THE EVOLUTION OF MITOCHONDRIAL GENOME STRUCTURE AND FUNCTION IN INSECTS by James Bruce Stewart B.Sc.H, University of Waterloo, Waterloo, Ontario, 1997 M.Sc. Simon Fraser University, Burnaby, British Columbia, 2001 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Molecular Biology and Biochemistry O James Bruce Stewart, 2005 SIMON FRASER UNIVERSITY Summer 2005 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. Approval Name: James Bruce Stewart Degree: Doctor of Philosophy Title of thesis: The Evolution of Mitochondria1 Genome Structure and Function in Insects Examining Committee: Chair: Dr. Nicholas Harden, Associate Professor, Department of Molecular Biology and Biochemistry Dr. Andrew T. Beckenbach, Professor Senior Supervisor Department of Biological Sciences Dr. David L. Baillie, Professor Supervisory Committee Member Department of Molecular Biology and Biochemistry Dr. Peter J. Unrau, Assistant Professor Supervisory Committee Member Department of Molecular Biology and Biochemistry Dr. Esther M. Verheyen, Associate Professor Public Examiner Department of Molecular Biology and Biochemstry Dr. Laurie S. Kaguni, Professor External Examiner Department of Biochemistry and Molecular Biology Michigan State University Date Approved: August 19,2005 SIMON FRASER UNIVERSITY PARTIAL COPYRIGHT LICENCE The author, whose copyright is declared on the title page of this work, has granted to Simon Fraser University the right to lend this thesis, project or extended essay to users of the Simon Fraser University Library, and to make partial or single copies only for such users or in response to a request from the library of any other university, or other educational institution, on its own behalf or for one of its users. 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Bennett Library Simon Fraser University Burnaby, BC, Canada Abstract The mitochondrial genomes (mt-genomes) of animals are very compact in structure, encoding thirteen protein genes involved in the production of ATP, and the key components of the translation system to express these proteins. The mitochondrial expression system, which functions separately from that of the nucleus, shows characteristics of both prokaryotic and eukaryotic expression systems, and has diverged greatly from that currently observed in the closest living relatives of mitochondria, the a-proteobacteria. Current understanding of transcript maturation is that large multi-gene transcripts are processed by the removal of intervening tRNA genes, leaving behind RNA templates to be matured into the functional mRNAs and rRNAs. One of the most striking features of insect mt-genomes has been the apparent replacement of a start codon with a stop codon for the essential mitochondrial gene cytochrome c oxidase subunit 1 (coxl). When first observed in Drosophila, Clary and Wolstenholme proposed a highly unusual four-base "ATAA" start codon. With the expanded sampling of mitochondrial sequence across the various insect orders, the data does not support the use of this aberrant initiation for coxl. At the initiation of this study, the diversity of insect groups represented by complete mt-genome sequence was very poor. To address this deficit, I undertook sequencing projects to increase the number of insect orders represented in the mitochondrial sequence databases. I report the complete mt-genome sequences for two insects, the spittlebug Philaenus spumarius, and the giant stonefly Pteronarcys princeps. The sequences are annotated and compared to other insect mt-genomes in the sequence databases. I report the cDNA sequences of Drosophila melanogaster mitochondrial mRNAs, rRNA subunits, and a population of pre-mRNA molecules that are intermediates of the RNA processing system. Models to explain mitochondrial transcript maturation in light of these new observations are proposed. Comparative analyses were undertaken to apply the information gained from the mitochondrial transcripts of D. melanogaster to the mitochondrial structure and annotations of mt-genomes from the other insects. These analyses suggest a 5' specific modification to the tRNA punctuation model for insect mitochondria. This modification may represent a further evolutionary simplification of the mitochondrial expression system. To Laila, for everything, And to You who we patiently await. In memory: Grandpa Bruce Stewart December 6,1922 - February 27,200 1 and Great-Grandpa Ted Baxter October 20,1897 May 5,1990 - Acknowledgements To my senior supervisor Andy Beckenbach, for allowing me the chance to explore this project and believing that I could get it done. The last few years have been a lot of fun, and I will miss our lab chats. A special thanks to my former lab-mate Russ Watkins for keeping in touch after moving on, and for supplying a key tip for my early RNA struggles. To my lab-mates Andrea Scouras, Dave Russell, Alison Cloutier, Karen Beckenbach, Denise Reichow, Cam Muir and Mike Vankoeveringe. Thanks for all the tips, advice and silliness. Thanks to the RNA-work troubleshooters Alex Ebhardt and Don Sinclair, and to the computer guys Jeff Bryer and Duncan Napier. To Jillian Smith, Darrell Bessette and Theresa Kitos for the help with cell culture. To my committee members Peter Unrau and Dave Baillie for their time, effort and advice over the course of my degree work. I would also like to thank Mike Smith for his role as unofficial committee member. Thank-you to Jamie Cates for the very important discussion of translation at a key time. To Laurie Kaguni and Esther Verheyen, my external examining committee members, for the useful, constructive and kind comments regarding my thesis. To my fellow graduate students in Biology and the MBB for all the fun and for the shop talk. I especially want to thank John Taylor, Ian Hamilton, Jeremy Mitchell, Tom Chapman, Lynnette Kuervers, Nigel O'Neil, Greg Vatcher, Steve Springer, Michelle Harrison, Susanne Kiihnholz, Richard Fahlman, Anat Feldman, Carrie Simms and Daniela Ginta for important discussions over the course of my study. My personal thanks to the Biergarten Entrepreneurs, past and present. Where else would the discussions take place? To Carlo Artieri, Leslie Mitchell and Connie Roth for seminar practice sessions. To our Vancouver friends; Annick, Kevin and Giselle; Cam; Cathy and Rasmus; David; Kym, Dana and Tayme; Kat and Greg; Tina and Olie; Jennifer and Stevo; Markus, Lisa and Anna; Cris and Diana; Anna, Jurgen, Max and Sophie; Nat, Dirk, Steffen and Gabby; Leah, Omar and Arnira; Melissa. From