A new reagent for in vivo structure probing of RNA G and U residues that improves RNA structure prediction alone and combined with DMS.

A key to understanding the roles of RNA in regulating gene expression is knowing their structures in vivo. One way to obtain this information is through probing the structures of RNA with chemicals. To probe RNA structure directly in cells, membrane-permeable reagents that modify the Watson-Crick (WC) face of unpaired nucleotides can be used. Although dimethyl sulfate (DMS) has led to substantial insight into RNA structure, it has limited nucleotide specificity in vivo, with WC face reactivity only at adenine (A) and cytosine (C) at neutral pH. The reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was recently shown to modify the WC face of guanine (G) and uracil (U). Although useful at lower concentrations in experiments that measure chemical modifications by reverse transcription (RT) stops, at higher concentrations necessary for detection by mutational profiling (MaP), EDC treatment leads to degradation of RNA. Here, we demonstrate EDC-stimulated degradation of RNA in Gram-negative and Gram-positive bacteria. In an attempt to overcome these limitations, we developed a new carbodiimide reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide (ETC), which we show specifically modifies unpaired Gs and Us in vivo without substantial degradation of RNA. We establish ETC as a probe for MaP and optimize the RT conditions and computational analysis in Escherichia coli Importantly, we demonstrate the utility of ETC as a probe for improving RNA structure prediction both alone and with DMS.

Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity.

Small nucleolytic ribozymes are RNAs that cleave their own phosphodiester backbone. While proteinaceous enzymes are regulated by a variety of known mechanisms, methods of regulation for ribozymes remain unclear. Twister is one ribozyme class for which many structural and catalytic properties have been elucidated. However, few studies have analyzed the activity of twister ribozymes in the context of a native flanking sequence, even though ribozymes as transcribed in nature do not exist in isolation. Interactions between the ribozyme and its neighboring sequences can induce conformational changes that inhibit self-cleavage, providing a regulatory mechanism that could naturally determine ribozyme activity in vivo and in synthetic applications. To date, eight twister ribozymes have been identified within the staple crop rice (Oryza sativa). Herein, we select several twister ribozymes from rice and show that they are differentially regulated by their flanking sequence using published RNA-seq data sets, structure probing, and cotranscriptional cleavage assays. We found that the Osa 1-2 ribozyme does not interact with its flanking sequences. However, sequences flanking the Osa 1-3 and Osa 1-8 ribozymes form inactive conformations, referred to here as "ribozymogens", that attenuate ribozyme self-cleavage activity. For the Osa 1-3 ribozyme, we show that activity can be rescued upon addition of a complementary antisense oligonucleotide, suggesting ribozymogens can be controlled via external signals. In all, our data provide a plausible mechanism wherein flanking sequence differentially regulates ribozyme activity in vivo. More broadly, the ability to regulate ribozyme behavior locally has potential applications in control of gene expression and synthetic biology.

Structure-seq of tRNAs and other short RNAs in droplets and in vivo.

There is a multitude of small (<100nt) RNAs that serve diverse functional roles in biology. Key amongst these is transfer RNA (tRNA), which is among the most ancient RNAs and is part of the translational apparatus in every domain of life. Transfer RNAs are also the most heavily modified class of RNAs. They are essential and their misregulation, due to mutated sequences or loss of modification, can lead to disease. Because of the severe phenotypes associated with mitochondrial tRNA defects in particular, the desire to deliver repaired tRNAs via droplets such as lipid nanoparticles or other compartments is an active area of research. Here we describe how to use our tRNA Structure-seq method to study tRNAs and other small RNAs in two different biologically relevant contexts, peptide-rich droplets and in vivo.

In vivo-like nearest neighbor parameters improve prediction of fractional RNA base-pairing in cells.

