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

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• 作者：Satyaprakash Pandey ; Prachi Agarwala ; Gopal G. Jayaraj ; Raimundo Gargallo ; Souvik Maiti
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：December 8, 2015
• 年：2015
• 卷：54
• 期：48
• 页码：7067-7078
• 全文大小：528K
• ISSN：1520-4995

文摘

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鈥搇oop structures. The equilibrium between alternate structures within RNA may occur and influence its functionality. We tested the equilibrium between G-quadruplex and stem鈥搇oop structure in RNA and its effect on biological processes using pre-miRNA as a model system. Dicer enzyme recognizes canonical stem鈥搇oop structures in pre-miRNA to produce mature miRNAs. Deviation from stem鈥搇oop 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鈥搇oop 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鈥搇oop structure inside the cell and its effect on miRNA maturation in a comprehensive manner.