SPLICEOSOMAL INTRON and SPLICEOSOME EVOLUTION in GIARDIA LAMBLIA and OTHER DIPLOMONADS ANDREW JOHN HUDSON B.Sc. University of Le
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SPLICEOSOMAL INTRON AND SPLICEOSOME EVOLUTION IN GIARDIA LAMBLIA AND OTHER DIPLOMONADS ANDREW JOHN HUDSON B.Sc. University of Lethbridge, 2009 A Thesis Submitted to the School of Graduate Studies of the University of Lethbridge in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY IN BIOMOLECULAR SCIENCE Biological Sciences University of Lethbridge LETHBRIDGE, ALBERTA, CANADA © Andrew John Hudson, 2014 PREPARATION OF THESIS ANDREW JOHN HUDSON Date of Defence: December 8, 2014 Dr. Anthony (Tony) Russell Assistant Professor Ph.D. Co-supervisor Dr. Cameron Goater Associate Professor Ph.D. Co-supervisor Dr. Roy Golsteyn Associate Professor Ph.D. Thesis Examination Committee Member Dr. Ute Wieden-Kothe Associate Professor Ph.D. Thesis Examination Committee Member Dr. Nehalkumar Thakor Assistant Professor Ph.D. Internal Examiner Dr. Staffan Svärd Professor Ph.D. External Examiner Uppsala University Uppsala, Sweden Dr. James Thomas Professor Ph.D. Chair, Thesis Examination Committee Dedication This thesis is dedicated to Becky, Audrey and my parents Ann and Rob… …for all your support and unconditional love. iii Abstract Spliceosomal introns interrupt protein coding genes in all characterized eukaryotic nuclear genomes and are removed by a large RNA-protein complex termed the spliceosome. Diplomonads are diverse unicellular eukaryotes that display compact genomes with few spliceosomal introns. My thesis objectives were to explore spliceosomal intron and spliceosome diversity as well as RNA processing mechanisms in the diplomonads Giardia lamblia and Spironucleus spp. Surprisingly, G. lamblia was found to contain a proportionally large number of fragmented spliceosomal introns that are spliced in trans from separate pre-mRNA molecules. Next, both evolutionarily divergent and conventional spliceosomal small nuclear RNAs were identified in G. lamblia and Spironucleus spp. and an RNA 3ʹ end motif was determined to be involved in processing of both non-coding RNAs and trans-introns in G. lamblia. These findings shed light on spliceosome and spliceosomal intron evolution in eukaryotes undergoing severe genomic reduction and potentially complete loss of their spliceosomal introns. iv Acknowledgements Firstly, I would like to express my most sincere gratitude to my supervisor and mentor Dr. Tony Russell for the opportunity to perform studies in his laboratory and allowing me independence in choosing my research directions. Whatever success I have had, I link closely with the guidance and encouragement I have received from Dr. Russell over the years. Truly, I will never forget all of the stimulating ‘what if’ conversations we have had regarding RNA-protein evolution and I look forward to more of these in the future. I would also like to thank the other members of my Ph. D. supervisory committee: Drs. Ute Kothe, Roy Golsteyn and Cam Goater who have so patiently provided me with critical advice and guidance through the years of my graduate studies. I am also extremely grateful (and honored) that one of world’s most prolific Giardia researchers, Professor Staffan Svärd, was willing to travel from Uppsala, Sweden to serve as my external examiner – particularly on such short notice! Indeed, much of my research project has relied heavily on diplomonad genomic and transcriptomic databases which were generated in a large part by Professor Svärd’s group and for that, I cannot thank you enough. I also thank Dr. Nehal Thakor for accepting to be my internal-external examiner and Dr. Jim Thomas for serving as my Ph. D. defence chair. I have left my lab mates Ashley and David late in this list, however, I cannot express how thankful (and lucky) I feel to have been able to work with you over the years. In addition to your useful suggestions, you have made all of the challenges associated with pursuing a graduate degree more bearable and the successes even more joyous! I will forever cherish our ‘Lab-opoly’ nights and the countless (and ridiculous) puns spouted off (admittedly mostly by yours’ truly) in the Russell Lab. However, I also go forward with a v sense of excitement for both of you: David, I see a promising young research scientist and Ashley, you will soon be an instructor at RDC! In addition to my ‘core’ lab mates, I also want to thank the many Russell Lab undergraduate students who have contributed to my success (and many who have become my friends): Dave Elniski (the ‘E-pun coli’ and banjo master), Ashlee Matkin (Lab-opoly extraordinaire and future MD), Kenzie Visser (Witty pun-ner and future MD), John Stimson (You’re good at EVERYTHING and future MD), Colleen Chen (a not so ‘underwhelming’ DVM), Shayne Rybchinski, Ben