Visualizing transient low-populated structures of RNA

Dethoff, Elizabeth A.; Petzold, Katja; Chugh, Jeetender; Casiano-Negroni, Anette; Al‐Hashimi, Hashim M.

doi:10.1038/nature11498

Cited by 186 publications

(339 citation statements)

References 53 publications

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“…This is strikingly similar to the intrinsic dynamic behavior of RNA, which is used in riboswitches where metabolite-based effectors such as amino acids and nucleotides possess a similar chaperone activity to direct different folding pathways. 92 In addition, mutations in the intronic region near the 5 splice site of exon 10 in tau pre-mRNA have been associated with frontotemporal dementia and parkinsonism. 93 These mutations are found in the putative stem loop that destabilize the structure, altering the tau protein isoforms by alternative splicing.…”

Section: Figurementioning

confidence: 99%

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters

et al. 2016

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Activation of human telomerase reverse transcriptase (hTERT) is necessary for limitless replication in tumorigenesis. Whereas hTERT is transcriptionally silenced in normal cells, most tumor cells reactivate hTERT expression by alleviating transcriptional repression through diverse genetic and epigenetic mechanisms. Transcription-activating hTERT promoter mutations have been found to occur at high frequencies in multiple cancer types. These mutations have been shown to form new transcription factor binding sites that drive hTERT expression, but this model cannot fully account for differences in wildtype (WT) and mutant promoter activation and has not yet enabled a selective therapeutic strategy. Here we demonstrate a novel mechanism by which promoter mutations activate hTERT transcription, which also sheds light on a unique therapeutic opportunity. Promoter mutations occur in a core promoter region that forms tertiary structures consisting of a pair of G-quadruplexes involved in transcriptional silencing.We show that promoter mutations exert a detrimental effect on the folding of one of these Gquadruplexes, resulting in a nonfunctional silencer element that alleviates transcriptional repression. We have also identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, GTC365, that acts at an early step in the G-quadruplex folding pathway to redirect mutant promoter G-quadruplex misfolding, partially reinstate the correct folding pathway, and reduce hTERT activity through transcriptional repression. This transcription-mediated repression effects cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERT's role in regulating apoptosis-related proteins and inducing senescence by decreasing telomerase activity and telomere length.We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress hTERT.3

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Section: Figurementioning

confidence: 99%

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters

et al. 2016

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“…Such secondary structural transitions entail large kinetic barriers, so they are often catalyzed by RNA-binding proteins (9), ATP-dependent chaperones (10), or otherwise occur by modulating cotranscriptional folding (5,11). Recently, NMR R1ρ relaxation dispersion experiments (12)(13)(14)(15) in concert with mutagenesis (16) have helped uncover more labile RNA secondary structural transitions that can take place without assistance from external cofactors at rates that are 2-4 orders of magnitude faster than larger-scale secondary structural rearrangements. These transitions entail excursions away from the energetically favorable ground state (GS) toward lowpopulated (typically populations <15%) and short-lived (lifetime < milliseconds) species often referred to as "excited states" (ES) (12,13).…”

mentioning

confidence: 99%

“…These invisible RNA ES feature localized reshuffling of base pairing in and around noncanonical motifs such as bulges, internal loops, and apical loops (16) which can also expose or sequester functionally important residues or promote ATPindependent large-scale changes in secondary structure (14,15). These faster and more localized changes in secondary structure may meet unique demands in RNA-based regulatory functions (16).…”

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confidence: 99%

Invisible RNA state dynamically couples distant motifs

Lee

Dethoff

Al‐Hashimi

2014

Proc. Natl. Acad. Sci. U.S.A.

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102

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Using on-and off-resonance carbon and nitrogen R1ρ NMR relaxation dispersion in concert with mutagenesis and NMR chemical shift fingerprinting, we show that the transactivation response element RNA from the HIV-1 exists in dynamic equilibrium with a transient state that has a lifetime of ∼2 ms and population of ∼0.4%, which simultaneously remodels the structure of a bulge, stem, and apical loop. This is accomplished by a global change in strand register, in which bulge residues pair up with residues in the upper stem, causing a reshuffling of base pairs that propagates to the tip of apical loop, resulting in the creation of three noncanonical base pairs. Our results show that transient states can remodel distant RNA motifs and possibly give rise to mechanisms for rapid long-range communication in RNA that can be harnessed in processes such as cooperative folding and ribonucleoprotein assembly.NMR spectroscopy | dynamics | R1ρ relaxation dispersion | nucleic acids I t is now well-established that RNA sequences do not code for a single static structure, but rather, many conformations that populate energetic minima along a free-energy landscape (1, 2). Cellular inputs, ranging from changes in temperature and pH to the binding of proteins, other RNAs, and ligands, can preferentially stabilize select conformations along the landscape, resulting in dynamic changes in RNA structure that drive the multistep catalytic cycles of ribozymes (3), regulatory activities of riboswitches (4) and other RNA-based switches (5), and the dynamic assembly and disassembly of ribonucleoprotein (RNP) complexes (6).A common mode of RNA dynamics involves rearrangements in secondary structure that can melt or create entire hairpins, and thereby expose or sequester key regulatory elements that are several nucleotides long (1,4,7,8). Such secondary structural transitions entail large kinetic barriers, so they are often catalyzed by RNA-binding proteins (9), ATP-dependent chaperones (10), or otherwise occur by modulating cotranscriptional folding (5, 11). Recently, NMR R1ρ relaxation dispersion experiments (12-15) in concert with mutagenesis (16) have helped uncover more labile RNA secondary structural transitions that can take place without assistance from external cofactors at rates that are 2-4 orders of magnitude faster than larger-scale secondary structural rearrangements. These transitions entail excursions away from the energetically favorable ground state (GS) toward lowpopulated (typically populations <15%) and short-lived (lifetime < milliseconds) species often referred to as "excited states" (ES) (12, 13). These invisible RNA ES feature localized reshuffling of base pairing in and around noncanonical motifs such as bulges, internal loops, and apical loops (16) which can also expose or sequester functionally important residues or promote ATPindependent large-scale changes in secondary structure (14, 15). These faster and more localized changes in secondary structure may meet unique demands in RNA-based regulatory functions (16).Using...

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“…a) The 27 nt A‐site RNA mimic switches between two alternate secondary structures. R 1 ρ RD experiments conducted earlier gave an exchange rate of 4 kHz and an excited state population of 2.5 % 3a. The 13 C and/or 2 H modified residues are highlighted in orange.…”

mentioning

confidence: 89%

“…A detailed investigation using 13 C R 1ρ relaxation dispersion NMR was recently conducted on this RNA 3a. The RNA is involved in the selection process of cognate and near‐cognate tRNAs in the ribosome.…”

mentioning

confidence: 99%

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy

Juen

Wunderlich²,

Nußbaumer

et al. 2016

Angew Chem Int Ed

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In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear 13C and 1H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site‐specific 2H and 13C labeling, spin topologies are introduced into DNA and RNA that make 1H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A‐site RNA was previously shown to undergo a two site exchange process in the micro‐ to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV‐1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for 1H RD experiments of nucleic acids.

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Visualizing transient low-populated structures of RNA

Cited by 186 publications

References 53 publications

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters

Invisible RNA state dynamically couples distant motifs

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy

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