-
Molecular Cell Feb 2021The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data...
The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data (R2D2), a method to uncover details of cotranscriptional RNA folding. We model the folding of the Escherichia coli signal recognition particle (SRP) RNA and show that it requires specific local structural fluctuations within a key hairpin to engender efficient cotranscriptional conformational rearrangement into the functional structure. All-atom molecular dynamics simulations suggest that this rearrangement proceeds through an internal toehold-mediated strand-displacement mechanism, which can be disrupted with a point mutation that limits local structural fluctuations and rescued with compensating mutations that restore these fluctuations. Moreover, a cotranscriptional folding intermediate could be cleaved in vitro by recombinant E. coli RNase P, suggesting potential cotranscriptional processing. These results from experiment-guided multi-scale modeling demonstrate that even an RNA with a simple functional structure can undergo complex folding and processing during synthesis.
Topics: Escherichia coli; Escherichia coli Proteins; RNA Folding; RNA, Bacterial; Ribonuclease P; Signal Recognition Particle
PubMed: 33453165
DOI: 10.1016/j.molcel.2020.12.017 -
Biophysical Journal Feb 2022Fluorescent RNA aptamers have the potential to enable routine quantitation and localization of RNA molecules and serve as models for understanding biologically active...
Fluorescent RNA aptamers have the potential to enable routine quantitation and localization of RNA molecules and serve as models for understanding biologically active aptamers. In recent years, several fluorescent aptamers have been selected and modified to improve their properties, revealing that small changes to the RNA or the ligands can modify significantly their fluorescent properties. Although structural biology approaches have revealed the bound, ground state of several fluorescent aptamers, characterization of low-abundance, excited states in these systems is crucial to understanding their folding pathways. Here we use pressure as an alternative variable to probe the suboptimal states of the Mango III aptamer with both fluorescence and NMR spectroscopy approaches. At moderate KCl concentrations, increasing pressure disrupted the G-quadruplex structure of the Mango III RNA and led to an intermediate with lower fluorescence. These observations indicate the existence of suboptimal RNA structural states that still bind the TO1-biotin fluorophore and moderately enhance fluorescence. At higher KCl concentration as well, the intermediate fluorescence state was populated at high pressure, but the G-quadruplex remained stable at high pressure, supporting the notion of parallel folding and/or binding pathways. These results demonstrate the usefulness of pressure for characterizing RNA folding intermediates.
Topics: Aptamers, Nucleotide; Fluorescent Dyes; Mangifera; RNA; RNA Folding
PubMed: 34971617
DOI: 10.1016/j.bpj.2021.12.037 -
Journal of Molecular Biology Sep 2022Riboswitches are an outstanding example of genetic regulation mediated by RNA conformational switching. In these non-coding RNA elements, the occupancy status of a... (Review)
Review
Riboswitches are an outstanding example of genetic regulation mediated by RNA conformational switching. In these non-coding RNA elements, the occupancy status of a ligand-binding domain governs the mRNA's decision to form one of two mutually exclusive structures in the downstream expression platform. Temporal constraints upon the function of many riboswitches, requiring folding of complex architectures and conformational switching in a limited co-transcriptional timeframe, make them ideal model systems for studying these processes. In this review, we focus on the mechanism of ligand-directed conformational changes in one of the most widely distributed riboswitches in bacteria: the cobalamin family. We describe the architectural features of cobalamin riboswitches whose structures have been determined by x-ray crystallography, which suggest a direct physical role of cobalamin in effecting the regulatory switch. Next, we discuss a series of experimental approaches applied to several model cobalamin riboswitches that interrogate these structural models. As folding is central to riboswitch function, we consider the differences in folding landscapes experienced by RNAs that are produced in vitro and those that are allowed to fold co-transcriptionally. Finally, we highlight a set of studies that reveal the difficulties of studying cobalamin riboswitches outside the context of transcription and that co-transcriptional approaches are essential for developing a more accurate picture of their structure-function relationships in these switches. This understanding will be essential for future advancements in the use of small-molecule guided RNA switches in a range of applications such as biosensors, RNA imaging tools, and nucleic acid-based therapies.
Topics: Bacteria; Crystallography, X-Ray; Gene Expression Regulation, Bacterial; Ligands; RNA Folding; Riboswitch; Vitamin B 12
PubMed: 35427633
DOI: 10.1016/j.jmb.2022.167585 -
Wiley Interdisciplinary Reviews. RNA 2012Noncoding RNAs have emerged as important key players in the cell. Understanding their surprisingly diverse range of functions is challenging for experimental and... (Review)
Review
Noncoding RNAs have emerged as important key players in the cell. Understanding their surprisingly diverse range of functions is challenging for experimental and computational biology. Here, we review computational methods to analyze noncoding RNAs. The topics covered include basic and advanced techniques to predict RNA structures, annotation of noncoding RNAs in genomic data, mining RNA-seq data for novel transcripts and prediction of transcript structures, computational aspects of microRNAs, and database resources.
