Molecular Characterization of Animal microRNAs: Sequence, Expression, and Stability. by Nelson C. Lau B.S. Biochemistry and Molecular Biology University at Albany, SUNY, 1999 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY SEPTEMBER 2004 ( 2004 Nelson C. Lau. All Rights Reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part. Signature of Author: Department of Biology (June 30, 2004) Certified by: / David P. Bartel Professor of Biology Thesis Supervisor Accepted by: qtenhin P Rfll' Professor of Biology Chair, Biology Graduate Committee MASSACHUSETTS INSiEUTE OF TECHNOLOGY JUL 0 1 2004 1 LIBRARIES ARCHIVES Molecular Characterization of Animal microRNAs: Sequence, Expression, and Stability. by Nelson C. Lau Submitted to the Department of Biology On June 30, 2004 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ABSTRACT Multicellular organisms possess natural gene-regulatory pathways that employ small RNAs to negatively regulate gene expression. In nematodes, the small temporal RNAs (stRNAs), lin-4 and let-7, negatively regulate genes important in specifying developmental timing. A gene- silencing pathway present in plants, fungi and animals called RNA interference, involves the conversion of long double-stranded RNA into short interfering RNAs, which can serve to negatively regulate endogenous genes or suppress the replication of viruses and transposons. To investigate how wide a role small RNAs play in regulating gene expression in animals, we developed a RNA cloning procedure and first applied it to the cloning of small RNAs from the nematode, Caenorhabditis elegans. In addition to cloning lin-4 and let-7 sequences, our study revealed a large number of conserved and highly expressed small RNAs with features reminiscent of stRNAs. Because not all of these small RNAs were expressed in temporal fashion, we and others have referred to this novel class of tiny RNAs as microRNAs. We completed an extensive census of microRNA (miRNA) genes in C.elegans by cloning and bioinformatics searches to lay the groundwork for future functional studies. Our census marked the detection of nearly 90 C.elegans miRNAs, estimated an upper-bound of about 120 miRNA genes in C.elegans, and detailed the conservation and clustering of miRNA sequences. We also determined the high molecular abundance of several miRNAs in C.elegans and Hela cells. In an effort to understand the reason for the high molecular abundance of miRNAs, we constructed an inducible miRNA-expressing cell line to measure the stability of animal miRNAs. Time course measurements suggested a long (>24 hours) half-life for two well-conserved miRNAs. These cells lines may also be useful for other functional studies, such as validation of putative mRNA target genes. Thesis supervisor: David P. Bartel Title: Professor of Biology 2 Acknowledgements I would like to thank my advisor, David Bartel, for being an extraordinary mentor and teaching me the standard for rigorous biological research. I am truly grateful for his guidance and his sincerity, and I admire his brilliance, meticulousness, perseverance and enthusiasm for doing great science. Although I entered graduate school with a mind set on performing an in vitro selection of a ribozyme, I was fortunate to receive the direction from David to explore the new and exciting world of microRNAs. I thank the many past and present members of the Bartel Lab for collaborations, reagents, advice and other intangibles. I especially acknowledge Lee Lim, Matt Jones-Rhoades, Soraya Yekta, Aliaa Abdelhakim, and Earl Weinstein for collaborating on work in Chapters 1 and 2. I thank Craig Ceol, Peter Unrau, Erik Schultes, Chuck Merryman, Brenda Reinhart, and Alex Ensminger for advice with regards to work in Chapter 1. I thank I-hung Shih and Chang-Zheng Chen for advice and the Matsudaira lab for facilities with regards to work in Chapter 3. I also thank Scott Baskerville and Laura Resteghini for the things they do to make the drudgeries of labwork go more smoothly. The Bartel lab, the Whitehead Institute, and MIT, together, has been an exciting environment for me to study biology, and I will miss but remember this place well. I want to express my gratitude to my family for their support and for making me the person I am today. My father instilled in me a love for science, by teaching me constantly, engaging me with science experiments as a child, and giving me the inspiration to follow in his footsteps of obtaining a PhD. My mother has been courageous in supporting my father through his struggle with diabetes, and together they have placed their needs second so that I may succeed at my graduate career. My younger brother has been a close friend and also a fellow aspiring scientist. Last but not least, I thank my wife, Dianne, who is not only my peer, but is also my confidant and my soul mate, through science and in life. She has supported me in countless ways from undergraduate through graduate school, and she has always inspired me to reach further and accomplish more as a person. 