Loss of Sbds in Shwachman-Diamond Syndrome Murine Model Leads to Reduction of 80S Ribosomes and Altered Transcript Binding
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Loss of Sbds in Shwachman-Diamond Syndrome Murine Model Leads to Reduction of 80S Ribosomes and Altered Transcript Binding by Hongrui Liu A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Hongrui Liu 2016 Loss of Sbds in Shwachman-Diamond Syndrome Murine Model Leads to Reduction of 80S Ribosomes and Altered Transcript Binding Hongrui Liu Doctor of Philosophy Department of Molecular Genetics University of Toronto 2016 Abstract Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease characterized by growth retardation, exocrine pancreatic dysfunction, skeletal dysplasia, cognitive impairment and bone marrow failure. SDS is caused by mutations in SBDS (Shwachman- Bodian-Diamond syndrome). A recent model proposes that SBDS/Sbds functions together with EFL1/Efl1 to release EIF6/Eif6 from the pre-60S complex, enabling ribosomal subunit joining for translation initiation. To assess the protein synthesis deficiency that has been detected in SDS, I examined ribosomal profiles of murine fetal organs with the SDS-associated missense mutation, R126T (SbdsR126T/R126T). The SDS organ extracts revealed reduced 80S monosomes and preserved polysomes, with no ribosomal subunit imbalance compared to matched controls. Further, Eif6 was found to bind to both the 60S and 80S ribosome complexes in mutants in contrast to only 60S complexes in controls. To investigate these changes and to learn how the SDS translatome is affected, total and polysomal mRNAs of mutant and control samples were studied using cDNA microarray analyses. By comparing individual polysomal transcripts ii to respective total transcript levels, I found 799 transcripts (of 18,936 analyzed) with altered polysome loading in mutant fetal livers, with 634 being increased. Changes in polysome loading did not correspond with steady state protein levels, as indicated by proteome analysis using label-free mass spectrometry. Rather, these changes correlated with physical features of the transcripts, including 5' untranslated region composition as well as the lengths and nucleotide contents of the open reading frame and the 3' untranslated regions. Together, I conclude that the untimely release of Eif6 due to Sbds deficiency results in ribosomes with compromised translation initiation and that SDS disease phenotypes reflect protein synthesis insufficiency and the formation of sub- populations of ‘SDS ribosomes’ with non- or poor- translating capability. iii Acknowledgments I would like to express my deep appreciation and gratitude for my supervisor, Dr. Johanna M. Rommens’ patient guidance and mentorship over the years, from the first day I started as a rotation student in her lab just admitted into the graduate program to the completion of this degree. From her I learned not just the enthusiasm for academic pursuits, rigorous attitude for science, but also her genuinely good nature and infectious kindheartedness toward everyone around her. I am truly fortunate to have had the opportunity to work with her. I would like to thank my thesis committee members, Drs. Barbara Funnell and Craig Smibert, for their friendly guidance and thought-provoking suggestions that together nourished my intellectual growth over the years. I would also like to extend my thanks to staff from The Centre for Applied Genomics of The Hospital for Sick Children: Dr. Chao Lu and Xiaolin Wang for assistance with microarray preparation and analyses, to Jeff MacDonald for providing the transcript length and GC content data, and to Dr. Andrew Paterson and Dr. Pingzhao Hu for assistance and critical discussion of the statistical analyses of my data. Thank you to Paul Taylor and Dr. Jiefei Tong from the SPARC Biocentre of The Hospital for Sick Children for advice and support for mass spectrometry. I also thank the staff at the Toronto Centre for Phenogenomics for outstanding technical assistance. I am grateful to all members of the Rommens lab (past and present) for their technical expertise, stimulating discussions and above all, moral support during my studies. Special thanks to Dr. Marina Tourlakis, Rikesh Gandhi, and Fan Lin, who enriched my graduate study experience and made ‘Rommens lab’ have a special meaning to my life. Lastly, I would like to acknowledge my family for their ongoing support. Particularly to my husband Huapu Zhao; for his computer technical rescues during my many panicky moments, in addition to the hot meals awaiting me after long days of experiment. iv Table of Contents Acknowledgements……………………………………………………...………………..iv Table of Contents…………………………………………………………….……………v List of Tables…………………………………………………………………..