Topics: Animals; Computational Biology; Data Mining; Databases, Nucleic Acid; Genomics; Humans; MicroRNAs; Molecular Sequence Annotation; Nucleic Acid Conformation; RNA Folding; RNA, Untranslated
PubMed: 22991327
DOI: 10.1002/wrna.1134 -
Methods (San Diego, Calif.) May 2012Thermodynamic studies of G-quadruplex stability are an essential complement to structures obtained by NMR or X-ray crystallography. An understanding of the energetics of...
Thermodynamic studies of G-quadruplex stability are an essential complement to structures obtained by NMR or X-ray crystallography. An understanding of the energetics of quadruplex folding provides a necessary foundation for the physical interpretation of quadruplex formation and reactivity. While thermal denaturation methods are most commonly used to evaluate quadruplex stability, it is also possible to study folding using isothermal titration methods. G-quadruplex folding is tightly coupled to specific cation binding. We describe here protocols for monitoring the cation-driven quadruplex folding transition using circular dichroism or absorbance, and for determination of the distribution of free and bound cation using a fluorescence indicator. Together these approaches provide insight into quadruplex folding at constant temperature, and characterize the linkage between cation binding and folding.
Topics: Cations; Circular Dichroism; Crystallography, X-Ray; Fluorescence; G-Quadruplexes; Magnetic Resonance Spectroscopy; Nucleic Acid Conformation; Oligodeoxyribonucleotides; RNA Folding; Temperature; Thermodynamics
PubMed: 22525787
DOI: 10.1016/j.ymeth.2012.04.006 -
International Journal of Molecular... Apr 2021The genus comprises numerous, small, single positive-stranded RNA viruses, many of which are important human pathogens. To store all the information required for their... (Review)
Review
The genus comprises numerous, small, single positive-stranded RNA viruses, many of which are important human pathogens. To store all the information required for their successful propagation, flaviviruses use discrete structural genomic RNA elements to code for functional information by the establishment of dynamic networks of long-range RNA-RNA interactions that promote specific folding. These structural elements behave as true cis-acting, non-coding RNAs (ncRNAs) and have essential regulatory roles in the viral cycle. These include the control of the formation of subgenomic RNAs, known as sfRNAs, via the prevention of the complete degradation of the RNA genome. These sfRNAs are important in ensuring viral fitness. This work summarizes our current knowledge of the functions performed by the genome conformations and the role of RNA-RNA interactions in these functions. It also reviews the role of RNA structure in the production of sfRNAs across the genus , and their existence in related viruses.
Topics: Animals; Flavivirus; Genome, Viral; Humans; RNA Folding; RNA Stability; RNA, Viral
PubMed: 33916729
DOI: 10.3390/ijms22073738 -
Wiley Interdisciplinary Reviews. RNA 2015RNA conformation plays a significant role in stability, ligand binding, transcription, and translation. Single nucleotide variants (SNVs) have the potential to disrupt... (Review)
Review
RNA conformation plays a significant role in stability, ligand binding, transcription, and translation. Single nucleotide variants (SNVs) have the potential to disrupt specific structural elements because RNA folds in a sequence-specific manner. A riboSNitch is an element of RNA structure with a specific function that is disrupted by an SNV or a single nucleotide polymorphism (SNP; or polymorphism; SNVs occur with low frequency in the population, <1%). The riboSNitch is analogous to a riboswitch, where binding of a small molecule rather than mutation alters the structure of the RNA to control gene regulation. RiboSNitches are particularly relevant to interpreting the results of genome-wide association studies (GWAS). Often GWAS identify SNPs associated with a phenotype mapping to noncoding regions of the genome. Because a majority of the human genome is transcribed, significant subsets of GWAS SNPs are putative riboSNitches. The extent to which the transcriptome is tolerant of SNP-induced structure change is still poorly understood. Recent advances in ultra high-throughput structure probing begin to reveal the structural complexities of mutation-induced structure change. This review summarizes our current understanding of SNV and SNP-induced structure change in the human transcriptome and discusses the importance of riboSNitch discovery in interpreting GWAS results and massive sequencing projects.
Topics: Animals; Genome-Wide Association Study; Humans; Nucleic Acid Conformation; Polymorphism, Single Nucleotide; Precision Medicine; RNA; RNA Folding; Riboswitch
PubMed: 26115028
DOI: 10.1002/wrna.1291 -
Nature Communications Nov 2017The expression of the compact mammalian mitochondrial genome requires transcription, RNA processing, translation and RNA decay, much like the more complex chromosomal...