3 Table of Contents Abstract 2 Acknowledgements 3 Table of Contents 4 Introduction 5 Chapter I 62 An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans ChapterI has been publishedpreviously as: N.C. Lau, L.P. Lim, E.G. Weinstein, and D.P. Bartel, "An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans. " Science 294: 858-862. (2001) © American Association for the Advancement of Science. Chapter II 82 The microRNAs of Caenorhabditis elegans ChapterII has beenpublished previously as: LP. Lim, N.C. Lau, E.G. Weinstein, A. Abdelhakim, S. Yekta, M. W. Rhoades, C.B. Burge, and D.P. Bartel, "The microRNAs of Caenorhabditis elegans. " Genes & Development 17: 991-1008. (2003) © Cold Spring Harbor Laboratory Press. Chapter III 131 MicroRNA Stability Determined In an Inducible Cell Line Future Directions 166 Appendix A 181 Appendix A has been published previously as: N.C. Lau, and D.P. Bartel, "Censors of the Genome. " Scientific American 289: 34-41.(2003) © Scientific American Inc. Appendix B 189 Appendix B has been published previously as: N.H. Bergman, N.C. Lau, V. Lehnert, E. Westhof,, and D.P. Bartel, " The three- dimensional architecture of the class I ligase ribozyme. " RNA 10: 176-184. (2004) © Cold Spring Harbor Laboratory Press. Appendix C 198 Isolation and Preliminary Survey of C.elegans miRNA Deletion Mutants 4 Introduction 5 microRNAs: Nuggets of RNA Reveal a Goldmineof Biology Although microRNAs (miRNAs) remained hidden in the "genomic dirt" of intergenic regions and introns for more than 25 years after the first mutants were isolated [1, 2], they now represent a treasure trove of new biology. Not only are these small RNAs found in many branches of multi-cellular organisms, they also exert an extraordinary level of control on gene expression, and have refreshed our appreciation of RNA's functional diversity in eukaryotes. While the importance of miRNAs in development is undisputed, establishing the integral roles of each miRNA remains a challenge. Progress in miRNA research has been rapid and dramatic. The catalog of miRNAs in diverse organisms has swelled in recent years (Figure 1). Before 2001, only two miRNA genes were known, but quickly the discovery of other miRNAs surged as cloning and computational methods homed in on small RNA genes. Identification and prediction of target mRNAs regulated by miRNAs have recently seen similar increases. I will survey how genetic, biochemical, and genomic approaches have uncovered this rich repository of gene riboregulators, and review our expanding knowledge about the biogenesis and function of miRNAs. A Tale of Worm Mutants The current flood of miRNA knowledge began with research performed in Victor Ambros's lab and Gary Ruvkun's lab who were interested in the problem of heterochrony - the proper ordering and timing of developmental processes. What drove their research was a set of Caenorhabditis elegans mutants with various defects in the manner by which the cells should divide and differentiate according to their programmed lineage fates [3]. Of these lineage- defective, or lin mutants, two became a focus of study in determining how heterochronic genes 6 interact - lin-4 and lin-14. While the lin-4 loss-of-function (LOF) mutant contained somatic cells that reiterated early larval divisions and whose maturity was retarded, the lin-14 LOF mutants instead displayed cells that precociously differentiate [4]. Cloning and characterization of lin-14 by the Ruvkun lab indicated that proper division of those certain somatic cells depended on a downregulation of LIN-14 during development [5, 6]. The intriguing connection between lin-4 and lin-14 was first hinted by three observations: (1) lin-4 LOF mutants showed similar phenotypes to the lin-14 gain-of-function (GOF) mutants which failed to downregulate LIN-14; (2) accumulation of LIN-14 was also seen in the lin-4 mutant; and (3) lin-14 LOF mutants in a lin-4 LOF background partially suppressed the lin-4 heterochronic defects [7-10]. After the lin-14 gene was cloned, the gene was found to encode a protein that appeared localized to the nucleus and was temporally regulated [5, 6]. Characterization of the lin-14 GOF mutants revealed lesions in the 3' untranslated region (UTR) of the lin-14 mRNA which suggested sequence elements that might be acted upon by the lin-4 gene to negatively regulate LIN-14 levels [7-9, 11]. Although one might have expected that lin-4 could be an RNA-binding protein, such notions were dispelled when the Ambros lab cloned a -700 nt genomic fragment capable of rescuing the lin-4 mutant, and which could not encode a significant protein product [12].