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Stress promotes RNA G-quadruplex folding in human cells.

ABSTRACT: Guanine (G)-rich nucleic acids can fold into G-quadruplex (G4) structures under permissive conditions. Although many RNAs contain sequences that fold into RNA G4s (rG4s) in vitro, their folding and functions in vivo are not well understood. In this report, we showed that the folding of putative rG4s in human cells into rG4 structures is dynamically regulated under stress. By using high-throughput dimethylsulfate (DMS) probing, we identified hundreds of endogenous stress-induced rG4s, and validated them by using an rG4 pull-down approach. Our results demonstrate that stress-induced rG4s are enriched in mRNA 3'-untranslated regions and enhance mRNA stability. Furthermore, stress-induced rG4 folding is readily reversible upon stress removal. In summary, our study revealed the dynamic regulation of rG4 folding in human cells and suggested that widespread rG4 motifs may have a global regulatory impact on mRNA stability and cellular stress response.

SUBMITTER: Kharel P

PROVIDER: S-EPMC9839774 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Stress promotes RNA G-quadruplex folding in human cells.

Kharel Prakash P Fay Marta M Manasova Ekaterina V EV Anderson Paul J PJ Kurkin Alexander V AV Guo Junjie U JU Ivanov Pavel P

Nature communications 20230113 1

Guanine (G)-rich nucleic acids can fold into G-quadruplex (G4) structures under permissive conditions. Although many RNAs contain sequences that fold into RNA G4s (rG4s) in vitro, their folding and functions in vivo are not well understood. In this report, we showed that the folding of putative rG4s in human cells into rG4 structures is dynamically regulated under stress. By using high-throughput dimethylsulfate (DMS) probing, we identified hundreds of endogenous stress-induced rG4s, and validat ...[more]

PMID: 36639366

Similar Datasets

Stress promotes RNA G-quadruplex folding in human cells

Project description:Guanine (G)-rich nucleic acids can fold into G-quadruplex (G4) structures under permissive conditions. Although many RNAs contain sequences that fold into RNA G4s (rG4s) in vitro, their folding and functions in vivo are not well understood. Here, we showed that the folding of putative rG4s in human cells into rG4 structures was dynamically induced by stress. By using high-throughput dimethylsulfate probing, we identified hundreds of endogenous stress-induced rG4s. Our results demonstrated that stress-induced rG4s were enriched in mRNA 3′-untranslated regions and enhanced mRNA stability. Furthermore, stress-induced rG4 folding was readily reversible upon stress removal. In summary, our study revealed the dynamic regulation of rG4 folding in living human cells and suggests suggested that widespread rG4 motifs may have a global regulatory impact on mRNA stability and cellular stress response.

2022-12-15 | GSE200706 | GEO

Stress promotes RNA G-quadruplex folding in human cells

Project description:Stress promotes RNA G-quadruplex folding in human cells

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New scoring system to identify RNA G-quadruplex folding.

Project description:G-quadruplexes (G4s) are non-canonical structures involved in many important cellular processes. To date, the prediction of potential G-quadruplex structures (PG4s) has been based almost exclusively on the sequence of interest agreeing with the algorithm Gx-N-1-7-Gx-N1-7-Gx-N1-7-Gx (where x ≥ 3 and N = A, U, G or C). However, many sequences agreeing with this algorithm do not form G4s and are considered false-positive predictions. Here we show the RNA PG4 candidate in the 3'-untranslated region (UTR) of the TTYH1 gene to be one such false positive. Specifically, G4 folding was observed to be inhibited by the presence of multiple-cytosine tracks, located in the candidate's genomic context, that adopted a Watson-Crick base-paired structure. Clearly, the neighbouring sequences of a PG4 may influence its folding. The secondary structure of 12 PG4 motifs along with either 15 or 50 nucleotides of their upstream and downstream genomic contexts were evaluated by in-line probing. Data permitted the development of a scoring system for the prediction of PG4s taking into account the effect of the neighbouring sequences. The accuracy of this scoring system was assessed by probing 14 other novel PG4 candidates retrieved in human 5'-UTRs. This new scoring system can be used, in combination with the standard algorithm, to better predict the folding of RNA G4s.

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Structural dynamics of propeller loop: towards folding of RNA G-quadruplex.

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Telomere DNA G-quadruplex folding within actively extending human telomerase.

