National Institute for Medical Research Division of Molecular Structure The Role of Clp1 and Pcf11 in Transcription and pre-mRNA 3’-end Processing A thesis submitted by Joseph James Hedden In partial fulfilment of the requirements of University College London for the degree of Doctor of Philosophy in Structural and Molecular Biology August 2012 1 Declaration I, Joseph James Hedden, declare that the work presented in this thesis is my own. This work was carried out in the laboratory of Dr. Ian Taylor in the Division of Molecular Structure at the MRC National Institute for Medical Research. Where information has been derived from other sources, I confirm this has been indicated in the text. 2 Acknowledgments I would like to begin by thanking Dr. Ian Taylor for giving me the opportunity to work for my PhD in his lab. He found the right balance of showing me how to do things properly whilst letting me take the project in my own direction, which has been most beneficial to me and for which I am truly grateful. He was always on hand to offer help and advice, despite the many other scientists queued up outside his office door looking for answers. His colourful language and ‘subtle’ humour also brought homely qualities that made me feel I had never left my local pub, and indeed, it will be difficult to leave… I owe so much to many people along the way. I would like to thank Dr. Dave Goldstone for his tuition in all things protein, the many snooker matches and for being a good friend during my time at the NIMR. I would like to thank Valerie Ennis-Adeniran for also being a great teacher in the lab and for bringing laughter, pilfered lab equipment and hot chilli sauce, but not for supporting a certain second-rate North London football club. I am indebted to Dr. Marco Geymonat, who took me under his wing and taught me enough about yeast genetics to allow me to build the second half of my project. Without Marco the project would not have been possible. I am also very thankful for the help and guidance provided by Dr. Simon Pennel in radioactive matters, and for the critical reading of this thesis. To all of the members of the Taylor lab (past and present), you are wonderful people. Your support, encouragement and thirst for banter know no limits. To Laura, thank you for at times being a metaphorical punching bag and I am sorry for the constant stream of Scots-related abuse. However, I think we can all agree you give as good as you get. To Laurence, thank you for always bringing a sociable atmosphere, for getting me hooked on tasty ale, but not so much for accelerating the decline in the health of my liver. Finally I would like to thank my friends and family, who have been an essential and unwavering source of love and support throughout. 3 Abstract Eukaryotic transcripts require a number of complex cotranscriptional modifications and processing events before translation to protein. Clp1 and Pcf11 are subunits of cleavage factor IA (CFIA), an essential component of the Saccharomyces cerevisiae pre-mRNA 3’-end processing machinery. The crystal structure of a Clp1-Pcf11 complex was determined previously and revealed the binding of ATP to a highly-conserved P-loop motif and a tight Pcf11-Clp1 interaction facilitated by a number of highly-conserved Pcf11 residues. Nonetheless, the biological function of both Clp1-ATP binding and the Pcf11-Clp1 interaction was not well understood. The work in this thesis combines an in vitro and in vivo investigation of the Clp-ATP and Clp-Pcf11 interactions in an effort to understand the function of these factors in transcription and pre-mRNA 3’-end processing. It is demonstrated that the interaction of ATP and Pcf11 with Clp1 are linked events: Loss of Clp1-ATP binding results in the abrogation of the Pcf11-Clp1 interaction and leads to Clp1 instability in vitro, and similarly, mutations that directly uncouple the Pcf11-Clp1 interaction also disrupt Clp1-ATP binding and cause Clp1 instability in vitro. An in vivo mutational analysis in S. cerevisiae revealed that both Clp1-ATP binding and the Pcf11-Clp1 interaction are essential for yeast survival. Further cell and immunoprecipitation studies demonstrated that one essential function of Clp1 is as a chaperone of Pcf11, and RT-qPCR analysis of mRNA from a sample set of yeast genes points to a role for these proteins in transcription and transcription termination rather than in poly(A) site selection. 4 Table of contents Declaration……………………………………………………………. 2 Acknowledgments……………………………………………………. 3 Abstract……………………………………………………………….. 4 Table of contents……………………………………………………... 5 List of figures…………………………………………………………. 10 List of tables…………………………………………………………... 12 List of abbreviations…………………………………………………. 13 Saccharomyces cerevisiae nomenclature……………………………. 