Ontario, to Pete, Tina, Mason and Rowan; Jilly, Bomina, Susan and Carole; Scott, Kurt and Karl and Christine for continued support and friendship. Thanks to Cathy, Don, Mike, Kelly Joel, Ardis, Fred, and to Norm, Joe, Vicky, Rob, Tracy, Mary and all the rest of my family, for your patience, support, and well wishes over the years. Thank-you to Agneta and Zubin for welcoming me. Table of Contents .. ................................................................................................................................ .... Approval ..............................................................................................................................1.1. . Abstract .........................................................................................................................1.1.1.. . Dedication iv ................................................................................................................ Acknowledgements v .................................................................................................................. Table of Contents vi ........................................................................................................................ List of Tables ix ......................................................................................................................... List of Figures x .................................................................................................. . CHAPTER 1 Introduction 1 ...................................................................................................... 1.1. Mitochondria 1 ................................................................................... 1.2. Mitochondria1 Evolution 2 ....................................................................... 1.3. Animal Mitochondria1 Genomes 5 1.3.1. Animal mitochondria1 gene content ..................................................... 5 1.3.2. Animal mitochondria1 genome structure. ............................................. 9 1.3.3. Mitochondria1 genome sequencing ....................................................1 0 ............................................. 1.4. Mitochondria1 Transcription And Replication 14 1.4.1. Mitochondria1 replication in animals ................................................ 14 1.4.2. Variation in transcriptional systems between eukaryotic groups .......................................................................................................... 17 1.4.3. Mammalian mitochondria1 transcription ...........................................1 8 1.4.4. Transcript processing in mammalian mitochondria .......................... 19 1.4.5. Transcription in Drosophila mitochondria .......................................2 0 1.4.6. RNA editing and programmed frameshifting in animal mitochondria .............................................................................................2 3 .................................................................................................. 1.5. Thesis Outline 24 . CHAPTER 2 The Complete Mitochondrial Genome Sequence of the Meadow Spittlebug Philaenus spumarius (Hemiptera: Auchenorrhyncha: .................................................................................................................... Cercopoidae) 26 .................................................................................................... 2.1. Introduction 26 .................................................................................... 2.2. Materials and Methods 28 2.2.1. DNA extraction and PCR amplification ...........................................2. 8 2.2.2. Sequence assembly, annotation and analysis .....................................3 2 ..................................................................................... 2.3. Results and Discussion 33 2.3.1 . Genome content .................................................................................3 3 2.3.2. Protein coding genes ......................................................................3..9.. 2.3.3. Genomic comparisons. ...................................................................4..3. ...................................................................................................... 2.4. Conclusions 44 . CHAPTER 3 The Complete Mitochondrial Genome Sequence of a Giant Stonefly. ............................................................ Pieronarcysprinceps (Plecoptera; Pteronarcyidae) 47 ........................................................................................................ 3.1. Introduction 47 ...................................................................................... 3.2. Materials and Methods 51 3.2.1. Specimen collection and identification. ...............................................5 1 3.2.2. DNA isolation ................................................................................5..1.. .. 3.2.3. PCR and Long-PCR amplification. ...................................................5..2. 3.2.4. Purification of PCR products. ...........................................................5 4 3.2.5. DNA sequencing. ..............................................................................5..6. 3.2.6. Sequence assembly, annotation and analysis ....................................... 56 3.2.7. Sequence comparisons. ........................................................................ 57 ....................................................................................... 3.3. Results and Discussion 61 3.3.1 . Genome annotation. ..........................................................................6.1.. 3.3.2. Genome architecture. ........................................................................... 65 3.3.3. Nucleotide composition. .................................................................6..5.. . 3.3.4. Stonefly A+T rich regions ...............................................................7..4.. .......................................................................................................... 3.4 Conclusions 80 . CHAPTER 4 Characterization Of Mitochondrial Transcripts In Drosophila ............................................................................................................................ melanogaster 82 .................................................................................................... 4.1. Introduction 82 4.1 .1 . The cox1 initiation mystery in insect mitochondria. ............................ 