Engineering a direct and inducible protein-RNA interaction to regulate RNA biology.
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.
Belmont
Brian J
BJ
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, 02139, USA.
Niles
Jacquin C
JC
D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D052156:SELEX Aptamer Technique; D014158:Transcription, Genetic
D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D012313:RNA