EFFECTS OF CODON USAGE ON MRNA TRANSLATION AND DECAY by VLADIMIR PRESNYAK Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Adviser: Dr. Jeffery Coller Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May, 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Vladimir Presnyak Candidate for the Doctor of Philosophy degree*. Committee chair Timothy Nilsen Dissertation advisor Jeff Coller Committee members Maria Hatzoglou Donny Licatalosi Nathan Morris Date of defense March 18, 2015 * We also certify that written approval has been obtained for any proprietary material contained therein. 2 TABLE OF CONTENTS TABLE OF CONTENTS .................................................................................................... 3 INDEX OF FIGURES ........................................................................................................ 5 INDEX OF TABLES .......................................................................................................... 6 ACKNOWLEDGEMENTS ................................................................................................ 7 ABSTRACT ........................................................................................................................ 8 CHAPTER 1: BACKGROUND ....................................................................................... 10 Cellular context of mRNA decay .................................................................................. 10 Overview of mRNA decay ............................................................................................ 12 Pathways of mRNA decay ............................................................................................ 14 Quality control pathways .............................................................................................. 18 mRNA translation is intricately linked to decay ........................................................... 20 Mechanism is insufficient to explain decay rate ........................................................... 23 Codon bias and optimality ............................................................................................ 23 CHAPTER 2: RARE CODON ANALYSIS..................................................................... 26 Position of rare codons changes effects on decay ......................................................... 26 Destabilization by rare codons depends on translation ................................................. 28 Decay of reporters occurs through the major decay pathway ....................................... 29 Ribosome association of constructs remains unchanged .............................................. 32 CHAPTER 3: GENOMIC ANALYSIS ............................................................................ 34 Determination of half-lives by RNA-seq ...................................................................... 34 The search for features correlating with decay ............................................................. 37 Codon usage correlates with mRNA stability ............................................................... 39 Codon usage in genome falls into distinct patterns ...................................................... 45 Optimal codons increase average ribosome density ..................................................... 49 Related genes appear correlated through codon usage ................................................. 52 CHAPTER 4: EXPERIMENTAL VALIDATION ........................................................... 55 Changes in codon content leads to changes in stability ................................................ 55 Regulation through codon usage dominates over UTR regulation ............................... 56 Codon content affects the major decay pathway .......................................................... 58 HIS3 reporter system allows for fine tuning of mRNA stability .................................. 60 Codon content impacts translation beyond changes in mRNA .................................... 62 Affected step of translation is elongation ..................................................................... 65 3 Changes in codon content can impact cellular fitness .................................................. 68 CHAPTER 5: DISCUSSION ............................................................................................ 70 Overview ....................................................................................................................... 70 Considerations of rare codon experiments .................................................................... 71 Considerations of RNA-seq study ................................................................................ 72 Considerations of codon content experiments .............................................................. 75 Possible roles of DHH1 ................................................................................................ 76 Codon optimality in yeast and other organisms ............................................................ 77 Ribosome as monitor of all mRNA fates ...................................................................... 83 Future directions ........................................................................................................... 84 APPENDIX A: BIOINFORMATICS ............................................................................... 86 Half-life fitting .............................................................................................................. 86 CSC calculation ............................................................................................................ 88 APPENDIX B: MATERIALS AND METHODS ............................................................ 93 Yeast strains and growth ............................................................................................... 93 Plasmids and strain construction ................................................................................... 94 Northern RNA analysis ................................................................................................. 97 Polyribosome analysis .................................................................................................. 98 Asymmetric PCR probes............................................................................................... 99 Plating assays .............................................................................................................. 100 RNA-seq ..................................................................................................................... 100 Alignment and half-life calculation ............................................................................ 101 Statistical techniques ................................................................................................... 102 Heat map generation ................................................................................................... 102 Tables .......................................................................................................................... 103 BIBLIOGRAPHY ........................................................................................................... 111 4 INDEX OF FIGURES Figure 1: Overview of 5’-3’ decay ................................................................................. 15 Figure 2: Structure and features of the rare codon constructs .................................. 26 Figure 3: mRNAs bearing rare codons display reduction in stability dependent on the position of the rare codons ....................................................................................... 28 Figure 4: mRNA translation is required for position dependence ............................. 29 Figure 5: Inclusion of rare codons accelerates both deadenylation and degradation ........................................................................................................................................... 30 Figure 6: Decapping is required for position dependent mRNA destabilization by rare codons ...................................................................................................................... 31 Figure 7: mRNAs bearing rare codons do not change in ribosome distribution ...... 33 Figure 8: Sequencing of poly(A)+ enriched and total mRNA sets produces different halflives ............................................................................................................................ 35 Figure 9: Occurrence of codons correlates with differential effects on mRNA half- life ..................................................................................................................................... 39 Figure 10: Influence on stability correlated with optimality for virtually all codons ........................................................................................................................................... 41 Figure 11: Frequency of occurrence is not equivalent to optimality for codons ....... 42 Figure 12: Data from an independent source show codon effects similar to ours .... 43 Figure 13: Codon effects are dependent on reading frame ......................................... 44 Figure 14: Genes cluster into distinct patterns of codon usage .................................. 46 Figure 15: Individual gene clusters show effects of codon usage...............................