Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 114-131). / The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common regulatory mechanism uses inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this using a novel set of protein-RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from normal cellular physiology. We describe a ligand-responsive protein-RNA interaction module that can be used to target a specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, RNA aptamers binding to the bacterial Tet Repressor protein (TetR) with low- to sub- nanomolar affinities were identified. This interaction is reversibly controlled by tetracycline in a manner analogous to the interaction of TetR with its cognate DNA operator. Aptamer minimization and mutational analyses support a functional role for conserved sequence and structural motifs in TetR binding. We illustrate the utility of this chemically-inducible RNA-protein interaction to directly regulate translation in both a prokaryotic and eukaryotic organism. By genetically encoding TetR-binding RNA elements into the 5'-untranslated region (5'-UTR) of a given mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this modular system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from nonfunctional RNA-TetR interactions. We also demonstrate engineering this TetR-aptamer module to regulate subcellular mRNA localization. This is efficiently achieved by fusing TetR to proteins natively involved in localizing endogenous transcripts, and genetically encoding TetR-binding RNA aptamers into the target transcript. Using this platform, we achieve tetracycline-regulated enhancement of target transcript subcellular localization. We also systematically examine some rules for successfully forward engineering this RNA localization system. Altogether, these results define and validate an inducible protein-RNA interaction module that incorporates desirable aspects of a ubiquitous mechanism for regulating RNA function in Nature and that can be used as a foundation for functionally and reversibly controlling multiple fates of RNA in cells. / by Brian J. Belmont. / Ph.D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/78136 |
| Date | January 2012 |
| Creators | Belmont, Brian J. (Brian Joshua) |
| Contributors | Jacquin C. Niles., Massachusetts Institute of Technology. Dept. of Biological Engineering., Massachusetts Institute of Technology. Dept. of Biological Engineering. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 131 p., application/pdf |
| Rights | MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission., http://dspace.mit.edu/handle/1721.1/7582 |
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