Project description:Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase-DNA complex. Addition of the telomerase processivity factor POT1-TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1-TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

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Folding and unfolding pathways of the human telomeric G-quadruplex.

Project description:Sequence analogs of human telomeric DNA such as d[AGGG(TTAGGG)3] (Tel22) fold into monomeric quadruplex structures in the presence of a suitable cation. To investigate the pathway for unimolecular quadruplex formation, we monitored the kinetics of K(+)-induced folding of Tel22 by circular dichroism (CD), intrinsic 2-aminopurine fluorescence, and fluorescence resonance energy transfer (FRET). The results are consistent with a four-step pathway U ↔ I1 ↔ I2 ↔ I3 ↔ F where U and F represent unfolded and folded conformational ensembles and I1, I2, and I3 are intermediates. Previous kinetic studies have shown that I1 is formed in a rapid pre-equilibrium and may consist of an ensemble of "prefolded" hairpin structures brought about by cation-induced electrostatic collapse of the DNA. The current study shows that I1 converts to I2 with a relaxation time τ1=0.1s at 25 °C in 25 mM KCl. The CD spectrum of I2 is characteristic of an antiparallel quadruplex that could form as a result of intramolecular fold-over of the I1 hairpins. I3 is relatively slowly formed (τ2≈3700s) and has CD and FRET properties consistent with those expected of a triplex structure as previously observed in equilibrium melting studies. I3 converts to F with τ3≈750s. Identical pathways with different kinetic constants involving a rapidly formed antiparallel intermediate were observed with oligonucleotides forming mixed parallel/antiparallel hybrid-1 and hybrid-2 topologies {e.g. d[TTGGG(TTAGGG)3A] and d[TAGGG(TTAGGG)3TT]}. Aspects of the kinetics of unfolding were also monitored by the spectroscopic methods listed above and by time-resolved fluorescence lifetime measurements using a complementary strand trap assay. These experiments reveal a slow, rate-limiting step along the unfolding pathway.

| S-EPMC3969789 | biostudies-literature

RNA G-quadruplex folding is a multi-pathway process driven by conformational entropy.

Project description:The kinetics of folding is crucial for the function of many regulatory RNAs including RNA G-quadruplexes (rG4s). Here, we characterize the folding pathways of a G-quadruplex from the telomeric repeat-containing RNA by combining all-atom molecular dynamics and coarse-grained simulations with circular dichroism experiments. The quadruplex fold is stabilized by cations and thus, the ion atmosphere forming a double layer surrounding the highly charged quadruplex guides the folding process. To capture the ionic double layer in implicit solvent coarse-grained simulations correctly, we develop a matching procedure based on all-atom simulations in explicit water. The procedure yields quantitative agreement between simulations and experiments as judged by the populations of folded and unfolded states at different salt concentrations and temperatures. Subsequently, we show that coarse-grained simulations with a resolution of three interaction sites per nucleotide are well suited to resolve the folding pathways and their intermediate states. The results reveal that the folding progresses from unpaired chain via hairpin, triplex and double-hairpin constellations to the final folded structure. The two- and three-strand intermediates are stabilized by transient Hoogsteen interactions. Each pathway passes through two on-pathway intermediates. We hypothesize that conformational entropy is a hallmark of rG4 folding. Conformational entropy leads to the observed branched multi-pathway folding process for TERRA25. We corroborate this hypothesis by presenting the free energy landscapes and folding pathways of four rG4 systems with varying loop length.

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Linkage of cation binding and folding in human telomeric quadruplex DNA.

Project description:Formation of DNA quadruplexes requires monovalent cation binding. To characterize the cation binding stoichiometry and linkage between binding and folding, we carried out KCl titrations of Tel22 (d[A(GGGTTA)(3)]), a model of the human telomere sequence, using a fluorescent indicator to determine [K(+)](free) and circular dichroism to assess the extent of folding. At [K(+)](free)=5 mM (sufficient for >95% folding), the apparent binding stoichiometry is 3K(+)/Tel22; at [K(+)](free)=20 mM, it increased to 8-10K(+)/Tel22. Thermodynamic analysis shows that at [K(+)](free)=5 mM, K(+) binding contributes approximately -4.9 kcal/mol for folding Tel22. The overall folding free energy is -2.4 kcal/mol, indicating that there are energetically unfavorable contributions to folding. Thus, quadruplex folding is driven almost entirely by the energy of cation binding with little or no contribution from other weak molecular interactions.

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