Title
The RNA Stem−Loop to G‑Quadruplex Equilibrium Controls Mature MicroRNA Production inside the Cell
Author
Satyaprakash Pandey, Prachi Agarwala, Gopal G. Jayaraj, Raimundo Gargallo, Souvik Maiti
Year
2015
Journal
Biochemistry
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 Mg2+ 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.
Instrument
J-815
Keywords
Circular dichroism, Biochemistry