BIOINFORMATIC IDENTIFICATION OF SMALL RNA (AND THEIR TARGETS) IN THE TESTIS OF CLAWED FROGS ( XENOPUS AND SILURANA ): IMPLICATIONS FOR MALE FERTILITY by M.J. MADISON-VILLAR Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON December 2011 Copyright © M.J. Madison-Villar 2011 All Rights Reserved ACKNOWLEDGEMENTS I would like to thank all the frogs who died for this study. I would also like to thank Dr. Pawel Michalak. If it weren’t for him, I may have lived and died without ever once having set foot in the grate state of Texas. I also want to thank Dr. Paul Chippindale for adopting me in lieu of Dr. Michalak’s departure from UTA, as well as my other committee members, Drs. Esthér Betran, Jeff Demuth, and Shawn Christensen. You have all been great people. Further thanks to Drs. Daina Ma and Alie Koroma for being my soundboard and support system; Eldon Prince for assistance with the Ruby scripts; Becky Robinson and Mike Natishyn for being awesome study buddies and having all the stats answers I could ask for; and Dr. Mandy Keogh for just listening and being there when I needed someone to talk to. Last, but certainly not least, I want to thank (insert name here). November 11, 2011 iii ABSTRACT BIOINFORMATIC IDENTIFICATION OF SMALL RNA (AND THEIR TARGETS) IN THE TESTIS OF CLAWED FROGS ( XENOPUS AND SILURANA ): IMPLICATIONS FOR MALE FERTILITY M.J. Madison-Villar, PhD The University of Texas at Arlington, 2011 Supervising Professor: Supervising Professors Paul Chippindale and Pawel Michalak (VBI) Chapter one sets the stage for this project, which lies somewhere near the intersection of speciation, genome, and reproductive biology. Chapter two identifies miRNA with shared expression profiles ( Silurana and Xenopus ). These miRNA are pulled from a subset that has retained testis expression despite a hybridization induced WGD event early in the Xenopus lineage (~21-41 MYA). Allopolyploidization is a revolutionary mechanism of genome evolution, and this event should have afforded an opportunity for diversification and changes in the expression of what are generally highly conserved transcript regulators. It is inferred that the miRNA identified in this study retained testis expression due to strict selection, and that their targets should participate in key pathways, such as those that would ensure reproductive potential. This chapter moves on to identify the most likely targets of these miRNA, and then investigates their fates following WGD. Given the expectation of widespread genome disruption following allopolyploidization, it is hypothesizes that the miRNA targets (kdm2a, map3k9, kif23, smarca4, clasp1, eed, nob1, and abcb1 ) will, if retained, show evidence of sub/neofunctionalization. Most targets are clearly iv associated with fertility, and several are associated with early embryogenesis. This latter association suggests a possible role for male mediated miRNA outside of the male germline, where they may help to facilitate fertilization and/or help regulate maternally deposited mRNA. Chapter three identifies a subset of testis expressed miRNA that are misexpressed in the sterile hybrids, and associates these with miRNA known from the testis of mammals. This chapter proposes a subset of miRNA that may have a conserved role in ensuring male reproductive potential in all of vertebrates; it is currently in press with the Journal of Molecular Evolution. Chapter four investigates pachytene expressed pilRNA (PIWI-like RNA), and how the repeat and non-repeat subpopulations respond to hybridization. Part one describes the repeat associated population (rapilRNA): relative abundance, which transposable elements (TEs) they are associated with, how they map to both Class I and Class II elements, and which DNA strand they are derived from. Part two investigates the response of rapilRNA to hybridization: fold change between hybrids and parental taxa is quantified, and ping-pong amplification is considered, but rejected. Part three identifies misexpressed pilRNA from those having homology to previously identified piRNA, possible targets are identified, and the roles of these targets in male reproduction are investigated. Many of these recover association with SUMOlation, a post-translational modification previously linked to the same cellular locations as the protein (MAEL, MIWI) partners of piRNA (chromatoid bodies). v TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................... ……………..iii ABSTRACT ..................................................................................................................................... iv LIST OF FIGURES .......................................................................................................................... ix LIST OF TABLES ............................................................................................................................ xi Chapter Page 1. OVERVIEW AND BACKGROUND INFORMATION ....................................................... 1 1.1 Speciation ......................................................................................................... 1 1.2 Small RNA ........................................................................................................ 2 1.3 SmRNA, Epigenetics, and Male Reproduction ................................................ 4 1.3.1 Overview of Spermatogenesis ......................................................... 6 1.4 Hybridization, Genomic Shock, and Mobile Elements ..................................... 7 2. IDENTIFYING TESTIS-DERIVED MIRNA RETAINED IN LIGHT OF DYNAMIC GENOME RESTRUCTURING, AND INFERRING THE LONG-TERM EVOLUTIONARY (21-41 MY) TRAJECTORY OF REVOLUTIONARILY ACQUIRED (ALLOPOLYPLOID) MIRNA TARGETS ..................................................... 9 2.1 miRNA-mRNA Heteroduplex Dynamics ........................................................... 9 2.2 Materials and Methods ................................................................................... 13 2.2.1 cDNA Acquisition, Expression Profiling, and Cluster Analysis ...... 13 2.2.2 Identification of Gene Targets ........................................................ 15 2.2.3 Identification of Homeologs ............................................................ 16 2.2.4 Identification of Sub/Neofunctionalization ...................................... 17 2.2.5 Ka/Ks Measures of Selection ......................................................... 17 2.3 Results ........................................................................................................... 18 vi 2.3.1 MiRNA and Gene Target Identification .......................................... 18 2.3.2 Homeologs of kdm2a and Inferences of Sub/Neofunctionalization .............................................................. 21 2.3.3 Homeologs of eed and Inferences of Sub/Neofunctionalization ............................................................... 22 2.3.4 Homeologs of smarca4 and Inferences of Sub/Neofunctionalization .............................................................. 25 2.3.5 Homeologs of map3k9 and Inferences of Sub/Neofunctionalization .............................................................. 27 2.3.6 Homeologs of kif23 and Inferences of Sub/Neofunctionalization ............................................................... 28 2.3.7 Homeologs of clasp1 and Inferences of Sub/Neofunctionalization ............................................................... 30 2.3.8 Homeologs of nob1 and Inferences of Sub/Neofunctionalization ............................................................... 30 2.3.9 Homeologs of abcb1b and Inferences of Sub/Neofunctionalization ............................................................... 33 2.4 Discussion ...................................................................................................... 33 3. MISEXPRESSION OF TESTICULAR MICRORNA IN STERILE XENOPUS HYBRIDS POINTS TO TETRAPOD SPECIFRIC MICRORNAS ASSOCIATED WITH MALE FERTILITY ............................................................................................... 37 3.1 Materials and Methods ................................................................................... 38 3.2 Results ........................................................................................................... 39 3.3 Discussion ...................................................................................................... 43 3.4 Conclusions .................................................................................................... 47 4. REPEAT AND NON-REPEAT ASSOCAITED PIWI-LIKE (PILRNA) IN THE TESTIS OF FERTILE X. LAEVIS , X. MUELLERI , AND THEIR STERILE INTERSPECIFIC HYBRID ........................................................................................................................ 49 4.1 Materials and Methods ................................................................................... 51 4.1.1 Preparation of smRNA Library and Summarization of Reads ....... 51 4.1.2 Characterization and Identification of Repeat Associated smRNA .......................................................................................... 51 vii 4.1.3 Expression of Repeat Associated smRNA in Hybrid and Parental Taxa ................................................................................. 52 4.1.4 Quantification