RNA stem-loop to G-quadruplex equilibrium controls mature miRNA production inside the cell

Biological role for existence of overlapping structures in RNA is possible yet remains very less explored. G-rich tracts of RNA form G-quadruplexes while GC-rich sequences prefer stem-loop structures. Equilibrium between alternate structures within RNA may occur and influence its functionality. We tested 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 existence of 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-stem-loop equilibrium in a subset of pre-miRNAs. Our combined analysis thus provides evidence for formation of non-canonical G-quadruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner

Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control

Readthrough into the 3′ untranslated region (3′ UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3′ UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis

RNA stem-loop to G-quadruplex equilibrium controls mature miRNA production inside the cell

Biological role for existence of overlapping structures in RNA is possible yet remains very less explored. G-rich tracts of RNA form G-quadruplexes while GC-rich sequences prefer stem-loop structures. Equilibrium between alternate structures within RNA may occur and influence its functionality. We tested 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 existence of 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-stem-loop equilibrium in a subset of pre-miRNAs. Our combined analysis thus provides evidence for formation of non-canonical G-quadruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner

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

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. <i>In vitro</i> 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 <i>in cellulo</i> 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