We conducted a thermodynamic analysis of RNA stability in Eco80 artificial cytoplasm, which mimics in vivo conditions, and compared it to transcriptome-wide probing of mRNA. Eco80 contains 80% of Escherichia coli metabolites, with biological concentrations of metal ions, including 2 mM free Mg2+ and 29 mM metabolite-chelated Mg2+. Fluorescence-detected binding isotherms (FDBI) were used to conduct a thermodynamic analysis of 24 RNA helices and found that these helices, which have an average stability of -12.3 kcal/mol, are less stable by ΔΔGo37 ∼1 kcal/mol. The FDBI data was used to determine a set of Watson-Crick free energy nearest neighbor parameters (NNPs), which revealed that Eco80 reduces the stability of three NNPs. This information was used to adjust the NN model using the RNAstructure package. The in vivo-like adjustments have minimal effects on the prediction of RNA secondary structures determined in vitro and in silico, but markedly improve prediction of fractional RNA base pairing in E. coli, as benchmarked with our in vivo DMS and EDC RNA chemical probing data. In summary, our thermodynamic and chemical probing analyses of RNA helices indicate that RNA secondary structures are less stable in cells than in artificially stable in vitro buffer conditions.

RNA folding studies inside peptide-rich droplets reveal roles of modified nucleosides at the origin of life.

Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Here, we detail next-generation sequencing (NGS) experiments performed in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Notably, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life.

In vivo structure probing of RNA in Archaea: novel insights into the ribosome structure of Methanosarcina acetivorans.

Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure-function relationships. To date, such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) from Methanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing in M. acetivorans Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure within M. acetivorans.

Transcriptome-wide probing reveals RNA thermometers that regulate translation of glycerol permease genes in Bacillus subtilis.

RNA structure regulates bacterial gene expression by several distinct mechanisms via environmental and cellular stimuli, one of which is temperature. While some genome-wide studies have focused on heat shock treatments and the subsequent transcriptomic changes, soil bacteria are less likely to experience such rapid and extreme temperature changes. Though RNA thermometers (RNATs) have been found in 5' untranslated leader regions (5' UTRs) of heat shock and virulence-associated genes, this RNA-controlled mechanism could regulate other genes as well. Using Structure-seq2 and the chemical probe dimethyl sulfate (DMS) at four growth temperatures ranging from 23°C to 42°C, we captured a dynamic response of the Bacillus subtilis transcriptome to temperature. Our transcriptome-wide results show RNA structural changes across all four temperatures and reveal nonmonotonic reactivity trends with increasing temperature. Then, focusing on subregions likely to contain regulatory RNAs, we examined 5' UTRs to identify large, local reactivity changes. This approach led to the discovery of RNATs that control the expression of glpF (glycerol permease) and glpT (glycerol-3-phosphate permease); expression of both genes increased with increased temperature. Results with mutant RNATs indicate that both genes are controlled at the translational level. Increased import of glycerols at high temperatures could provide thermoprotection to proteins.

The Functional RNA Identification (FRID) Pipeline: Identification of Potential Pseudoknot-Containing RNA Elements as Therapeutic Targets for SARS-CoV-2.

The COVID-19 pandemic persists despite the development of effective vaccines. As such, it remains crucial to identify new targets for antiviral therapies. The causative virus of COVID-19, SARS-CoV-2, is a positive-sense RNA virus with RNA structures that could serve as therapeutic targets. One such RNA with established function is the frameshift stimulatory element (FSE), which promotes programmed ribosomal frameshifting. To accelerate identification of additional functional RNA elements, we introduce a novel computational approach termed the Functional RNA Identification (FRID) pipeline. The guiding principle of our pipeline, which uses established component programs as well as customized component programs, is that functional RNA elements have conserved secondary and pseudoknot structures that facilitate function. To assess the presence and conservation of putative functional RNA elements in SARS-CoV-2, we compared over 6,000 SARS-CoV-2 genomic isolates. We identified 22 functional RNA elements from the SARS-CoV-2 genome, 14 of which have conserved pseudoknots and serve as potential targets for small molecule or antisense oligonucleotide therapeutics. The FRID pipeline is general and can be applied to identify pseudoknotted RNAs for targeted therapeutics in genomes or transcriptomes from any virus or organism.

