RESUMO
7SK is a conserved noncoding RNA that regulates transcription by sequestering the transcription factor P-TEFb. 7SK function entails complex changes in RNA structure, but characterizing RNA dynamics in cells remains an unsolved challenge. We developed a single-molecule chemical probing strategy, DANCE-MaP (deconvolution and annotation of ribonucleic conformational ensembles), that defines per-nucleotide reactivity, direct base pairing interactions, tertiary interactions, and thermodynamic populations for each state in RNA structural ensembles from a single experiment. DANCE-MaP reveals that 7SK RNA encodes a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. The 7SK structural equilibrium shifts in response to cell growth and stress and can be targeted to modulate expression of P-TEFbresponsive genes. Our study reveals that RNA structural dynamics underlie 7SK function as an integrator of diverse cellular signals to control transcription and establishes the power of DANCE-MaP to define RNA dynamics in cells.
Assuntos
Fator B de Elongação Transcricional Positiva , Proteínas de Ligação a RNA , Sítios de Ligação/genética , Células HeLa , Humanos , Fator B de Elongação Transcricional Positiva/genética , RNA Nuclear Pequeno/genética , RNA não Traduzido , Proteínas de Ligação a RNA/genéticaRESUMO
RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having deficiencies. Here we convert the common reagent dimethyl sulfate into a useful probe of all 4 RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base-pairing interactions in cells. PAIR-MaP has superior resolution compared to alternative experiments, can resolve multiple sets of pairing interactions for structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and 2 bacterial messenger RNA 5' untranslated regions reveals functionally important and complex structures undetected by prior analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.