 1.10.2 Interactions between US snRNA and the spliceosome 22 1.10.3 Interaction of U6 and U2 snRNA 24 1.10.4 Interaction of U6 snRNA with the substrate 24 1.10.5 Tertiary Interactions 25 1.10.6 Catalysis of RNA confonnational change: the DEAD-box putative RNA helicases 25 1.10.7 The Active Sites 27 1.11 PRP8 28 viii 1.11.1 The Identification of PRPS 28 1.11.2 The Role of PRP8 29 1.1 1.3 The N-terminal Region of PRP8 31 1.12 Proline-rich Regions in Other Proteins 32 1.12.1 Other proline-rich Splicing Factors 34 1.13 This Thesis 35 1.14 An Overview of the Yeast Cell Cycle 37 1.14.1 The Cyclin Cascade 37 1.14.2 Transcription and the Cell Cycle 39 1.14.3 Replication Licensing Factors 40 CHAFFER 2: MATERIALS AND METHODS 2.1 MATERIALS 42 2.1.1 Suppliers of Laboratory Reagents 42 2.1.2 Growth Media 42 2.1.2.1 E.coli Media 42 2.1.2.2 Yeast Media 43 2.1.2.3 Use of Microtubule-directed Drugs 43 2.1.3 Bacterial Strains 43 2.1.4 Yeast Strains 44 2.1.5 Plasmid Vectors and Constructs 46 2.1.6 Synthetic Oligonucleotides 49 2.1.7 Antisera and Antibodies 52 2.1.8 Size Markers 52 2.1.9 Buffers 53 2.2 GENERAL METHODS 53 2.2.1 General Guidelines 53 2.2.2 Sterilisation Procedures 53 2.2.3 Deionisation of Solutions 54 2.2.4 Photography 54 2.2.5 Autoradiography 54 2.2.6 Phosphorimaging 54 2.3 MICROBIOLOGICAL METHODS 55 2.3.1 Propagation and Storage of E.coli 55 2.3.2 Transformation of E.coli 55 2.3.3 Propagation and Storage of Yeast 56 2.3.4 Sporulation and Spore Recovery 56 2.3.5 Transformation of Yeast 56 2.4 NUCLEIC ACID METHODS 57 ix 2.4.1 Plasmid DNA Preparations 58 2.4.1.1 Minipreps 58 2.4.1.2 Midipreps 58 2.4.1.3 Preparation of eDNA Library 60 2.4.1.4 Maxipreps 60 2.4.2 Sequencing 61 2.4.2.1 Preparation of Phagemid DNA 61 2.4.2.2 Production of Single-stranded DNA by Primer- Extension with a Thermostable Polymerase 62 2.4.2.3 Preparation of DNA for Direct, Double- stranded Sequencing 63 2.4.3 Extraction of Genomic DNA from Yeast 63 2.4.3.1 Plasmid Recovery from Yeast to E.coli 64 2.4.4 Extraction of RNA from Yeast 65 2.4.5 Labelling Restriction Fragments by Random Priming 66 2.4.6 5-end Labelling of Oligodeoxynucleotides 67 2.4.7 Denaturing Agarose Gel Electrophoresis of RNA 67 2.4.8 Denaturing Polyacrylamide Gel Electrophoresis of Nucleic Acids 68 2.4.9 Capillary Blotting of RNA and DNA 68 2.4.10 Electroblotting of RNA from Polyacrylamide Gels 69 2.4.11 Hybridisation of Southern and Northern Blots with Random-primed Probes 69 2.4.12 Hybridisation of Northern Blots with Oligodeoxynucleotide Probes 70 2.4.13 The Polymerase Chain Reaction 70 2.4.14 In vitro Transcription 71 2.4.15 Purification of Large Fragments for Cloning 72 2.5 PROTEIN AND IMMUNOLOGICAL METHODS 73 2.5.1 Quantitation of Proteins 73 2.5.2 SDS-Polyacrylamide Gel Electrophoresis of
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