14 1. Introduction……………………………………………………….. 15 1.1. Eukaryotic pre-mRNA processing………………………………………. 15 1.1.1. Pre-mRNA 5’-end capping…………………………………………… 15 1.1.2. Pre-mRNA splicing…………………………………………………… 17 1.1.3. Pre-mRNA 3’-end processing………………………………………… 18 1.1.4. mRNA export…………………………………………………………. 18 1.1.5. Timing of processing events………………………………………….. 19 1.2. Pre-mRNA 3’-end processing……………………………………………. 20 1.2.1. Sequence elements for mRNA 3’-end formation……………………... 20 1.2.2. Protein factors for pre-mRNA 3’-end processing…………………….. 22 1.3. Cleavage factor I (CFI)…………………………………………………… 23 1.3.1. Rna14 (CFIA)………………………………………………………… 23 1.3.2. Rna15 (CFIA)………………………………………………………… 25 1.3.3. Pcf11 (CFIA)…………………………………………………………. 29 1.3.4. Clp1 (CFIA)…………………………………………………………... 33 1.3.5. Hrp1 (CFIB)…………………………………………………………... 37 1.4. Cleavage and Polyadenylation Factor (CPF)…………………………… 40 1.4.1. The role of CPF in cleavage………………………………………….. 42 1.4.2. The role of CPF in polyadenylation…………………………………... 44 1.5. A model for pre-mRNA 3’-end processing in S. cerevisiae…………….. 47 1.6. Links between 3’-end processing and other processing events………… 49 1.6.1. 3’-end processing and 5’-end capping………………………………... 49 1.6.2. 3’-end processing and splicing………………………………………... 50 1.6.3. 3’-end processing and export…………………………………………. 50 5 1.7. Links between 3’-end processing and transcription………………………. 52 1.7.1. 3’-end processing and transcription termination……………………… 53 1.7.2. 3’-end processing and transcription initiation………………………… 38 1.8. Mammalian homologues of 3’-end processing factors…………………. 59 1.9. Research objectives……………………………………………………….. 62 2. Materials and Methods……………………………………………. 64 2.1. Bioinformatics…………………………………………………………….. 64 2.1.1. DNA and protein information………………………………………… 64 2.1.2. Multiple sequence alignments………………………………………… 64 2.2. Molecular biology techniques……………………………………………. 64 2.2.1. Bacterial strains………………………………………......................... 64 2.2.2. Yeast strains………………………………………………………....... 65 2.2.3. Plasmid construction………………………………………………….. 65 2.2.4. DNA manipulation and analysis……………………………………… 68 2.2.5. Polymerase chain reaction……...…………………………………….. 69 2.2.6. Restriction digests…………………………………………………….. 69 2.2.7. Ligation reactions……………………………………………………... 70 2.2.8. Transformations………………………………………………………. 71 2.2.9 Site-directed mutagenesis……………………………………………... 71 2.3. Protein expression and purification……………………………………... 74 2.3.1. Protein expression…………………………………………………….. 74 2.3.2. Bacterial cell lysis…………………………………………………….. 75 2.3.3. Purification of glutathione S-transferase fusion proteins……………... 75 2.3.4. Nickel affinity purification…………………………………………… 76 2.3.5. Size-exclusion chromatography……………………………………… 77 2.3.6. SDS-PAGE………………………………………………………........ 77 2.3.7. Protein concentration, storage and dialysis…………………………… 78 2.3.8. Determination of protein concentration………………………………. 78 2.4. Multiangle laser light scattering (MALLS)……………………………... 79 2.5. Reversed-phase high-performance liquid chromatography…………… 82 2.5.1. Reversed-phase nucleotide assay……………………………………... 82 2.5.2. Reversed-phase complex composition assay…………………………. 83 2.5.3. Integration of UV absorbance peaks and stoichiometry calculation…. 84 2.6. Incorporation of radiolabelled ATP and scintillation counting……….. 84 2.7. Circular dichroism spectroscopy………………………………………… 85 2.8. Yeast methods……………………………………………………………... 86 2.8.1. Yeast transformation………………………………………………….. 86 2.8.2. Construction of a haploid pcf11 yeast strain………………………... 87 2.8.3. 5-FOA Plasmid shuffle……………………………………………….. 88 2.8.4. Growth rate assays……………………………………………………. 90 6 2.8.5. Western blotting………………………………………………………. 91 2.8.6. Coimmunoprecipitation experiment………………………………….. 92 2.8.7. Determination of protein half-life…………………………………….. 93 2.8.8. Preparation of total RNA……………………………………………... 93 2.9. Poly(A) tail-length assay………………………………………………….. 94 2.9.1. Poly(A) tail 3’-end labelling………………………………………….. 94 2.9.2. Poly(A) tail digestion and purification……………………………….. 95 2.9.3. Visualisation of poly(A) tails…………………………………………. 96 2.9.4. Modifications to poly(A) tail-length assay protocols………………… 96 2.9.5. DNA ladder preparation……………………………………………… 97 2.10. Quantitative real-time PCR…………………………………………….. 97 2.10.1. Reaction setup……………………………………………………….. 98 2.10.2. ACT1 Poly(A) site selection assay…………………………………... 100 2.10.3. Analysis of TDH2, ACT1, ADH1, CYC1 and YPT1 transcription…... 102 2.10.4. ACT1 Transcription readthrough assay……………………………… 102 3. in vitro analysis of recombinant Clp1-ATP binding mutants…... 104 3.1. Introduction and overview……………………………………………….. 104 3.2. Mutation of the Clp1-ATP binding pocket affects protein stability…… 105 3.2.1. Design of Clp1 mutants………………………………………………. 105 3.2.2. Size-exclusion