Engineering a Direct and Inducible Protein-RNA Interaction To Regulate RNA Biology
Author(s)Belmont, Brian Joshua; Niles, Jacquin
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The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common mechanism uses inducible protein−RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this regulatory mode in cells using a novel set of protein−RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from endogenous cellular physiology. Achieving this outcome, however, has previously proven challenging. Here, we describe a ligand-responsive protein−RNA interaction module, which 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 subnanomolar affinities were obtained. 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 two conserved sequence motifs in TetR binding. As an initial illustration of using this system to achieve protein-based regulation of RNA function in living cells, insertion of a TetR aptamer into the 5′-UTR of a reporter mRNA confers post-transcriptionally regulated, ligand-inducible protein synthesis in E. coli. 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 can be used as a foundational interaction for functionally and reversibly controlling the multiple fates of RNA in cells.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering
ACS Chemical Biology
American Chemical Society
Belmont, Brian J., and Jacquin C. Niles. “Engineering a Direct and Inducible Protein−RNA Interaction To Regulate RNA Biology.” ACS Chemical Biology 5.9 (2010): 851-861.
Author's final manuscript