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Unexpected Repeat Associated Proteins Unexpected Repeat Associated Proteins A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Brian Ballard Gibbens IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ADVISOR: Laura P.W. Ranum, Ph.D. FEBRUARY, 2011 © BRIAN BALLARD GIBBENS, 2011 ACKNOWLEDGEMENTS I would like to thank all those who helped me achieve this goal. I would like to start by thanking the Ranum Lab and all of its past and current members. First and foremost, I would like to thank my advisor, Dr. Laura Ranum for her support, guidance, and thought provoking ideas. I would also like to acknowledge my fellow graduate students for fostering a productive lab environment. Our shared sense of purpose and comradery really made it a joy to come in each day. I am very grateful to Dr. Melinda Moseley for listening to my ideas and for giving me a few of her own. I would also like to express my deep appreciation to Dr. Tao Zu for his help and advice on countless occasions. His unparalleled skill and patience is truly inspiring. Finally, I‟d like to thank the technicians and undergrads who work tirelessly to ensure that the lab can keep functioning. I would also like to thank Lisa Duvick and Shaojin Lai in the Orr lab for their help and advice with Western blots and tissue culture. In addition, I‟d like to thank the Dubinsky lab for their kind gift of Huntington‟s disease (HD) constructs. Last but not least, I would like to thank my family including my parents, my in- laws, my wife Ying and my son Wally whose love and support has made all of this possible and worthwhile. i ABSTRACT Spinocerebellar ataxia type 8 (SCA8) is one of a number of dominantly inherited disorders caused by triplet CTG•CAG repeat expansions (2). While investigating the mechanisms of SCA8, Dr. Ranum‟s lab made the surprising discovery that CAG•CTG expansion constructs express homopolymeric polyglutamine, polyalanine and polyserine expansion proteins without an ATG start codon (3). This repeat associated non-ATG (RAN) translation occurs in transfected cells and lenti-viral transduced cells and brains. Additionally, in vivo mouse and human data demonstrate that RAN-translation across human SCA8 and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins (3). RAN-translation can occur across CAG expansions in a number of different sequence contexts, but the mechanism of this newly discovered phenomenon, which does not follow the previously described rules of translational regulation, is completely unknown. To gain a better understanding of the mechanisms of RAN-translation and their potential role in microsatellite disorders, I chose to test the hypothesis that RNA sequence variations within and outside of the repeat affect the efficiency of RAN- translation. Data described in my thesis support the following conclusions: 1) the efficiency of RAN-translation in different frames can be positively and negatively affected by the nucleotide sequence within and around the repeat tract; 2) non-ATG translation in rabbit reticulocyte lysates (RRLs) is much less permissive than in HEK293T and N2a cells, initiates with methionine, and requires close cognate start codons (e.g. ATT and ATC); 3) RAN-translation in multiple frames can occur in the ii presence or absence of an ATG-initiated open reading frame; 4) cellular factors found in HEK293T and N2a cells substantially enhance RAN-translation compared to cell free RRLs; 5) RAN-translation is enhanced across repeat motifs which form hairpin structures. These data have led me to propose two models for RAN-translation: the “stalling model” and the “IRES-like model”. The stalling model proposes that scanning ribosomes are stalled by repeat-containing hairpins until translation is initiated and that permissive initiation is increased when hairpins or RNA binding proteins interact with the ribosome and its associated translation initiation factors. The IRES-like model proposes that repeat containing hairpins facilitate RAN-translation by mimicking an internal ribosome entry site (IRES) to recruit the ribosome and initiation factors and initiate translation at non-ATG sites. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................. i ABSTRACT ....................................................................................................................... ii LIST OF TABLES .......................................................................................................... vii LIST OF FIGURES ....................................................................................................... viii ABREVIATIONS ............................................................................................................. ix CHAPTER 1: INTRODUCTION TO RAN-TRANSLATION..................................... 1 REPEAT EXPANSION DISORDERS ........................................................................... 1 Polyglutamine Diseases and Protein Gain-of-Function Mechanisms ........................ 3 Myotonic Dystrophy and the RNA Gain-of-Function Mechanism .............................. 6 Spinocerebellar Ataxia Type 8 (SCA8) ........................................................................ 9 RAN-TRANSLATION ................................................................................................. 13 SUMMARY .................................................................................................................. 17 HYPOTHESIS .............................................................................................................. 18 CHAPTER 2: SEQUENCE AND INITIATION REQUIREMENTS OF RAN- TRANSLATION ............................................................................................................. 28 INTRODUCTION ......................................................................................................... 28 Scanning Mechanism of Translation Initiation ......................................................... 28 IRES Mechanism of Translation Initiation ................................................................ 29 Alternative Start Codons ........................................................................................... 30 Thesis Aims ................................................................................................................ 32 RESULTS...................................................................................................................... 33 Unspliced/unedited Transcripts are Capable of RAN-Translation ........................... 33 RAN-Translation in RRL Requires Alternative Start Codons ................................... 34 Methionine is Used to Initiate Non-ATG Translation in RRL ................................... 36 RAN-Translation Can Occur in an Existing ATG-Initiated PolyGln ORF ............... 37 DISCUSSION ............................................................................................................... 39 Unspliced/unedited Transcripts are Capable of RAN-Translation ........................... 39 RAN-Translation in RRL Requires Alternative Start Codons ................................... 40 iv Methionine is Used to Initiate Non-ATG Translation in RRL ................................... 41 RAN-Translation Can Occur in an Existing ATG-Initiated PolyGln ORF ............... 42 CHAPTER 3: EFFECT OF HAIRPIN SECONDARY STRUCURE ON RAN- TRANSLATION ............................................................................................................. 54 INTRODUCTION ......................................................................................................... 54 Hairpin Secondary Structure and RAN-Translation ................................................. 54 Thesis Aims ................................................................................................................ 57 RESULTS...................................................................................................................... 57 RAN-Translation across Hairpin Forming and Non-Hairpin Forming Repeats ...... 57 RAN-Translation across a CAG/CAA Hybrid Repeat ............................................... 62 RAN-Translation across Clamped Constructs .......................................................... 62 RAN-Translation across SCA1 Constructs ................................................................ 64 DISCUSSION ............................................................................................................... 65 RAN-Translation across Hairpin Forming and Non-Hairpin Forming Repeats ...... 65 RAN-Translation across a CAA/CAG Hybrid Repeat ............................................... 68 RAN-Translation across Clamped Constructs .......................................................... 69 RAN-Translation across SCA1 Constructs ................................................................ 69 General Findings and Conclusions ........................................................................... 70 CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS ............................... 85 RESULTS SUMMARY AND CONCLUSIONS ......................................................... 85 POSSIBLE MECHANISMS OF RAN-TRANSLATION ............................................ 88 The Stalling Mechanism of RAN-Translation ............................................................ 88 The IRES-Like Mechanism
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