Copyright by Yen-I Grace Chen 2007 The Dissertation Committee for Yen-I Grace Chen Certifies that this is the approved version of the following dissertation: Proteomic Analysis of the Pre-mRNA Splicing Machinery Utilizing Chromosomal Locus Epitope Tagging in Metazoans Committee: Scott Stevens, Supervisor Henry Bose Robert Krug Arlen Johnson David Hoffman Proteomic Analysis of the Pre-mRNA Splicing Machinery Utilizing Chromosomal Locus Epitope Tagging in Metazoans by Yen-I Grace Chen, M.D. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August, 2007 Dedication To my loving family Acknowledgements I offer sincere thanks to my advisor, Professor Scott Stevens for providing me the opportunity to join his laboratory. His enthusiasm and patience contributed to the success of my research. I also wish to acknowledge professors Robert Krug, Henry Bose, Arlen Johnson, and David Hoffman for serving as my dissertation committee and for their constructive comments and expertise during my dissertation work; professors Phil Tucker and Karen Artzt for helpful conversations, and William Kuziel for plasmids. I would especially thank Shanna Maika and Deb Surman for their persistence and painstaking work in generating our transgenic mice, breeding, and maintenance; Xin Yao and Chad McKee for technical assistance. I also wish to thank all my colleagues in the Stevens lab: Josh Combs, Adam Roth, Champ Gupton, Rea Lardelli, Jennifer Hennigan and former members Dannialle Clayton and Colin Chu, for helpful discussions and establishing a fun environment for research. I would also like to thank Roger Moore, Helen Ge, and Terry Lee for mass spectrometry expertise and Jimi Lynn Brandon for mouse tissue processing. My gratitude also extends to all members of Bose lab for chicken DT40 cell advice and tissue culture help and all members of Johnson lab for their suggestions and friendship over the years. v Lastly, I would like to thank my dear husband, Edward, for his encouragement and love during my graduate studies; my parents for their support and belief in my achievement and my sister, for sharing laughs and tears with me in Austin. vi Proteomic Analysis of the Pre-mRNA Splicing Machinery Utilizing Chromosomal Locus Epitope Tagging in Metazoans Publication No._____________ Yen-I Grace Chen, Ph.D. The University of Texas at Austin, 2007 Supervisor: Scott W. Stevens Epitope tagging in metazoans is an important tool for biochemical analyses and is generally accomplished by using trans-genes with in-frame epitope tags. However, protein levels from trans-genes are rarely representative of native levels. To overcome the shortcomings using trans-genes, epitope tags were introduced by homologous recombination technology, termed CLEP tagging (Ch romosomal Locus EP itope tagging), immediately upstream of the stop codon of targeted genes in chicken B cell line DT40 and mouse embryonic stem (ES) cells. I first demonstrated the feasibility and promise of this technique in DT40 cells by purifying low abundance polypeptides and factors loosely associated with the SmD3 protein, a core protein participating in pre-mRNA splicing and mRNA turnover, with a TAP (tandem affinity purification) tag. Glycerol gradient separation was performed to further characterize the SmD3-associated protein complexes from the 200S fractions, corresponding to the supraspliceosomes. The purification included all five spliceosomal snRNAs. Most known snRNP-associated vii proteins, 5’ end binding factors, 3’ end processing factors, mRNA export factors, hnRNPs, and other RNA binding proteins were identified from the protein components. Intriguingly, the purified supraspliceosomes also contained a number of structural proteins, nucleoporins, chromatin remodeling factors, and several novel proteins that were absent from splicing complexes assembled in vitro . I showed that the in vivo analyses provide a more comprehensive list of polypeptides associated with pre-mRNA splicing apparatus as well as those that coupled transcription to the pre-mRNA processing steps. With similar techniques, the TAP tag was inserted into the chromosomal locus of a pre-mRNA splicing factor component, mSART-1 in live mice. Surprisingly, a profound autoimmune response was induced in homozygous-modified mice, due likely to an inappropriate stimulation of the immune system. I believe these mice will serve as a valuable tool for the studies of mammalian autoimmune diseases, especially those resulting from the generation of autoantibodies against RNP components. viii Table of Contents List of Tables ....................................................................................................... xiv List of Figures....................................................................................................... xv List of Illustrations.............................................................................................. xvii Chapter 1: Introduction............................................................................................1 1.1 Gene expression in eukaryotes..................................................................1 1.1.1 Overview.......................................................................................1 1.1.2 The carboxyl-terminal domain (CTD) of RNA Pol II: an assembly platform for pre-mRNA processing factors ..................................1 1.1.3 Coupling between transcription and the pre-mRNA processing machinery......................................................................................4 1.1.3.1 5’ end capping...................................................................4 1.1.3.2 Recruitment of the spliceosome........................................4 1.1.3.3 3’ end formation................................................................5 1.1.3.4 Recruitment of mRNA export factors...............................5 1.2 Pre-mRNA splicing and spliceosome assembly .......................................6 1.2.1 Small nuclear RNAs (snRNA)......................................................6 1.2.1.1 The discovery of snRNAs.................................................6 1.2.1.2 The biogenesis of snRNAs................................................7 1.2.2 Small nuclear ribonucleoproteins (snRNP) ..................................8 1.2.2.1 The isolation of snRNPs ...................................................8 1.2.2.2 A link between snRNPs and splicing................................9 1.2.3 The splicing reaction.....................................................................9 1.2.3.1 Pre-mRNA splicing machinery, spliceosome...................9 1.2.3.2 Splicing signals ...............................................................10 1.2.3.3 The catalysis of splicing..................................................12 1.2.3.4 The spliceosome assembly cycle ....................................13 1.2.3.5 Structure and composition of snRNPs ............................16 1.2.3.6 ATP-dependent RNA helicases ......................................17 ix 1.2.4 Large nuclear ribonucleoprotein particle (lnRNP), the pre-mRNA processing machine.....................................................................18 1.3 Affinity tools for the purification of ribonucleoproteins in yeast...........19 1.3.1 Application of affinity chromatography for the purification of epitope-tagged proteins...............................................................19 1.3.1.1 Gene targeting and affinity chromatography in yeast.....20 1.3.1.2 Purification of protein complexes from metazoan cells .20 1.3.2 Purification of salt stable yeast snRNPs .....................................21 1.3.3 The discovery of penta-snRNP ...................................................22 1.4 Dissertation objectives............................................................................24 Chapter 2: Gene targeting in cultured vertebrate cells...........................................26 2.1 Background.............................................................................................26 2.1.1 A vertebrate cell line useful for gene targeting...........................26 2.1.2 A human cell line useful for gene targeting................................27 2.2 Materials and methods ............................................................................28 2.2.1 The design of targeting vector ....................................................28 2.2.2 DT40 cell culture and targeting vector transfection ...................31 2.2.2.1 Growth condition and media preparation .......................31 2.2.2.2 Cryopreservation of cells ................................................32 2.2.2.3 Recovery of frozen cultured cells ...................................32 2.2.3 DNA transfection into DT40 cells ..............................................32 2.2.4 Screening for tagged gene by RT-PCR analysis.........................33 2.2.5 Screening for tagged gene by western blot analysis ...................33 2.2.6 Extract preparation......................................................................34 2.2.7 TAP purification procedure ........................................................34 2.2.8 Mass spectrometry analysis ........................................................35 2.3 Results.....................................................................................................36 2.3.1 DT40 cells carrying