MeltR software provides facile determination of nucleic acid thermodynamics.

Thermodenaturation (melting) curves of macromolecules are used to determine folding thermodynamic parameters. Notably, this insight into RNA and DNA stability underlies nearest neighbor theory and diverse structure prediction tools. Analysis of UV-detected absorbance melting curves is complex and multivariate, requiring many data preprocessing, regression, and error analysis steps. The absorbance melting curve-fitting software MeltWin, introduced in 1996, provided a consistent and facile melting curve analysis platform used in a generation of folding parameters. Unfortunately, MeltWin software is not maintained and relies on idiosyncratic choices of baselines by the user. Herein, we provide MeltR, an open-source, curve-fitting package for analysis of macromolecular thermodynamic data. The MeltR package provides the facile conversion of melting curve data to parameters provided by MeltWin while offering additional features including global fitting of data, auto-baseline generation, and two-state melting analysis. MeltR should be a useful tool for analyzing the next generation of DNA, RNA, and nonnucleic acid macromolecular melting data.

RNA folding studies inside peptide-rich droplets reveal roles of modified nucleosides at the origin of life.

Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically-plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Herein, we detail Next-Generation Sequencing (NGS) experiments performed for the first time in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Strikingly, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life. One Sentence Summary: We demonstrate that RNA folds into native secondary and tertiary structures in protocell models and that this is favored by covalent modifications, which is critical for the origins of life.

Rock, scissors, paper: How RNA structure informs function.

RNA can fold back on itself to adopt a wide range of structures. These range from relatively simple hairpins to intricate 3D folds and can be accompanied by regulatory interactions with both metabolites and macromolecules. The last 50 yr have witnessed elucidation of an astonishing array of RNA structures including transfer RNAs, ribozymes, riboswitches, the ribosome, the spliceosome, and most recently entire RNA structuromes. These advances in RNA structural biology have deepened insight into fundamental biological processes including gene editing, transcription, translation, and structure-based detection and response to temperature and other environmental signals. These discoveries reveal that RNA can be relatively static, like a rock; that it can have catalytic functions of cutting bonds, like scissors; and that it can adopt myriad functional shapes, like paper. We relate these extraordinary discoveries in the biology of RNA structure to the plant way of life. We trace plant-specific discovery of ribozymes and riboswitches, alternative splicing, organellar ribosomes, thermometers, whole-transcriptome structuromes and pan-structuromes, and conclude that plants have a special set of RNA structures that confer unique types of gene regulation. We finish with a consideration of future directions for the RNA structure-function field.

The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability.

We examined the complex network of interactions among RNA, the metabolome, and divalent Mg2+ under conditions that mimic the Escherichia coli cytoplasm. We determined Mg2+ binding constants for the top 15 E. coli metabolites, comprising 80% of the metabolome by concentration at physiological pH and monovalent ion concentrations. These data were used to inform the development of an artificial cytoplasm that mimics in vivo E. coli conditions, which we term "Eco80". We empirically determined that the mixture of E. coli metabolites in Eco80 approximated single-site binding behavior toward Mg2+ in the biologically relevant free Mg2+ range of ∼0.5 to 3 mM Mg2+, using a Mg2+-sensitive fluorescent dye. Effects of Eco80 conditions on the thermodynamic stability, chemical stability, structure, and catalysis of RNA were examined. We found that Eco80 conditions lead to opposing effects on the thermodynamic and chemical stabilities of RNA. In particular, the thermodynamic stability of RNA helices was weakened by 0.69 ± 0.12 kcal/mol, while the chemical stability was enhanced ∼2-fold, which can be understood using the speciation of Mg2+ between weak and strong Mg2+-metabolite complexes in Eco80. Overall, the use of Eco80 reflects RNA function in vivo and enhances the biological relevance of mechanistic studies of RNA.

Upstream Flanking Sequence Assists Folding of an RNA Thermometer.

