Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4-P6 RNA by smFRET

Abstract Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4-P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na + and K + , to the RNA tetralooptetraloop receptor (TL-TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state

Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4-P6 RNA by smFRET

Abstract Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4-P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na + and K + , to the RNA tetralooptetraloop receptor (TL-TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state

Does Cation Size Affect Occupancy and Electrostatic Screening of the Nucleic Acid Ion Atmosphere?

Electrostatics are central to all aspects of nucleic acid behavior, including their folding, condensation, and binding to other molecules, and the energetics of these processes are profoundly influenced by the ion atmosphere that surrounds nucleic acids. Given the highly complex and dynamic nature of the ion atmosphere, understanding its properties and effects will require synergy between computational modeling and experiment. Prior computational models and experiments suggest that cation occupancy in the ion atmosphere depends on the size of the cation. However, the computational models have not been independently tested, and the experimentally observed effects were small. Here, we evaluate a computational model of ion size effects by experimentally testing a <i>blind</i> prediction made from that model, and we present additional experimental results that extend our understanding of the ion atmosphere. Giambasu et al. developed and implemented a three-dimensional reference interaction site (3D-RISM) model for monovalent cations surrounding DNA and RNA helices, and this model predicts that Na⁺ would outcompete Cs⁺ by 1.8–2.1-fold; i.e., with Cs⁺ in 2-fold excess of Na⁺ the ion atmosphere would contain an equal number of each cation (<i>Nucleic Acids Res.</i> <b>2015</b>, <i>43</i>, 8405). However, our ion counting experiments indicate that there is no significant preference for Na⁺ over Cs⁺. There is an ∼25% preferential occupancy of Li⁺ over larger cations in the ion atmosphere but, counter to general expectations from existing models, no size dependence for the other alkali metal ions. Further, we followed the folding of the P4–P6 RNA and showed that differences in folding with different alkali metal ions observed at high concentration arise from cation–anion interactions and not cation size effects. Overall, our results provide a critical test of a computational prediction, fundamental information about ion atmosphere properties, and parameters that will aid in the development of next-generation nucleic acid computational models

Hidden Structural Modules in a Cooperative RNA Folding Transition

Summary: Large-scale, cooperative rearrangements underlie the functions of RNA in RNA-protein machines and gene regulation. To understand how such rearrangements are orchestrated, we used high-throughput chemical footprinting to dissect a seemingly concerted rearrangement in P5abc RNA, a paradigm of RNA folding studies. With mutations that systematically disrupt or restore putative structural elements, we found that this transition reflects local folding of structural modules, with modest and incremental cooperativity that results in concerted behavior. First, two distant secondary structure changes are coupled through a bridging three-way junction and Mg2+-dependent tertiary structure. Second, long-range contacts are formed between modules, resulting in additional cooperativity. Given the sparseness of RNA tertiary contacts after secondary structure formation, we expect that modular folding and incremental cooperativity are generally important for specifying functional structures while also providing productive kinetic paths to these structures. Additionally, we expect our approach to be useful for uncovering modularity in other complex RNAs. : Gracia et al. dissect a complex RNA folding transition with changes in secondary and tertiary structure. They show that modular structures fold and interact with limited, incremental cooperativity. This cooperativity is sufficient to give a concerted folding transition and may balance competing biological demands on RNA stability and folding efficiency. Keywords: folding cooperativity, group I intron, kinetics, RNA folding, RNA structure probing, RNA tertiary structure, tetrahymena ribozym

MicroRNA 3′-compensatory pairing occurs through two binding modes, with affinity shaped by nucleotide identity and position

MicroRNAs (miRNAs), in association with Argonaute (AGO) proteins, direct repression by pairing to sites within mRNAs. Compared to pairing preferences of the miRNA seed region (nucleotides 2–8), preferences of the miRNA 3′ region are poorly understood, due to the sparsity of measured affinities for the many pairing possibilities. We used RNA bind-n-seq with purified AGO2–miRNA complexes to measure relative affinities of &gt;1000 3′-pairing architectures for each miRNA. In some cases, optimal 3′ pairing increased affinity by &gt;500 fold. Some miRNAs had two high-affinity 3′-pairing modes—one of which included additional nucleotides bridging seed and 3′ pairing to enable high-affinity pairing to miRNA nucleotide 11. The affinity of binding and the position of optimal pairing both tracked with the occurrence of G or oligo(G/C) nucleotides within the miRNA. These and other results advance understanding of miRNA targeting, providing insight into how optimal 3′ pairing is determined for each miRNA.</jats:p

RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo

© 2019 Elsevier Inc. Wesselhoeft et al. find that exogenous circular RNAs are able to bypass RNA sensors, thereby avoiding antiviral defense induction upon cellular entry. They report that nanoformulated, synthetic protein-coding circRNA can be translated in mouse tissues, providing evidence for the potential of circRNA as a vector for therapeutic gene expression