The expression of the compact mammalian mitochondrial genome requires transcription, RNA processing, translation and RNA decay, much like the more complex chromosomal systems, and here we use it as a model system to understand the fundamental aspects of gene expression. Here we combine RNase footprinting with PAR-CLIP at unprecedented depth to reveal the importance of RNA-protein interactions in dictating RNA folding within the mitochondrial transcriptome. We show that LRPPRC, in complex with its protein partner SLIRP, binds throughout the mitochondrial transcriptome, with a preference for mRNAs, and its loss affects the entire secondary structure and stability of the transcriptome. We demonstrate that the LRPPRC-SLIRP complex is a global RNA chaperone that stabilizes RNA structures to expose the required sites for translation, stabilization, and polyadenylation. Our findings reveal a general mechanism where extensive RNA-protein interactions ensure that RNA is accessible for its biological functions.
Topics: Animals; Binding Sites; Fibroblasts; Genome, Mitochondrial; Humans; Male; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Molecular Chaperones; Neoplasm Proteins; Polyadenylation; Protein Binding; Protein Biosynthesis; Protein Footprinting; RNA Folding; RNA Stability; RNA, Messenger; RNA-Binding Proteins; Recombinant Proteins; Sequence Analysis, RNA; Transcriptome
PubMed: 29146908
DOI: 10.1038/s41467-017-01221-z -
Nature Structural & Molecular Biology Sep 2020Small interfering RNAs (siRNAs) promote RNA degradation in a variety of processes and have important clinical applications. siRNAs direct cleavage of target RNAs by...
Small interfering RNAs (siRNAs) promote RNA degradation in a variety of processes and have important clinical applications. siRNAs direct cleavage of target RNAs by guiding Argonaute2 (AGO2) to its target site. Target site accessibility is critical for AGO2-target interactions, but how target site accessibility is controlled in vivo is poorly understood. Here, we use live-cell single-molecule imaging in human cells to determine rate constants of the AGO2 cleavage cycle in vivo. We find that the rate-limiting step in mRNA cleavage frequently involves unmasking of target sites by translating ribosomes. Target site masking is caused by heterogeneous intramolecular RNA-RNA interactions, which can conceal target sites for many minutes in the absence of translation. Our results uncover how dynamic changes in mRNA structure shape AGO2-target recognition, provide estimates of mRNA folding and unfolding rates in vivo, and provide experimental evidence for the role of mRNA structural dynamics in control of mRNA-protein interactions.
Topics: Argonaute Proteins; Cell Line; HEK293 Cells; Humans; Nucleic Acid Conformation; RNA Cleavage; RNA Folding; RNA, Messenger; Ribosomes
PubMed: 32661421
DOI: 10.1038/s41594-020-0461-1 -
Journal of the American Chemical Society Jan 2023We have developed and used single-molecule field-effect transistors (smFETs) to characterize the conformational free-energy landscape of RNA stem-loops. Stem-loops are...
We have developed and used single-molecule field-effect transistors (smFETs) to characterize the conformational free-energy landscape of RNA stem-loops. Stem-loops are one of the most common RNA structural motifs and serve as building blocks for the formation of complex RNA structures. Given their prevalence and integral role in RNA folding, the kinetics of stem-loop (un)folding has been extensively characterized using both experimental and computational approaches. Interestingly, these studies have reported vastly disparate timescales of (un)folding, which has been interpreted as evidence that (un)folding of even simple stem-loops occurs on a highly rugged conformational energy landscape. Because smFETs do not rely on fluorophore reporters of conformation or mechanical (un)folding forces, they provide a unique approach that has allowed us to directly monitor tens of thousands of (un)folding events of individual stem-loops at a 200 μs time resolution. Our results show that under our experimental conditions, stem-loops (un)fold over a 1-200 ms timescale during which they transition between ensembles of unfolded and folded conformations, the latter of which is composed of at least two sub-populations. The 1-200 ms timescale of (un)folding we observe here indicates that smFETs report on complete (un)folding trajectories in which unfolded conformations of the RNA spend long periods of time wandering the free-energy landscape before sampling one of several misfolded conformations or the natively folded conformation. Our findings highlight the extremely rugged landscape on which even the simplest RNA structural elements fold and demonstrate that smFETs are a unique and powerful approach for characterizing the conformational free-energy of RNA.
Topics: RNA; Molecular Conformation; RNA Folding; Nucleic Acid Conformation; Thermodynamics; Protein Folding; Kinetics
PubMed: 36547391
DOI: 10.1021/jacs.2c10218