Many heat shock genes in bacteria are regulated through a class of temperature-sensitive stem-loop (SL) RNAs called RNA thermometers (RNATs). One of the most widely studied RNATs is the Repression Of heat Shock Expression (ROSE) element associated with expression of heat shock proteins. Located in the 5'UTR, the RNAT contains one to three auxiliary hairpins upstream of it. Herein, we address roles of these upstream SLs in the folding and function of an RNAT. Bradyrhizobium japonicum is a nitrogen-fixing bacterium that experiences a wide range of temperatures in the soil and contains ROSE elements, each having multiple upstream SLs. The 5'UTR of the messenger (mRNA) for heat shock protein A (hspA) in B. japonicum has an intricate secondary structure containing three SLs upstream of the RNAT SL. While structure-function studies of the hspA RNAT itself have been reported, it has been unclear if these auxiliary SLs contribute to the temperature-sensing function of the ROSE elements. Herein, we show that the full length (FL) sequence has several melting transitions indicating that the ROSE element unfolds in a non-two-state manner. The upstream SLs are more stable than the RNAT itself, and a variant with disrupted base pairing in the SL immediately upstream of the RNAT has little influence on the melting of the RNAT. On the basis of these results and modeling of the co-transcriptional folding of the ROSE element, we propose that the upstream SLs function to stabilize the transcript and aid proper folding and dynamics of the RNAT.

Genome-wide analysis of the in vivo tRNA structurome reveals RNA structural and modification dynamics under heat stress.

RNA structure plays roles in myriad cellular events including transcription, translation, and RNA processing. Genome-wide analyses of RNA secondary structure in vivo by chemical probing have revealed critical structural features of mRNAs and long ncRNAs. Here, we examine the in vivo secondary structure of a small RNA class, tRNAs. Study of tRNA structure is challenging because tRNAs are heavily modified and strongly structured. We introduce "tRNA structure-seq," a new workflow that accurately determines in vivo secondary structures of tRNA. The workflow combines dimethyl sulfate (DMS) probing, ultra-processive RT, and mutational profiling (MaP), which provides mutations opposite DMS and natural modifications thereby allowing multiple modifications to be identified in a single read. We applied tRNA structure-seq to E. coli under control and stress conditions. A leading folding algorithm predicts E. coli tRNA structures with only ∼80% average accuracy from sequence alone. Strikingly, tRNA structure-seq, by providing experimental restraints, improves structure prediction under in vivo conditions to ∼95% accuracy, with more than 14 tRNAs predicted completely correctly. tRNA structure-seq also quantifies the relative levels of tRNAs and their natural modifications at single nucleotide resolution, as validated by LC-MS/MS. Our application of tRNA structure-seq yields insights into tRNA structure in living cells, revealing that it is not immutable but has dynamics, with partial unfolding of secondary and tertiary tRNA structure under heat stress that is correlated with a loss of tRNA abundance. This method is applicable to other small RNAs, including those with natural modifications and highly structured regions.

Biological solution conditions and flanking sequence modulate LLPS of RNA G-quadruplex structures.

Guanine-rich regions of DNA or RNA can form structures with two or more consecutive G-quartets called G-quadruplexes (GQ). Recent studies reveal the potential for these structures to aggregate in vitro. Here, we report effects of in vivo concentrations of additives-amino acids, nucleotides, and crowding agents-on the structure and solution behavior of RNAs containing GQ-forming sequences. We found that cytosine nucleotides destabilize a model GQ structure at biological salt concentrations, while free amino acids and other nucleotides do not do so to a substantial degree. We also report that the tendency of folded GQs to form droplets or to aggregate depends on the nature of flanking sequence and the presence of additives. Notably, in the presence of biological amounts of polyamines, flanking regions on the 5'-end of the RNA drive more droplet-like phase separation, while flanking regions on the 3'-end, as well as both the 5'- and 3'-ends, induce more condensed, granular structures. Finally, we provide an example of a biological sequence in the presence of polyamines and show that crowders such as PEG and dextran can selectively cause its phase separation. These findings have implications for the participation of GQS in LLPS in vivo.

Phase-specific RNA accumulation and duplex thermodynamics in multiphase coacervate models for membraneless organelles.