Vuong (future DVM), Peter Van Herk, Thomas Scott and Ryan Taylor (future MD). I also want to acknowledge members of the Wieden-Kothe and Wieden Labs (particularly Raja, Laura, Evan, Fan, Luke, Harland and Dylan) who have been helpful in providing advice and sharing equipment over the years. I would also like to acknowledge the sources of financial support provided to me during my graduate studies: Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Lethbridge, School of Graduate Studies (SGS). I hope you know that your financial support has made a tremendous contribution to my success. Lastly, I would like to thank my family and friends for supporting me in this long (and selfish) endeavor of pursuing a doctoral degree and giving me nothing but love, support and encouragement throughout my whole life. Mom and Dad, I hope you see this as the ‘end’ of my studentship and beginning of my career in science! Becky, this work is as much your accomplishment as it is mine. Joey, thank you for everything! Chad and Sharon, thanks for all your encouragement and inspiration. Christin and Ryan, thank you for your friendship and support over the years. There are also so many others who I would like to thank (e.g. previous mentors: Dr. Kevin Floate and Paul Coglin) but don’t have the space and thus I can only say – thank you all for your support! vi Table of Contents Thesis Exam Committee Members…………………………………………………….. ii Dedication……………………………………………………………………………….. iii Abstract………………………………………………………………………………….. iv Acknowledgements……………………………………………………………………….v List of Tables…………………………………………………………………………….. x List of Figures…………………………………………………………………………… xi List of Abbreviations………………………………………………………………….. xiii Chapter 1 – Introduction………………………………………………………………... 1 1.0 Genes in Pieces…………………………………………………………………. 1 1.1 Introns and their Mechanisms of Splicing……………………………………… 3 1.1.1 Group II introns…………………………………………………………. 3 1.1.2 Spliceosomal introns…………………………………………………….. 6 1.1.3 U2-type and U12-type introns…………………………………………… 9 1.1.4 Intron structures of ‘intron-rich’ vs. ‘intron-poor’ eukaryotes…………. 11 1.1.5 Other intron types………………………………………………………. 12 1.1.6 Alternative splicing……………………………………………………...13 1.1.7 RNA trans-splicing……………………………………………………...14 1.2 The Spliceosome……………………………………………………………….15 1.2.1 Spliceosomal snRNAs………………………………………………….. 16 1.2.2 Spliceosomal proteomes………………………………………………... 21 1.2.3 Spliceosomal snRNP biogenesis……………………………………….. 23 1.2.4 The spliceosome cycle…………………………………………………. 24 1.3 Spliceosomal Intron and Spliceosome Evolution……………………………... 27 1.3.1 ‘Introns early’ vs. ‘introns late’………………………………………… 27 1.3.2 Reconstruction of ancestral exon-intron structures…………………….. 28 1.3.3 Origins of spliceosomal introns and the spliceosome………………….. 29 1.3.4 Emergence of U2- and U12-dependent spliceosomes………………….. 31 1.4 Diplomonads as Model Organisms…………………………………………….32 1.5 Objectives……………………………………………………………………... 36 Chapter 2 – Numerous Fragmented Spliceosomal Introns, AT–AC Splicing, and an Unusual Dynein Gene Expression Pathway in Giardia lamblia………………. 37 2.1 Introduction…………………………………………………………………… 37 2.2 Materials and Methods…………………………………………………………39 2.2.1 Identification and comparative genomics of G. lamblia introns……….. 39 2.2.2 PCR and RT-PCR mediated confirmation of trans-splicing and G. lamblia genome annotation………………………………………….. 41 2.3 Results and Discussion………………………………………………………... 42 2.3.1 Cis and trans-spliced introns in Giardia……………………………….. 42 2.3.2 Extensive fragmentation of the DHC β gene…………………………… 46 2.3.3 Confirmation of trans-splicing………………………………………..... 47 2.3.4 Utilization of atypical splice boundaries in the DHC γ gene……………48 2.3.5 Base pairing and trans-splicing: evolutionary implications……………. 51 2.4 Conclusions…………………………………………………………………….53 vii Chapter 3 – Evolutionarily Divergent snRNAs and a Conserved Non-coding RNA Processing Motif in Giardia lamblia…………………………………………….. 54 3.1 Introduction…………………………………………………………………….54 3.2 Materials and Methods…………………………………………………………58 3.2.1 RNA motif identification and characterization………………………….58 3.2.2 Identification of new Giardia ncRNAs………………………………… 58 3.2.3 RT-PCR experiments…………………………………………………… 61 3.2.4 Primer extension and northern blotting………………………………… 62 3.2.5 RNA end-mapping by random amplification of cDNA ends (RACE)…. 63 3.2.6 In vitro U4/U6 snRNA complex formation…………………………….. 64 3.3 Results………………………………………………………………………… 65 3.3.1 Identification of a conserved sequence motif in Giardia ncRNA genes.. 65 3.3.2 The conserved motif mediates 3ʹ end formation of G. lamblia ncRNAs. 69 3.3.3 The conserved sequence motif has a role in the novel Giardia mRNA trans-splicing pathway…………………………………………………. 73 3.3.4 A G. lamblia telomerase RNA candidate………………………………. 76 3.3.5 Identification of novel Giardia U1 and U6 snRNA candidates………..