An Investigation of miRNA Repertoires in Bdelloid Rotifers By Anupriya Dutta B.A., Rutgers University, 2002 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Division of Biology and Medicine at Brown University Providence, Rhode Island May 2013 © Copyright 2013 by Anupriya Dutta This dissertation by Anupriya Dutta is accepted in its present form by the Division of Biology and Medicine as satisfying the dissertation requirement for the degree of Doctor of Philosophy. Date___________ ___________________________________ David Mark Welch, Ph.D., Advisor Recommended to the Graduate Council Date___________ ___________________________________ Michael Mckeown, Ph.D., Reader Date___________ ___________________________________ Richard Bennett, Ph.D., Reader Date___________ ___________________________________ Irina Arkhipova, Ph.D., Reader Date___________ ___________________________________ Kevin Chen, Ph.D., Reader Rutgers University Approved by the Graduate Council Date___________ ___________________________________ Peter Weber Dean of the Graduate School iii Acknowledgements First and foremost I want to thank the many people who have mentored and inspired me throughout my life. The following pages are a product of the your effort and influence. I am deeply grateful for your guidance. Specifically, I would like to thank my advisor, David Mark Welch, for all the helpful discussions and feedback. I would also like to express my gratitude to my thesis committee members made up of Richard Bennett, Irina Arkhipova, and Mike Mckeown. They have also been immensely helpful throughout this experience. I thank Kevin Peterson for introducing me to a project that transformed the course of my thesis work. I thank my outside reader, Kevin Chen. I thank the Brown-MBL program for their support. I gratefully acknowledge the two funding sources that supported this work: NSF and the Watson Fellowship. I thank my family for their support. I found the strength to overcome challenges thanks to a group of caring people I am fortunate enough to call friends. This journey would have been a lot tougher without their comfort, concern and companionship. Lastly, I thank my computer and car for surviving this long. I realize I have not been easy on you these past couple of years, but you have dutifully kept going and that has helped keep me going iv Table of Contents List of Figures ............................................................................................................................ 1 List of Tables ............................................................................................................................. 1 Abstract ....................................................................................................................................... 2 Chapter I ..................................................................................................................................... 4 Introduction .............................................................................................................................. 4 Introduction to microRNAs .................................................................................................. 5 The Discovery of microRNAs ......................................................................................................... 6 miRNA Biogenesis ............................................................................................................................. 8 miRNA Target Interaction .............................................................................................................. 9 Mechanisms of miRNA Regulation ............................................................................................ 11 Implications of miRNA Regulation ............................................................................................ 14 miRNA Evolution ............................................................................................................................. 16 Introduction to Bdelloid Rotifers .................................................................................... 18 Overview of Bdelloid Rotifers ..................................................................................................... 18 Comparison of Bdelloids and Monogononts .......................................................................... 21 Bdelloid Genome Structure ......................................................................................................... 23 Chapter II ................................................................................................................................. 35 General Methods ................................................................................................................... 35 Chapter III ............................................................................................................................... 50 miRNA informatics ............................................................................................................... 50 Chapter IV .............................................................................................................................. 105 Loss of widely conserved miRNAs let-7 and miR-100 in bdelloid rotifers ...... 105 Chapter V ............................................................................................................................... 140 FreQuent substitutions in some Adineta vaga miRNA suggest RNA editing ... 140 Chapter VI .............................................................................................................................. 156 Future Directions ............................................................................................................... 156 Appendix A ............................................................................................................................ 162 Secondary Structures of rotifer miRNAs .................................................................... 162 Appendix A-1 ................................................................................................................................. 163 A.vaga secondary structures .................................................................................................... 163 Appendix A-2 ................................................................................................................................. 168 B.manjavacas secondary structures ...................................................................................... 168 v List of Figures Figure 1-1 Overview of the miRNA pathway ____________________________ 8 Figure 3-1 let-7 family members in nematodes. ________________________ 78 Figure 3-2 let-7 sequences of flatworms _____________________________ 79 Figure 3-3 Processing pipeline of SMD _______________________________ 80 Figure 3-4 Schematic depiction of SMD accessory programs _____________ 81 Figure 3-5 Mixed top hits returned by BLAST __________________________ 96 Figure 3-6 Advantage of the SMD algorithm in identifying correct miRNA homolog match for queried Dme miRNA ______________________________ 97 Figure 3-7 Gap tolerance beyond the seed sequence to minimize substitutions within the match chosen by SMD ___________________________________ 98 Figure 3-8 SMD return of divergent miRNA homologs ___________________ 99 Figure 3-9 The conserved let-7 cluster in B. manjavacas ________________ 101 Figure 3-10 SMD vs. miRBase(BLAST) _____________________________ 102 Figure 3-11 Seed shift in a rotifer miRNA homolog. ____________________ 103 Figure 3-12 Sample output from SMD accessory programs ______________ 103 Figure 4-1 Alignment of rotifer miRNAs ______________________________ 135 Figure 4-2 Ct tables of qPCR reactions ______________________________ 136 Figure 4-3 qPCR amplification of miRNAs ___________________________ 137 Figure 4-4 Northern for let-7 and miR-87 _____________________________ 138 Figure 4-5 Reduced stringency search for A. vaga let-7 _________________ 139 Figure 4-6 Alignment of miR-100/let-7/miR-125 genomic cluster __________ 140 Figure 4-7 Sequences from miRbase of miR-100 and let-7 ______________ 136 Figure 4-8 lin-41 binding sites in B. manjavacas _______________________ 143 Figure 4-9 lin-41 binding sites in A. vaga ____________________________ 144 Figure 4-10 Binding sites on hbl-1 and dicer-1 ________________________ 145 Figure 5-1 miR-125 editing in A. vaga. ______________________________ 152 Figure 5-2 miR-125 reads from B. manjavacas ________________________ 153 Figure 5-3 Alignment of isomirs corresponding to conserved miRNAs from SMD output _______________________________________________________ 154 Figure 5-4 Lack of C-to-U substitutions in miR-1175 and miR-315 _________ 155 List of Tables Table 3-1 Summary of SMD and BLAST identification of Dme miRNAs _____ 82 Table 3-2 SMD and BLAST identification of Dme miRNAs ________________ 84 Table 3-3 SMD and BLAST assignment of Dme miRNAs _______________ 100 Table 4-1 Sequences of qPCR probes ______________________________ 126 Table 4-2 Abundance of conserved miRNAs from small RNA libraries _____ 127 Table 4-3 Sequences of conserved miRNA homologs from rotifer surveys __ 128 Table 4-4 Genomic environment of miR-125 loci in A. vaga. _____________ 135 1 Abstract An exceptional group of aquatic invertebrates makes up Class Bdelloidea. Bdelloid rotifers are the only group of ancient asexual animals known.