Molecular architecture of SF3B and the structural basis of splicing modulation Dissertation for the award of the degree “Doctor rerum naturalium” (Dr. rer. nat.) in the Molecular Biology Graduate Program Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen submitted by Constantin Cretu born in Chisinau Göttingen 2018 Members of the thesis committee: Dr. Vladimir Pena (1st reviewer) Research group Macromolecular Crystallography, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Patrick Cramer (2nd reviewer) Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Henning Urlaub Research group Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen Further members of the Examination Board: Prof. Dr. Reinhard Lührmann Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Ralf Ficner Department of Molecular Structural Biology, Institute for Microbiology and Genetics, Göttingen Alexis Caspar (Alex) Faesen, PhD Research group Biochemistry of Signal Dynamics, Max Planck Institute for Biophysical Chemistry, Göttingen Date of submission of the thesis: March 31, 2018 Date of the oral examination: June 26, 2018 ii Affidavit I hereby declare that this thesis has been written independently and with no other sources and aids other than quoted. This thesis has not been submitted elsewhere for any academic degree or qualification. Constantin Cretu Göttingen, 2018 iii This work is dedicated to my beloved grandfather iv “Să vezi departe e ceva, să ajungi acolo e altceva.” “To see far is one thing, going there is another.” (Constantin Brancusi) v Table of Contents 1 Summary ................................................................................................................................1 2 Introduction ............................................................................................................................4 2.1 The central dogma of molecular biology ............................................................................4 2.2 Split genes and pre-mRNA splicing ...................................................................................4 2.3 The chemistry of pre-mRNA splicing ................................................................................9 2.4 snRNPs – the building blocks of the spliceosome ............................................................ 11 2.5 The splicing cycle – lessons from recent cryo-EM structures ........................................... 17 2.6 The multimeric SF3B complex and the recognition of the branch-site region ................... 23 2.7 Splicing factor mutations in cancers................................................................................. 29 2.8 Therapeutic targeting of the spliceosome with small-molecule compounds ...................... 33 2.9 About this work ............................................................................................................... 36 3 Results ................................................................................................................................... 38 3.1 Molecular architecture of SF3b and structural consequences of its cancer-related mutations (published manuscript) .......................................................................................................... 39 3.2 Structural basis of splicing modulation by antitumor macrolide compounds (published manuscript) ........................................................................................................................... 72 4 Discussion and Perspectives ............................................................................................... 102 4.1 Molecular architecture and structural dynamics of the human SF3B complex ................ 102 4.1.1 The extended SF3B1’s NTD domain is a protein-protein interaction hub ................ 103 vi 4.1.2 SF3B1’s HEAT domain has a unique superhelical conformation............................. 104 4.1.3 SF3B1’s HEAT domain and recognition of the branch-site region .......................... 106 4.1.4 Is SF3B6/p14 a branch-site interacting protein? ...................................................... 108 4.1.5 SF3B3 – a multipurpose molecular scaffold ............................................................ 109 4.1.6 Structural insights into SF3B1’s cancer-related mutations ....................................... 113 4.2 Molecular insights into splicing modulation by antitumor SF3B inhibitors .................... 117 4.2.1 A pipeline for structure-based discovery of next-generation splicing modulators..... 117 4.2.2 Revisiting the common pharmacophore hypothesis ................................................. 119 4.2.3 Splicing modulators binding site reveals a conformational switch in SF3B1 ........... 122 4.2.4 Novel structural insights into spliceosome assembly ............................................... 125 4.2.5 Splicing modulators as competitive branch-site antagonists .................................... 129 5 Conclusions and Outlook ................................................................................................... 133 References.............................................................................................................................. 135 Appendix ............................................................................................................................... 148 List of Figures........................................................................................................................ 150 Abbreviations ........................................................................................................................ 151 Acknowledgments ................................................................................................................. 152 vii 1 | Summary 1 Summary During splicing non-coding introns are excised from the transcribed pre-messenger RNA (pre- mRNA), and the protein-coding exons are ligated to generate the mature mRNA. In cells, the pre- mRNA splicing reaction is catalyzed by the spliceosome, a highly dynamic molecular machine composed of five small nuclear ribonucleoprotein particles (snRNPs) and additional non-snRNP factors (Wahl et al., 2009; Will and Luhrmann, 2011). At the earlier stages of spliceosome assembly, the U2 snRNP is recruited to the 3’ region of the intron for the U2 snRNA to base-pair with the branch-site (BS), in a complex and insufficiently understood process (Wahl et al., 2009). SF3B is the largest U2 subcomplex, and several of its seven subunits, including SF3B1, contact both the U2 snRNA and the intron near the BS, stabilizing the U2/BS base-pairing interaction. Recurrent somatic cancer mutations in SF3B1, and in several related splicing factors, reduce the accuracy of BS selection and, finally, lead to aberrant splicing (Dvinge et al., 2016). The compromised function of SF3B1 affects splicing of many different transcripts and thus translates to global changes in cancer cell transcriptome (Alsafadi et al., 2016; Darman et al., 2015). SF3B is also targeted by several small-molecule splicing modulators, regarded as promising chemotherapeutic agents (Bonnal et al., 2012; Effenberger et al., 2017). At the start of this project, it was unclear how SF3B is organized prior to its incorporation in the spliceosome, what structural features of human SF3B are perturbed in cancers, and how the antitumor compounds act on SF3B to modulate splicing. In the first part of this thesis work, we carried out a thorough structural analysis of the human SF3B complex (Cretu et al., 2016). Firstly, we have defined a structurally stable SF3B core complex (~254 kDa), composed of SF3B1’s C-terminal HEAT domain, SF3B3, SF3B5, and 1 1 | Summary PHF5A, and determined its structure by X-ray crystallography. The crystal structure of the SF3B core complex revealed that the 20 HEAT repeats of SF3B1 adopt a distinctive superhelical conformation and share extensive contacts with the other three core subunits (Cretu et al., 2016). SF3B3 exhibits a triple β-propeller fold and accommodates the three alpha helices of the SF3B5 subunit in a deep, clam-shaped cleft (Cretu et al., 2016). Organized as a compact knot composed of three zinc finger motifs, PHF5A bridges the terminal repeats of SF3B1’s HEAT domain, contributing to the unique conformation of the superhelix. Using a set of orthogonal mass spectrometry approaches, we showed that SF3B1-PHF5A together with the more mobile SF3B6/p14 subunit form a multipartite RNA binding platform that, in spliceosomes, stabilizes the U2/BS helix and the downstream 3’ end of the intron (Fica and Nagai, 2017; Shi, 2017). Comparative analyses with recent cryo-EM structures of yeast spliceosomes (Fica and Nagai, 2017) show that the cancer-related residues of SF3B1 map to a basic groove of the HEAT superhelix where, likely, the 3’ pyrimidine-rich region of the intron binds. Altogether, our analyses suggest how changes in SF3B1 structure and interactome may lead to a compromised BS selection, thus providing insights into the molecular mechanism of SF3B1-driven cancers. In the follow-up work (Cretu et al., 2018, unpublished data), we determined co-crystal structures of SF3B core variants in complex with different compounds that modulate splicing, including some approved for clinical trials. Our work shows that splicing modulators from the pladienolide and herboxidiene