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Signature of Author Computational and experimental analysis of plant microRNAs by Matthew W. Jones-Rhoades B.A., Chemistry (1999) Grinnell College Submitted to the Department of Biology in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biology MASSACHUSETTSINSTATE at the OF TECHNOLOGY Massachusetts Institute of Technology MAY 2 7 2005 June 2005 LIBRARIES © 2005 Massachusetts Institute of Technology All rights reserved Signatureof Author.................................... ................. ................. Department of Biology x.. '"---.~ May 20, 2005 Certified by ......... · .................................................... David P. Bartel Professor of Biology Thesis Supervisor Acceptedby ... ......... Stephen P. Bell Professor of Biology Chairman, Graduate Student Committee ,' tti v ;S Computational and experimental analysis of plant microRNAs Matthew W. Jones-Rhoades Submitted to the Department of Biology on May 20, 2005 in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biology ABSTRACT MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that mediate gene regulation in plants and animals. We demonstrated that Arabidopsis thaliana miRNAs are highly complementary (0-3 mispairs in an ungapped alignment) to more mRNAs than would be expected by chance. These mRNAs are therefore putative regulatory targets of their complementary miRNAs. Many miRNA complementary sites are conserved to the monocot Oryza sativa (rice), implying evolutionary conservation based on function at the nucleotide level. The majority of predicted miRNA targets encode for transcription factors and other proteins with known or inferred roles in developmental patterning, implying that the miRNAs themselves are high-level regulators of development. Our findings indicated that miRNAs are key components of numerous regulatory circuits in plants and set the stage for numerous additional experiments to investigate in depth the significance of miRNA-mediated regulation for particular target families and genes. We developed a comparative genomics approach to identify miRNAs and miRNA targets conserved between Arabidopsis and Oryza. Seven previously unknown miRNAs families were experimentally verified, bringing the total number of known miRNA genes in Arabidopsis to 92, representing 22 families. We expanded the range of functionalities known to be regulated by miRNAs to include F-box proteins, laccases, superoxide dismutases, and ATP-sulfurylases. The expression of miR395, which targets sulfate metabolizing enzymes, is induced by sulfate- starvation, demonstrating that miRNA expression can be responsive to growth conditions. We investigated the biological role of miR394-mediated regulation of Atlg27340, an F-box gene of previously unknown function. Transgenic plants expressing a miR394-resistant version of Atlg27340 displayed a range of developmental abnormalities, including radialized and fused cotyledons, absent shoot apical meristems, curled and radialized leaves, and abortive flowers. The severity of these abnormalities correlated with the overaccumulation of Atlg27340 mRNA. These findings confirm the biological relevance of the interaction between miR394 and Atlg27340, and represent the first insights into the roles of miRNA-mediated regulation of F-box genes. Our results establish that both MIR394 and Atlg27340 are important regulators of meristem identity, and suggest that Atlg27340 targets an activator of class III HD-ZIP function for ubiquitination and proteolysis. Thesis Supervisor: David Bartel Title: Professor of Biology 2 Acknowledgements I would like to thank David Bartel for being a tremendous advisor, role model, and friend throughout my time at MIT. I would like to thank all the past and present members of the Bartel lab, especially Mike Axtell, Scott Baskerville, Nelson Lau, Ben Lewis, Lee Lim, Allison Mallory, Ramya Rajagopalan, Brenda Reinhart, I-hung Shih, Herv6 Vaucheret, and Soraya Yekta. You have been a joy to work and play with, and a continual source of reagents, help, and advice. I would also like to thank Bonnie Bartel, without whom the analysis of plant miRNAs would have been ten times harder. I would like to thank my family, especially my parents, for always believing in me and supporting me. Most importantly, I would like to thank my wife Melinda for being my partner and my best friend. Thanks for putting up with all the late nights and odd-hours; you were always there to encourage me when things went well and to give me perspective when they didn't. 3 Table of contents Abstract 2 Acknowledgements 3 Table of contents 4 Introduction 5 Chapter I 42 Prediction of plant microRNA targets ChapterI has been publishedpreviously as: Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, and Bartel