………….x List of Figures…………………………………………………………………………..xi List of Appendices………………………………………………….…………………xiii List of Abbreviations……………………………………………………………………xiv Chapter 1 Shwachman-Diamond Syndrome: a Ribosomopathy Due to Loss of SBDS……………………………………………….……………………………………. 1 1 Shwachman-Diamond Syndrome: a Ribosomopathy Due to Loss of SBDS…………2 1.1 Shwachman-Diamond syndrome and natural history……………………...……2 1.1.1 Clinical features of Shwachman-Diamond syndrome………………….…2 1.1.1.1 Neutropenia and other hematological abnormalities…………….….2 1.1.1.2 Exocrine pancreatic dysfunction…………………………….………3 1.1.1.3 Skeletal abnormalities…………………………………………….…4 1.1.1.4 Neuro-developmental issues………………………………...………4 1.1.1.5 Liver features………………………………………………..………5 1.1.1.6 Other features………………………………………………………..5 1.1.1.7 Diagnosis and management of SDS……………………………..…..6 1.1.2 Molecular basis of SDS…………………………………………...………6 1.1.2.1 Identification of SBDS………………………………………………6 1.1.2.2 Structure and function of SBDS and relation to EIF6………………7 1.1.2.3 Models of SDS……………………………………………………...9 1.2 Ribosomes and translation…………………………………………………..…13 1.2.1 Ribosomopathies…………………………………………………….…...13 1.2.2 Translation overview…………………………………………….………18 v 1.2.3 Transcription and processing of rRNA……………………………..……22 1.2.4 Building ribosomes and ribosome structure…………………………..…23 1.2.4.1 Ribosomal proteins…………………………………………...……23 1.2.4.2 Regulation of RPs and extra-ribosomal functions…………………24 1.2.4.3 Ribosome structure…………………………………………...……26 1.2.5 The translation of mRNAs………………………………………….……28 1.2.5.1 Translation initiation………………………………………….……28 1.2.5.2 Translation elongation……………………………………..………33 1.2.5.3 Translation termination and recycling…………………………..…35 1.2.6 Contribution of mRNAs to translational control…………………...……36 1.2.6.1 5' Untranslated regions (5' UTRs) ………….………………...……36 1.2.6.2 Open reading frames (ORFs) ………………………………...……39 1.2.6.3 3' Untranslated regions (3' UTRs) ……….……………….….…….40 1.3 Thesis objectives……………………………………………………….………40 Chapter 2 Loss of Sbds Function Results in Abnormal Polysome Profiles with 80S Reduction and Abnormal Eif6 Binding……..……………………………………43 2 Loss of Sbds Function Results in Abnormal Polysome Profiles with 80S Reduction and Abnormal Eif6 Binding …………………………………………………………44 2.1 Summary…………………………………………………………………………44 2.2 Background………………………………………………………………………45 2.3 Materials and methods……………………………………………………...……46 2.3.1 Mice………………………………………………………...……………46 2.3.2 Polysome profiling and peak quantification…………………….….……46 2.3.3 Ribosome run-off profiling and quantification analyses...………………47 2.3.4 Western immunoblottings and quantification analyses………….………49 2.3.4.1 Protein precipitation from polysome profile fractions…….………49 2.3.4.2 Protein preparation from frozen tissues……………………………49 2.3.4.3 Western immunoblotting from prepared protein extracts…………49 2.3.5 Statistical analyses ………………………………………………………50 2.4 Results……………………………………………………………………………52 vi 2.4.1 Loss of Sbds leads to reduced 80S and persistent polysomes in multiple murine fetal organs………………………………………………………52 2.4.2 No ribosome subunit imbalance observed with Sbds deficiency in vivo……………………………………………………………………….52 2.4.3 Persistent high molecular complexes observed in SDS mutant polysome profiles represent polysomes…………………………………….…….…57 2.4.4 Aberrant polysome profiles observed in SbdsR126T/- fetal livers……….…57 2.4.5 Loss of Sbds function leads to aberrant association of Eif6 with 80S..…58 2.5 Discussion……………………………………………………………………..…67 Chapter 3 The Preserved Polysomes Associated with Sbds Deficiency Result From Altered mRNA Association………...……………………………………………70 3 The Preserved Polysomes Associated with Sbds Deficiency Result From Altered mRNA Association…………………………………………………………………..71 3.1 Summary…………………………………………………………………………71 3.2 Background………………………………………………………………………72 3.3 Materials and methods……………………………………………………...……73 3.3.1 Mice…………………………………………………………...…………73 3.3.2 Polysome profiles and RNA extraction…………………………………73 3.3.3 cDNA microarray and analyses…………………………………………74 3.3.4 Quantitative real-time RT-qPCR…………………………………...……75 3.3.5 Western immunoblottings and quantification analyses…………………78 3.3.6 Functional classification of transcripts and proteins……………………78 3.3.7 Physical characterization of transcripts with altered polysome loading……………………………………………………………………78 3.3.7.1 Transcript length and GC content analyses……………………..…78 3.3.7.2 uORF, TOP and IRES analyses UTRs……………………….……80 3.3.8 Label-free mass spectrometry and analyses………………………...……80 3.3.9 Compilation of protein synthesis and related genes……………..………81 3.3.10 Statistical analyses……………………………………………….………81 3.4 Results……………………………………………………………………………82 vii 3.4.1 Altered steady state total mRNA and polysome loading……………..…82 3.4.1.1 General work flow of microarray gene expression analyses………82 3.4.1.2 Microarray