The RNA Stem-Loop to G-Quadruplex Equilibrium Controls Mature MicroRNA Production inside the Cell

Biochemistry. 2015 Dec 8;54(48):7067-78. doi: 10.1021/acs.biochem.5b00574. Epub 2015 Nov 19.

Abstract

The biological role of the existence of overlapping structures in RNA is possible yet remains very unexplored. G-Rich tracts of RNA form G-quadruplexes, while GC-rich sequences prefer stem-loop structures. The equilibrium between alternate structures within RNA may occur and influence its functionality. We tested the equilibrium between G-quadruplex and stem-loop structure in RNA and its effect on biological processes using pre-miRNA as a model system. Dicer enzyme recognizes canonical stem-loop structures in pre-miRNA to produce mature miRNAs. Deviation from stem-loop leads to deregulated mature miRNA levels, providing readout of the existence of an alternate structure per se G-quadruplex-mediated structural interference in miRNA maturation. In vitro analysis using beacon and Dicer cleavage assays indicated that mature miRNA levels depend on relative amounts of K(+) and Mg(2+) ions, suggesting an ion-dependent structural shift. Further in cellulo studies with and without TmPyP4 (RNA G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated by G-quadruplex to stem-loop equilibrium in a subset of pre-miRNAs. Our combined analysis thus provides evidence of the formation of noncanonical G-quadruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • G-Quadruplexes*
  • Gene Expression Regulation
  • Humans
  • MCF-7 Cells
  • MicroRNAs / chemistry*
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Ribonuclease III / metabolism*
  • Ribonuclease T1 / metabolism
  • Transcription, Genetic

Substances

  • MIRN27 microRNA, human
  • MicroRNAs
  • mirnlet7 microRNA, human
  • Ribonuclease III
  • Ribonuclease T1