Engineering a direct and inducible protein-RNA interaction to regulate RNA biology

ACS Chem Biol. 2010 Sep 17;5(9):851-61. doi: 10.1021/cb100070j.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aptamers, Nucleotide / genetics
  • Aptamers, Nucleotide / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Gene Expression Regulation, Bacterial*
  • Molecular Sequence Data
  • Protein Binding
  • Protein Biosynthesis
  • RNA / genetics
  • RNA / metabolism
  • RNA, Bacterial / genetics*
  • RNA, Bacterial / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • SELEX Aptamer Technique*
  • Transcription, Genetic

Substances

  • Aptamers, Nucleotide
  • Bacterial Proteins
  • RNA, Bacterial
  • RNA, Messenger
  • Repressor Proteins
  • tetracycline resistance-encoding transposon repressor protein
  • RNA