A Computational and Evolutionary Approach to Understanding Cryptic Unstable Transcripts in Yeast By Jessica M. Vera B.S. University of Wisconsin-Madison, 2007 A thesis submitted to the Faculty of the Graduate School in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular, Cellular, and Developmental Biology 2015 This thesis entitled: A Computational and Evolutionary Approach to Understanding Cryptic Unstable Transcripts in Yeast written by Jessica M. Vera has been approved for the Department of Molecular, Cellular, and Developmental Biology Tom Blumenthal Robin Dowell Date The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline iii Vera, Jessica M. (Ph.D., Molecular, Cellular and Developmental Biology) A Computational and Evolutionary Approach to Understanding Cryptic Unstable Transcripts in Yeast Thesis Directed by Robin Dowell Cryptic unstable transcripts (CUTs) are a largely unexplored class of nuclear exosome degraded, non-coding RNAs in budding yeast. It is highly debated whether CUT transcription has a functional role in the cell or whether CUTs represent noise in the yeast transcriptome. I sought to ascertain the extent of conserved CUT expression across a variety of Saccharomyces yeast strains to further understand and characterize the nature of CUT expression. To this end I designed a Hidden Markov Model (HMM) to analyze strand-specific RNA sequencing data from nuclear exosome rrp6Δ mutants to identify and compare CUTs in four different yeast strains: S288c, Σ1278b, JAY291 (S.cerevisiae) and N17 (S.paradoxus). My RNA-seq based method has greatly expanded upon previous CUT annotations in S.cerevisiae, underscoring the extensive and pervasive nature of unstable transcription. Utilizing a four-way genomic alignment I identified a large population of CUTs with conserved syntenic expression across all four strains. Furthermore I observed that certain configurations of gene-CUT pairs, where CUT expression originates from a gene 5’ or 3’ nucleosome free region, correlate with distinct expression trends for the associated gene. Bidirectional gene-CUT pairs correlate with higher expression of genes, and antisense gene-CUT pairs correlate with reduced gene expression. Interestingly these effects on gene expression are most prevalent in the presence of conserved CUT expression. Additionally I have shown that CUTs lack a well-defined 3’ nucleosome free region that is commonly observed at protein-coding genes, and suggests that 3’ NFRs are not characteristic of Sen1-dependent terminated transcripts. iv Table of Contents Chapter I - Introduction ........................................................................................................... 1 Yeast ncRNAs ........................................................................................................................................... 2 The Basics of Transcription ....................................................................................................................... 3 Alternative RNAP II Transcription Termination Pathways......................................................................... 4 TRAMP (Trf4/Air2/Mtr4 polymerase complex) .......................................................................................... 7 Nuclear Exosome ...................................................................................................................................... 8 Evolutionary Context of Sen1-dependent Termination ........................................................................... 10 Cryptic Unstable Transcripts (CUTs) ...................................................................................................... 11 Conclusion ............................................................................................................................................... 12 Figures .................................................................................................................................................... 14 Chapter II - Survey of Cryptic Unstable Transcripts in Yeast ..............................................16 Authors' Contributions ............................................................................................................................. 16 Introduction .............................................................................................................................................. 16 Results and Discussion ........................................................................................................................... 18 Explicit duration HMM identifies CUTs de novo from RNA-seq data .................................................. 18 CUTs lack a defined 3’ nucleosome free region ................................................................................. 20 A large set of CUTs show conserved expression between S.cerevisiae and S.paradoxus ............... 22 Distinct trends of gene expression correlate with CUT expression in specific architectures with genes ............................................................................................................................................................ 24 Antisense CUT expression shows evidence of transcriptional interference on sense strand ............ 25 Divergent CUT expression correlates with higher gene expression ................................................... 27 Conclusion ............................................................................................................................................... 28 Methods ................................................................................................................................................... 29 Strain construction .............................................................................................................................. 29 Genome sequences and annotations ................................................................................................. 29 RNA-sequencing libraries ................................................................................................................... 30 Explicit duration hidden Markov model ............................................................................................... 30 CUT identification ................................................................................................................................ 31 Annotation overlap and significance test ............................................................................................ 31 Nucleosome occupancy and metagene analysis ................................................................................ 32 CUT transcription start site comparisons ............................................................................................ 32 Pecan whole genome alignment ......................................................................................................... 32 Conserved CUT expression ................................................................................................................ 33 CUT expression validation by RT-qPCR ............................................................................................. 33 NFR sharing between CUTs and protein-coding genes ..................................................................... 34 Figures .................................................................................................................................................... 35 Tables ...................................................................................................................................................... 49 v Chapter III - What Mechanisms Govern CUT Expression? ................................................................... 52 Introduction .............................................................................................................................................. 52 Results .................................................................................................................................................... 53 Discussion ............................................................................................................................................... 56 Methods ................................................................................................................................................... 57 Unique CUT Expression ...................................................................................................................... 57 Nucleosome Occupancy and Metagene Analysis............................................................................... 57 Promoter Nucleosome Occupancy Profile Clustering ......................................................................... 57 Figures .................................................................................................................................................... 58 Chapter IV - Assessment of Nascent CUT Expression by NET-qPCR ................................................. 64 Introduction .............................................................................................................................................. 64 Results ...................................................................................................................................................