DP, "Predictionof plant microRNAtargets. " Cell 110(4):513-520 (2002) © CellPress. Chapter II 65 Computational identification of plant microRNAs and their targets, including a stress-induced miRNA ChapterII has been publishedpreviously as: Jones-RhoadesMW, and BartelDP, "Computationalidentification of plant microRNAsand their targets,including a stress-inducedmiRNA. " MolecularCell 14(6): 787-799 (2004) © Cell Press. Chapter III 101 MicroRNA-mediated regulation of an F-box gene is required for embryonic, floral, and vegetative development Appendix A 120 Arabidopsis, Oryza, and Populus miRNA complementary sites Appendix B 128 Appendix B has been published previously as: ReinhartBJ, WeinsteinEG, RhoadesMW, BartelB, and BartelDP, "MicroRNAs in plants. "Genes & Development 16(13): 1616-1626 © Cold Spring HarborLaboratory Press. Appendix C 139 Appendix C has been published previously as: Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, and Burge CB, "Prediction of mammalian microRNA targets. " Cell 115(7): 787-798 (2003) © Cell Press. 4 Introduction The biology of multicellular organisms requires a complex network of gene regulatory pathways. MicroRNAs (miRNAs) are key components of this network which had been overlooked until recently. Initially discovered as regulators of developmental timing in C. elegans, miRNAs are now known to serve in a variety of regulatory roles in both plants and animals. The hallmark of a miRNA is a short (-20-24 nt), endogenously expressed non-coding RNA which is processed by RNaseIII proteins such as Dicer from a longer ssRNA precursor that contains a stem-loop secondary structure (reviewed in (11)). MicroRNAs are chemically and functionally similar to short interfering RNAs (siRNAs), which are processed by Dicer from long dsRNA precursor and which are central to the related phenomenon of RNA interference (RNAi), post-transcriptional gene silencing (PTGS), and transcriptional gene silencing (TGS). Mature miRNAs are incorporated into RNAi-induced silencing complexes (RISCs), in which the miRNA guides repression of target genes. Although miRNAs are deeply conserved with both the plant and animal kingdoms, there are substantial differences in the mechanism and scope of miRNA-mediated gene regulation between the two kingdoms, several of which have been instrumental in the rapid increase in our understanding of plant miRNA biology. Plant miRNAs are highly complementary to conserved target mRNAs, a fact which has allowed for the rapid and confident bioinformatic identification of plant miRNA targets (46, 113). Plant miRNAs guide the cleavage of their complementary mRNA targets, an activity which is readily assayed in vitro and in vivo (49, 76, 126). In addition, Arabidopsis is a genetically tractable model organism, which has enabled the study of the genetic pathways which underlie miRNA-mediated regulation and the phenotypic consequences of perturbing miRNA-mediated gene regulation. The picture emerging from this recent research is that plant miRNAs are master regulators of genetic pathways: the majority of genes regulated by plant miRNAs are themselves regulators such as transcription factors, F-box proteins, and RNAi related proteins. MicroRNAs: like siRNAs, but different Before discussing miRNAs, it is useful to consider a highly similar class of small RNAs, the small interfering RNAs (siRNAs). In Arabidopsis, siRNAs are the majority of small RNAs (75, 112, 124, 138), and have been implicated in a variety of pathways, including defense against 5 viruses, the establishment of heterochromatin, silencing of transposons and transgenes, and the post-transcriptional regulation of mRNAs (reviewed in (13)). MicroRNAs and siRNAs have much in common; both types of small RNAs are 20-24 nucleotides long, and both are processed from longer RNA precursors by Dicer ribonucleases (15, 38, 43, 50). Both are incorporated into ribonucleoprotein (RNP) complexes in which the small RNAs, through their base pairing potential, guide repression of target genes, and, as discussed below, the mechanisms by which they repress target genes are also similar. The fundamental difference between the two classes, then, is nature of their precursors; siRNAs are processed from long, dsRNAs, whereas miRNAs are processed from RNAs that are single-stranded but contain imperfect stem-loop secondary structures. In addition, there are a number of general, if not absolute, characteristics that set miRNAs apart from siRNAs. Many miRNAs are conserved between related organisms, whereas most endogenously expressed siRNAs are not(57, 61, 63, 112). Many (but not all) siRNAs target the gene from which they are derived. In contrast, a miRNA regulates genes unrelated to the locus from which the miRNA was derived. In addition,
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