PATTERN AND DISTRIBUTION OF RNA EDITING IN LAND PLANT RBCL AND NAD5 TRANSCRIPTS A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Traci L. Branch December, 2006 PATTERN AND DISTRIBUTION OF RNA EDITING IN LAND PLANT RBCL AND NAD5 TRANSCRIPTS Traci L. Branch Thesis Approved: Accepted: _______________________________ _________________________________ Advisor Dean of the College Dr. Robert Joel Duff Dr. Ronald F. Levant _______________________________ _________________________________ Committee Member Dean of the Graduate School Dr. Richard Londraville Dr. George R. Newkome _______________________________ _________________________________ Committee Member Date Dr. Francisco B. Moore _______________________________ Committee Member Dr. Amy Milsted _______________________________ Department Chair Dr. Bruce Cushing ii ABSTRACT RNA editing is a process that occurs in both the chloroplast and mitochondrial genome of plants. However, little is know about the patterns and distribution of RNA editing among land plant lineages. To date, any investigations utilizing comparisons between multiple taxonomic groups have only looked at a small number of samples, typically within three lineages. More importantly, the data collected in the previous studies were generally restricted to sequences available on GenBank making it problematic due to the small number of plants sequenced. Therefore, to resolve questions unanswered thus far, it was crucial to perform a more extensive study including samples representing all five land plant lineages. To further examine this, one chloroplast gene, rbcL, and one mitochondrial gene, nad5, were studied in detail. Fourteen DNA and 22 cDNA sequences of rbcL were generated for a diverse group of land plants. In addition, 1 DNA and 8 cDNA sequences of nad5 were generated. RNA editing sites were identified through direct comparison of cDNA/DNA sequences and prediction methods using an alignment of 126 samples (newly generated and obtained from Genbank) representing all plant lineages. A total of 122 editing sites were predicted within 1335 nucleotides of rbcL. A total of 123 (out of 1107nt) editing sites were detected for the mitochondrial gene, nad5. The majority of the editing sites initially predicted by this method were actually observed. iii The 143 amino acid changes predicted resulted in a variety of 39 different types of amino acid conversions. S-L, S-F, and F-L were the most frequent amino acid changes noted for rbcL, respectively. A definite T_A context bias was detected at the -1/+1 nucleotide positions of editing sites that is absent in non-edited sites. The rate and distribution of RNA editing sites varied greatly among the taxa sampled. In several cases, individual taxa showed higher or lower rates of editing than the rest of their group within one or even both organelles. The findings of the T_A context bias support the suggestion that there are sequence recognition factors necessary for editing to take place. The absence of shared editing sites among selected taxa, for rbcL, raise a number of interesting evolutionary questions. In order for these questions to be resolved, future research needs to focus on improving both the number of genes (or better whole organelle genmones) and the number of plant groups examined. iv DEDICATION For my mother, Peg: I am left speechless at how much her support and unconditional love has taught me. And, without whom I would not have strived to reach and achieve my dreams… For Nate: there are not enough words to express my gratitude for the amount of love, encouragement, and support you have shown me, your patience is inexhaustible… v ACKNOWLEDGMENTS I want to extend my sincerest appreciation to my advisor Dr. Robert Joel Duff who has patiently guided and supported me in my scientific endeavors. To the members of my committee, Dr. Richard Londraville, Dr. Francisco “Paco” Moore, and Dr. Amy Milsted, each of whom have not only shared their knowledge and support but have also inspired me with their own excitement, to explore the scientific process. In addition, I would like to thank the other people who have helped me along the way: Laurie Kay and Laura Peterson for easing my transition into our laboratory; Lauren Smith for burning the midnight oil along side of me; Chiara Benvenuto and Sadie Reed for their never ending guidance and encouragement in and out of the lab; Hope Ball for her ability to keep me laughing through the obstacles I faced; Sue Robinson for all of her help and incredible “paper-pushing” ability; and Nate Manning for all the hours of editing and brainstorming. For the company spent with our noses dug deep into our biochemistry and molecular texts, I would like to thank Hope Ball and Patty Taylor. To my beloved group of Algal Troopers, I would like to thank you for the most beautiful week in San Salvador, Bahamas. Who better to spend a week of snorkeling and eating conch fritters? Finally and with much gratitude, I would like to thank the faculty in the Department of Biology at the University of Akron for all of their support, guidance, and their individual awe-inspiring approaches to science. vi TABLE OF CONTENTS Page LIST OF TABLES……………………………………………………………………….xi LIST OF FIGURES…………………………………………………………………...