A Divalent Metal Cation-Metabolite Interaction Model Reveals Cation Buffering and Speciation.

I present the perspective that the divalent metalome and the metabolome can be modeled as a network of chelating interactions instead of separate entities. I review progress in understanding the complex cellular environment, in particular recent contributions to modeling metabolite-Mg interactions. I then demonstrate a simple extension of these strategies based approximately on intracellular concentrations.

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.

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 Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability.

We examined the complex network of interactions among RNA, the metabolome, and divalent Mg under conditions that mimic the cytoplasm. We determined Mg binding constants for the top 15 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 conditions, which we term "Eco80".

Genome-wide analysis of the 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.

RNA sequence and structure control assembly and function of RNA condensates.

Intracellular condensates formed through liquid-liquid phase separation (LLPS) primarily contain proteins and RNA. Recent evidence points to major contributions of RNA self-assembly in the formation of intracellular condensates. As the majority of previous studies on LLPS have focused on protein biochemistry, effects of biological RNAs on LLPS remain largely unexplored.

Functional Roles of Chelated Magnesium Ions in RNA Folding and Function.

RNA regulates myriad cellular events such as transcription, translation, and splicing. To perform these essential functions, RNA often folds into complex tertiary structures in which its negatively charged ribose-phosphate backbone interacts with metal ions. Magnesium, the most abundant divalent metal ion in cells, neutralizes the backbone, thereby playing essential roles in RNA folding and function.