{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/78136"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/78136","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Engineered mRNA regulation using an inducible protein-RNA aptamer interaction","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Belmont, Brian J. (Brian Joshua)"],"institution":"Massachusetts Institute of Technology","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Dept. of Biological Engineering.","school":null,"contributors":[],"advisors":["Jacquin C. Niles."],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012","date_published":"2012","updated_at":"2026-07-22T22:21:58Z","subjects":["Biological Engineering."],"languages":["eng"],"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."],"rights_urls":["http://dspace.mit.edu/handle/1721.1/7582"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1721.1/78136","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Jacquin C. Niles."]},{"key":"dc:contributor.department","label":"Department","values":["Massachusetts Institute of Technology. Dept. of Biological Engineering."]},{"key":"dc:contributor.other","label":"Dc Contributor Other","values":["Massachusetts Institute of Technology. Dept. of Biological Engineering."]},{"key":"dc:creator","label":"Author","values":["Belmont, Brian J. 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They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://dspace.mit.edu/handle/1721.1/7582"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1721.1/78136"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.","Cataloged from PDF version of thesis.","Includes bibliographical references (p. 114-131)."]},{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["Engineered mRNA regulation using an inducible protein-RNA aptamer interaction"]}]}],"canonical_facts":{"dc:contributor.advisor":["Jacquin C. Niles."],"dc:contributor.department":["Massachusetts Institute of Technology. Dept. of Biological Engineering."],"dc:contributor.other":["Massachusetts Institute of Technology. Dept. of Biological Engineering."],"dc:creator":["Belmont, Brian J. (Brian Joshua)"],"dc:date.accessioned":["2013-03-28T18:07:12Z"],"dc:date.available":["2013-03-28T18:07:12Z"],"dc:date.issued":["2012"],"dc:description":["Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.","Cataloged from PDF version of thesis.","Includes bibliographical references (p. 114-131)."],"dc:description.abstract":["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."],"dc:description.degree":["Ph.D."],"dc:identifier.uri":["http://hdl.handle.net/1721.1/78136"],"dc:language.iso":["eng"],"dc:publisher":["Massachusetts Institute of Technology"],"dc: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."],"dc:rights.uri":["http://dspace.mit.edu/handle/1721.1/7582"],"dc:subject":["Biological Engineering."],"dc:title":["Engineered mRNA regulation using an inducible protein-RNA aptamer interaction"],"dc:type":["Thesis"]},"updated_at":"2026-07-22T22:21:58Z"}