Structural and functional studies of the spliceosomal RNP remodeling enzyme Brr2 Dissertation for the award of the degree "Doctor of Philosophy" Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen submitted by Karine Santos from São José do Rio Preto, Brazil Göttingen, 2012 Thesis committee Prof. Dr. Markus C. Wahl (reviewer) Freie University Berlin Institute for Chemistry and Biochemistry Department of Structural Biochemistry Berlin, Germany Prof. Dr. Ralf Ficner (reviewer) Georg August University Göttingen Institute for Microbiology and Genetics Department of Molecular Structural Biology Göttingen, Germany Prof. Dr. Detlef Doenecke Georg August University Göttingen Institute for Biochemistry and Molecular Cell Biology Department of Molecular Biology Göttingen, Germany Prof. Dr. Dirk Fasshauer University of Lausanne Faculty of Biology and Medicine Department of Cellular Biology and Morphology Lausanne, Switzerland Prof. Dr. Marina Rodnina Georg August University Göttingen Max Planck Institute for Biophysical Chemistry Department of Physical Biochemistry Göttingen, Germany Prof. Dr. Kai Tittmann Georg August University Göttingen Albrecht-von-Haller Institute Department of Bioanalytics Göttingen, Germany Date of oral examination: 20.11.2012 Affidavit I hereby declare that my thesis entitled "Structural and functional studies of the spliceosomal RNP remodeling enzyme Brr2" has been written independently and with no other sources and aids than quoted. This thesis (wholly or in part) has not been submitted elsewhere for any academic award or qualification. Karine Santos September, 2012 Göttingen Related publications Parts of this thesis were published in: Weber, G., Cristao, V.F., de L Alves, F., Santos, K.F., Holton, N., Rappsilber, J., Beggs, J.D., Wahl, M.C. (2011) Mechanism for Aar2p function as a U5 snRNP assembly factor. Genes Dev. 25(15):1601-12. DOI: 10.1101/gad.635911. Santos, K. F., Mozaffari-Jovin, S., Weber, G., Pena, V., Lührmann, R., Wahl, M. C. (2012) Structural basis for functional cooperation between tandem helicase cassettes in Brr2- mediated remodeling of the spliceosome. Proc. Natl. Acad. Sci. USA, 109(43):17418-23. DOI: 10.1073/pnas.1208098109. Mozaffari-Jovin, S., Santos, K.F., Hsiao, H.H., Urlaub, H., Wahl, M.C., Lührmann, R. (2012) The Prp8 RNase H-like domain inhibits Brr-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA. Genes Dev. 26(21):2422-34. DOI: 10.1101/gad.200949.112. Weber, G., Cristao, V.F., Santos, K.F., Jovin, S.M., Heroven, A.C., Holton, N., Lührmann, R., Beggs, J.D., Wahl, M.C. (2013) Structural Basis for dual roles of Aar2p in U5 snRNP assembly. Genes Dev. 27(5):525-40. DOI: 10.1101/gad.213207.113. Mozaffari-Jovin, S.*, Wandersleben, T.*, Santos, K.F.*, Will, C., Lührmann, R., Wahl, M.C. (2013) Inhibition of RNA helicase Brr2 by the C-terminal tail of the spliceosomal protein Prp8. Science. DOI:10.1126/science.1237515. *These authors contributed equally to this work. Abstract ..................................................................................................................................... 1 1. Introduction ...................................................................................................................... 3 1.1 pre-mRNA splicing .................................................................................................................................... 3 1.1.1 The mechanism of nuclear pre-mRNA splicing ............................................................................. 6 1.1.2 Components of the spliceosome ..................................................................................................... 9 1.1.3 Stepwise assembly of the spliceosome ......................................................................................... 12 1.1.4 Rearrangements of the RNA-RNA network during the splicing cycle.......................................... 15 1.1.5 Compositional changes within the spliceosome .......................................................................... 18 1.2 Helicases: enzymatic motors of the spliceosome ..................................................................................... 21 1.2.1 RNA helicases .............................................................................................................................. 21 1.2.1.1 DEAD-box family ................................................................................................................................ 23 1.2.1.2 DEAH/RHA family .............................................................................................................................. 24 1.2.1.3 Ski2-like family .................................................................................................................................... 24 1.2.2 Spliceosomal RNA helicases ........................................................................................................ 25 1.2.3 Brr2: a helicase essential for spliceosome catalytic activation ................................................... 27 1.2.4 Brr2 and Renititis pigmentosa ..................................................................................................... 31 1.2.5 Structural studies of spliceosomes and their components ........................................................... 32 1.3 Aim of this study ...................................................................................................................................... 33 2. Materials and methods ....................................................................................................... 37 2.1 Materials .................................................................................................................................................. 37 2.1.1 Chemicals .................................................................................................................................... 37 2.1.2 Buffer solutions and media components ...................................................................................... 39 2.1.3 Consumables ............................................................................................................................... 40 2.1.4 Chromatographic resins and columns ......................................................................................... 41 2.1.5 Molecular biology kits ................................................................................................................. 42 2.1.6 Nucleotides .................................................................................................................................. 42 2.1.7 Crystallization screens and kits ................................................................................................... 42 2.1.8 Instrumentation ........................................................................................................................... 43 2.1.9 Enzymes and proteins .................................................................................................................. 45 2.1.10 DNA oligonucleotides ................................................................................................................ 46 2.1.11 Plasmids .................................................................................................................................... 49 2.1.12 Insect cell lines .......................................................................................................................... 51 2.1.13 Bacterial strains ........................................................................................................................ 51 2.1.14 Software ..................................................................................................................................... 52 2.2 Methods ................................................................................................................................................... 53 2.2.1 Nucleic acid methods ................................................................................................................... 53 2.2.1.1 Determination of nucleic acid concentration ........................................................................................ 53 2.2.1.2 Agarose gel electrophoresis for DNA ................................................................................................... 53 2.2.1.3 DNA purification using agarose gel electrophoresis ............................................................................. 53 2.2.1.4 Polymerase chain reaction (PCR) ......................................................................................................... 53 2.2.1.5 Site-directed mutagenesis ..................................................................................................................... 54 2.2.1.6 Restriction digestion and ligation of DNA ............................................................................................ 54 2.2.1.7 Plasmid isolation from Escherichia coli cells ....................................................................................... 55 2.2.1.8 Plasmid verification .............................................................................................................................. 55 2.2.1.9 Radioactive labeling of the 5'-end of RNA oligonucleotides ................................................................ 55 2.2.2 Cell and cell culture methods .....................................................................................................