Regulation of BLM Nucleolar Localization THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Larissa Jordan Tangeman, M.S. Graduate Program in Integrated Biomedical Science Program The Ohio State University 2017 Master's Examination Committee: Joanna Groden, PhD, Advisor Jeffrey Parvin, MD, PhD Copyright by Larissa Jordan Tangeman 2017 Abstract Defects in coordinated ribosomal RNA (rRNA) transcription in the nucleolus cause cellular and organismal growth deficiencies. Bloom’s syndrome, an autosomal recessive human disorder caused by mutated recQ-like helicase BLM, presents with growth defects suggestive of underlying defects in rRNA transcription. Our previous studies showed that BLM facilitates rRNA transcription and interacts with RNA polymerase I and topoisomerase I (TOP1) in the nucleolus. The mechanisms regulating localization of BLM to the nucleolus are unknown. In this study, we identify the TOP1-interaction region of BLM by co-immunoprecipitation of in vitro transcribed and translated BLM segments and show that this region includes the highly conserved nuclear localization sequence (NLS) of BLM. Biochemical and nucleolar co-localization studies using site- specific mutants show that two serines within the NLS (S1342 and S1345) are critical for nucleolar localization of BLM but do not affect the functional interaction of BLM with TOP1. Mutagenesis of both serines to aspartic acid (phospho-mimetic), but not alanine (phospho-dead), results in approximately 80% reduction in nucleolar localization of BLM while retaining the biochemical functions and nuclear localization of BLM. Our studies suggest a role for this region in regulating nucleolar localization of BLM via modification of the two serines within the NLS. ii Acknowledgments Thank you to my advisor Joanna Groden for supporting me for the past four years. Thank you to the current and former Groden lab members for their exceptional technical assistance, experimental suggestions, and company, especially Michael McIlhatton, William Hankey, Alaina Martinez, Zeenia Kaul, Julia Harris, and April Sandy Gocha. I would also like to thank the members of the Parvin lab for making our room a pleasant, congenial place to work. Thank you to my committee members, Jeff Parvin, Kay Huebner, and Mark Parthun, for guidance throughout my project. I would like to thank Samir Acharya for being an excellent friend and mentor for the past four years and for all of the thought-provoking conversations on almost every topic imaginable. I will miss our coffee and ice cream breaks. Finally, I would like to thank my family, especially my daughter Gabby, for their love and support. iii Vita May 2005 .......................................................Minster High School 2009................................................................B.S. Biology, Wright State University 2011................................................................M.S. Microbiology and Immunology, Wright State University 2011 to present ..............................................Graduate Research Associate, Biomedical Sciences Graduate Program, The Ohio State University Publications Tangeman, L., McIlhatton, M.A., Grierson, P., Groden, J., and Acharya, S. (2016) Regulation of BLM nucleolar localization. Genes (Basel), 7, e69. Tangeman, L., Wyatt, C.N., Brown, T.L. (2012) Knockdown of AMP-activated protein kinase alpha 1 and alpha 2 catalytic subunits. J. RNAi Gene Silencing, 8, 470-478. iv Lapalombella, R., Sun, Q., Williams, K., Tangeman, L., Jha, S., Zhong, Y., Goettl, V., Mahoney, E., Berglund, C., Gupta, S., Farmer, A., Mani, R., Johnson, A.J., Lucas, D., Mo, X., Daelemans, D., Sandanayaka, V., Shechter, S., McCauley, D., Shacham, S., Kauffman, M., Chook, Y.M., Byrd, J.C. (2012) Selective inhibitors of nuclear export show that CRM1/XPO1 is a target in chronic lymphocytic leukemia. Blood, 120, 4621-4634. Fields of Study Major Field: Integrated Biomedical Science Program v Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iii Vita ..................................................................................................................................... iv List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Chapter 1: Literature Review ............................................................................................. 1 Ribosome Biogenesis ...................................................................................................... 1 Ribosomal RNA Transcription and Cell Growth ............................................................ 2 Nucleolar Localization Mechanisms ............................................................................... 3 Bloom’s Syndrome .......................................................................................................... 4 BLM’s Roles in the Nucleolus ........................................................................................ 6 Chapter 2: Serines 1342 and 1345 Regulate BLM Nucleolar Localization but not DNA Topoisomerase I Interaction ............................................................................................. 10 I. Introduction ................................................................................................................ 10 II. Materials and Methods ............................................................................................. 11 Cell Lines ................................................................................................................... 11 vi Cloning ...................................................................................................................... 11 In Vitro Transcription and Translation ...................................................................... 13 Protein Purification .................................................................................................... 14 Helicase Assays ......................................................................................................... 16 Alignment and In Silico Phosphorylation Prediction ................................................ 17 Immunofluorescence ................................................................................................. 18 III. Results ..................................................................................................................... 19 BLM Amino Acids 1332-1349 Are Required for Binding to TOP1. ........................ 19 Deletion of Amino Acids 1332–1349 in BLM Abolishes TOP1-Mediated Unwinding of RNA:DNA substrate. ............................................................................................ 22 Amino Acids in the Region 1332–1349 are Critical for Nucleolar Targeting of BLM. ................................................................................................................................... 30 Alanine or Aspartic acid Mutagenesis of S1342 and S1345 Does Not Alter the Functional Interaction of BLM and TOP1. ............................................................... 37 IV. Discussion and Conclusions.................................................................................... 42 Chapter 3. Thesis Summary and Future Directions .......................................................... 47 References ......................................................................................................................... 53 vii List of Tables Table 1. Primers used in PCR. .......................................................................................... 12 viii List of Figures Figure 1. BLM amino acids 1332–1349 are required for binding to TOP1. .................... 21 Figure 2. Purification of recombinant BLM and TOP1 proteins. ..................................... 22 Figure 3. Specific activity of purified BLM proteins. ...................................................... 24 Figure 4. Unwinding of RNA:DNA substrate by BLM-Δ1332–1349 is not stimulated by TOP1. ................................................................................................................................ 26 Figure 5. Unwinding of RNA:DNA substrate by BLM-WT is stimulated by TOP1. ...... 28 Figure 6. Unwinding of RNA:DNA substrate by BLM-Δ1332–1349 is not stimulated by TOP1 at any protein concentration. .................................................................................. 29 Figure 7. Unwinding of DNA:DNA substrate by BLM-Δ1332–1349 is stimulated by TOP2A. ............................................................................................................................. 30 Figure 8. BLM-Δ1332–1349 does not localize to the nucleus. ........................................ 31 Figure 9. The TOP1-interaction domain of BLM is highly conserved in mammals. ....... 33 Figure 10. S1342 and S1345 determine nucleolar localization of BLM. ......................... 35 Figure 11. S1342 and S1345 do not affect PML body localization of BLM. ................... 36 Figure 12. Nucleolar localization of BLM is affected by phospho-mimetic