…xii CHAPTER I. INTRODUTION………………………………………………………………………..1 Phylogenetic distribution of RNA editing…………………………………….......2 Frequency of editing in subcellular organelles……………………………………4 RNA editing among hornworts……........................................................................6 Research questions………………………………………………………………...8 Specific Problem…………………………………………………………………10 II. MATERIALS AND METHODS………………………………………………….....12 Materials………………………………………………………………………....12 Isolation of Nucleic Acids and Synthesis of cDNA……………………………...12 Amplification of DNA and cDNA……………………………………………….14 Purification of PCR Products………………………………………………….....16 Cloning of cDNA and DNA PCR products……………………………………...16 Sequencing Reactions……………………………………………………………17 Determination of RNA Editing Sites…………………………………………….18 vii III. RESULTS…………………………………………………………………………...19 Results of Sequence Acquisition for rbcL……………………………………….19 Total Number of RNA Editing Sites for rbcL: all groups……………………....22 Results of Group Comparisons of Shared Editing Sites…………………………24 Results of the Affect of RNA Editing on Amino Acid Composition………...….27 RNA Editing in the Mitochondrial Gene, nad5……………………...…………..28 IV. DISCUSSION……………………………………………………………………...32 Describing Editing Sites: a conservative approach……………………………....32 Predicting RNA Edited Sites: problems encountered…………………………....32 Distribution of RNA Editing Sites: predicted and observed, in and between groups ……………………………………………………………….....34 RNA Editing Rate Across Land Plants and between Chloroplast and Mitochondrial………………………………………………………………….....36 Selection of RNA Editing Sites: what drives targeting of editing sites?...............38 Upstream and Downstream Base Preferences and RNA Editing in Land Plants…………………………………………………………………………..…41 Second Base Sites and RNA Editing…………………………………………….44 The evolution and maintenance of RNA editing in plant organelles…………….47 Conclusions and Future Research ……………………………………………….49 REFERENCES………………………………………………………………………….51 APPENDICES…………………………………………………………………………...55 viii APPENDIX A. ALLIGNMENT OF SELECTED TAXA DNA AND CDNA SEQUENCES FOR RBCL ………………………………………………………69 APPENDIX B. POSITION, AMINO ACID CHANGE AND TYPE OF EDITING SITES FOUND IN RBCL FOR SELECTED LAND PLANTS…..…72 ix LIST OF TABLES Table Page 1 A comparison of the known rates of RNA editing in plant organelles………4 2 Collection information for taxa included in this study. Genbank numbers for DNA sequences collected as part of this study are bolded. Genbank numbers represent samples for which prior DNA sequences had been generated but are being used in this study……………………… 13 3 PCR primers used for PCR amplifications and sequencing reactions. In some cases, multiple combinations of external or internal primers were used to obtain completed sequences…………………………………………………….15 4 Sequence reaction parameters used for each of the sequences that were generated during this study………………………………………………. .17 5 Characteristics of RNA editing sites in rbcL sequences for selected land plant groups. Data include taxa for which cDNAs were obtained and for which RNA edited sites were predicted from DNA sequence comparisons only…………………………………………………………………………24 6 Numbers of edited sites shared between selected plant groups compared to the total number of edited sites between the two groups. Abbreviations of plant names are as follows: hornworts (HW), lycophytes (LY), ferns (FN), mosses (MS), and Takakia (TK)……25 7 Characteristics of RNA editing sites in nad5 sequences for selected land plant groups and data compiled from literature. * indicates data has been obtained from Steinhauser et al. (1999), while the remaining data has been collected for the purpose of this study. Bold print indicates that cDNAs were available for direct comparison and edited sites reported for those samples are observed rather than predicted. A (?) indicates samples for which the T-C sites are only predicted……….30 8 Comparison of RNA editing rate between the chloroplast gene, rbcL, and the mitochondrial gene, nad5, for selected taxa as observed and predicted in this study. RNA editing rates for both single taxa and plant groups are listed. * indicates data has been obtained from Steinhauser et al. (1999)…………………………………………………..31