Liquid-liquid phase separation has emerged as an important means of intracellular RNA compartmentalization. Some membraneless organelles host two or more compartments serving different putative biochemical roles. The mechanisms for, and functional consequences of, this subcompartmentalization are not yet well understood. Here we show that adjacent phases of decapeptide-based multiphase model membraneless organelles differ markedly in their interactions with RNA. Single- and double-stranded RNAs preferentially accumulate in different phases within the same droplet, and one phase is more destabilizing for RNA duplexes than the other. Single-phase peptide droplets did not capture this behaviour. Phase coexistence introduces new thermodynamic equilibria that alter RNA duplex stability and RNA sorting by hybridization state. These effects require neither biospecific RNA-binding sites nor full-length proteins. As such, they are more general and point to primitive versions of mechanisms operating in extant biology that could aid understanding and enable the design of functional artificial membraneless organelles.

Experimental demonstration and pan-structurome prediction of climate-associated riboSNitches in Arabidopsis.

BACKGROUND: Genome-wide association studies (GWAS) aim to correlate phenotypic changes with genotypic variation. Upon transcription, single nucleotide variants (SNVs) may alter mRNA structure, with potential impacts on transcript stability, macromolecular interactions, and translation. However, plant genomes have not been assessed for the presence of these structure-altering polymorphisms or "riboSNitches." RESULTS: We experimentally demonstrate the presence of riboSNitches in transcripts of two Arabidopsis genes, ZINC RIBBON 3 (ZR3) and COTTON GOLGI-RELATED 3 (CGR3), which are associated with continentality and temperature variation in the natural environment. These riboSNitches are also associated with differences in the abundance of their respective transcripts, implying a role in regulating the gene's expression in adaptation to local climate conditions. We then computationally predict riboSNitches transcriptome-wide in mRNAs of 879 naturally inbred Arabidopsis accessions. We characterize correlations between SNPs/riboSNitches in these accessions and 434 climate descriptors of their local environments, suggesting a role of these variants in local adaptation. We integrate this information in CLIMtools V2.0 and provide a new web resource, T-CLIM, that reveals associations between transcript abundance variation and local environmental variation. CONCLUSION: We functionally validate two plant riboSNitches and, for the first time, demonstrate riboSNitch conditionality dependent on temperature, coining the term "conditional riboSNitch." We provide the first pan-genome-wide prediction of riboSNitches in plants. We expand our previous CLIMtools web resource with riboSNitch information and with 1868 additional Arabidopsis genomes and 269 additional climate conditions, which will greatly facilitate in silico studies of natural genetic variation, its phenotypic consequences, and its role in local adaptation.

RNA multimerization as an organizing force for liquid-liquid phase separation.

RNA interactions are exceptionally strong and highly redundant. As such, nearly any two RNAs have the potential to interact with one another over relatively short stretches, especially at high RNA concentrations. This is especially true for pairs of RNAs that do not form strong self-structure. Such phenomena can drive liquid-liquid phase separation, either solely from RNA-RNA interactions in the presence of divalent or organic cations, or in concert with proteins. RNA interactions can drive multimerization of RNA strands via both base-pairing and tertiary interactions. In this article, we explore the tendency of RNA to form stable monomers, dimers, and higher order structures as a function of RNA length and sequence through a focus on the intrinsic thermodynamic, kinetic, and structural properties of RNA. The principles we discuss are independent of any specific type of biomolecular condensate, and thus widely applicable. We also speculate how external conditions experienced by living organisms can influence the formation of nonmembranous compartments, again focusing on the physical and structural properties of RNA. Plants, in particular, are subject to diverse abiotic stresses including extreme temperatures, drought, and salinity. These stresses and the cellular responses to them, including changes in the concentrations of small molecules such as polyamines, salts, and compatible solutes, have the potential to regulate condensate formation by melting or strengthening base-pairing. Reversible condensate formation, perhaps including regulation by circadian rhythms, could impact biological processes in plants, and other organisms.