82 4.1.2. Mitochondria1 transcription and RNA processing in Drosophila ........ 84 4.1.3. 5' amplification and sequencing. .......................................................... 86 ...................................................................................... 4.2. Materials and methods 89 4.2.1. Blast searches for nuclear copies of the cox1 gene in Drosophila. ......8 9 4.2.2. Maintenance of Drosophila fly stocks. ................................................8 9 4.2.3. Maintenence of Drosophila Schneider-2 cell lines ..............................9 0 4.2.4. Mitochondria1 isolation protocols. ....................................................9..1. 4.2.5. RNA extraction protocol .................................................................9..3.. . 4.2.6. Reverse-transcription pcr protocols. ..................................................9.6. 4.2.7. Protocol for 5'-RACE amplification. ................................................1. 00 4.2.8. Protocol for 3'-RACE amplicication. ...............................................1.0. 1 4.2.9. RNA circularization for RT-PCR ....................................................1..0 1 4.2.10. Cloning of amplification products. .................................................. 102 4.2.1 1. Sequencing of cDNAs and sequence alignment. ............................. 104 ............................................................................................................... 4.3. Results 104 4.3.1. Search for a nuclear copy of cox1. ...................................................1..0 4 4.3.2. RNA extraction from mitochondria. ................................................1.0. 6 4.3.3. Detection of unprocessed pre-mRNA molecules in flies ..................1 07 4.3.4. Mapping of mRNA and rRNA boundaries by RACE .......................1 07 4.3.5. Detection of RACE amplified RNAs from Schneider-2 cells ........... 111 4.3.6. Characterization of RNAs by CR-RT-PCR ......................................1. 11 vii ......................................................................................................... 4.4. Discussion 117 4.4.1. Schnieder-2 cells versus adult fly data .............................................1. 17 4.4.2. Detection of pre-mRNA molecules. .................................................1. 18 4.4.3. Description of mitochondria1 mRNAs .............................................1..1 9 4.4.4. Intitiation of the cox1 gene ..............................................................1..2 7 4.4.5. Characterization of the ribosomal RNAs ....................................1 29 .............................................................. 4.4.6. Polyadenylated pre-mRNAs 130 . ....................................................................... CHAPTER 5 Conclusions And Discussion 136 ........................... 5.1. Transcript Characterization in Drosophila Mitochondria 136 5.1.1. Characterization of mt-rRNAs. .......................................................... 136 5.1.2. Characterization of mt-mRNAs .......................................................1..3 8 5.1.3. Processing of tRNAs versus mRNAs in insect mitochondria ............1 41 ...................................................................... 5.2. Insect Mitochondria1 Genomics 143 5.2.1. Two insect mitochondria1 genomes ................................................... 143 5.2.2. Annotation of mitochondria1 genes ..................................................1..4 3 5.2.3. Genome structure and rearrangements. ............................................1.4 5 ............................................................................................. 5.3. Future Directions 148 5.3.1. Modification of the CR-RT-PCR protocol. ....................................... 148 5.3.2. Transcrption of A+T rich region sequence. .......................................1 49 5.3.3. Further characterization of nuclear transcription and processing factors .......................................................................................................... 151 . .............................................. APPENDIX A Genbank Flatfile for Philaenus spumarius 155 . .............................................. APPENDIX B Genbank Flatfile for Pteronarcys princeps 165 .................................................................................................................... REFERENCES 174 ... Vlll List of Tables Table 2.1. List of Primer Pairs Utilized in the Amplification of the Mitochondrial .......................................................................... Genome of Philaenus spumarius 30 Table 2.2. Insect Mitochondrial Genomes Utilized in Alignments and Comparative Analyses. .............................................................................................................-.3 5 Table 2.3. Nucleotide Composition of Meadow Spittlebug (P. spumarius) Genome Features. ...........................................................................................................4..0.. . Table 2.4. Relative Synonymous Codon Usage for Meadow Spittlebug. the Kissing Bug and the Migratory Locust Mitochondria1 Genomes ......................................4 5 Table 3.1 Primers Used in the Amplification of the Mitochondrial Genome of the Giant Stonefly Pteronarcys princeps .................................................................... 53 Table 3.2. Complete and Near-Complete Hexapod Mitochondrial Genome Sequences Utilized in this Study. with their GenBank Accession Numbers .........................5. 9 Table 3.3. Nucleotide Composition of Stonefly Mitochondria] Genome Features. ............... 66 Table 3.4. Comparison of Relative Synonymous Codon Usage for the Giant Stonefly. a Silverfish and a Locust. .....................................................................................6. 8 Table 3.5. Positional Nucleotide Bias in the Mt-Genome of the Giant Stonefly and Other Representative Insects. ......................................................................7..3. Table 4.1. Primers Used in 5' and 3' RACE Amplification .................................................9..7 Table 4.2. Primers Utilized in CR-RT-PCR Reactions. ......................................................9..8. Table 4.3. Results of tBlastn Searches for a Nuclear cod Gene .......................................... 105 Table 4.4. Positional Annotation of Transcribed RNA Nucleotides as Determined Through cDNA Sequencing ...........................................................................1.. ..1 5