What is RNA?

A polymeric molecule called ribonucleic acid (RNA) is essential for many biological processes, including the coding, decoding, control, and expression of genes. Nucleic acids include RNA and deoxyribonucleic acid (DNA). Nucleic acids are one of the four primary macromolecules required for all known forms of life, along with lipids, proteins, and carbohydrates.

The role that RNA will play within the cell is determined by its kind. Other cellular RNA components, besides the coding region of messenger RNA (mRNA) molecules that will be translated into proteins, are involved in a variety of cellular functions, such as the transcriptional and post-transcriptional control of genetic material, temperature and ligand sensing, translation control, and RNA turnover.

Can RNA fold itself?

The most crucial mechanism underlying RNA function is RNA folding. Understanding the mechanisms causing RNA folding in vitro has advanced significantly, but research into the principle guiding the development of intracellular RNA structure is still infancy.

Nearly all cellular activities depend heavily on RNA molecules. RNAs require certain three-dimensional structures to carry out their function. The transformation of an RNA molecule from its unfolded, chaotic state to its native, functional conformation is known as folding. Intensive research has been done on in vitro RNA folding, mostly employing catalytic RNAs as model systems. Investigating ribozyme folding benefits greatly from measuring the development of the native structure as a function of catalysis.

RNA undergoes two main folding issues:

  1. Due to their propensity for misfolding, RNA molecules can get stuck in inactive, frequently long-lasting conformations, whose escape becomes rate-limiting during the folding process.                                                          
  2. The natural, functional RNA conformation could not be thermodynamically preferred over other intermediate structures, necessitating the aid of a particular RNA-binding protein (or high salt) for stabilization of the tertiary structure.

Discovering In Vivo RNA Folding Pathways

Intermediate folding states are scattered across the hierarchical path leading to the functional structure. Most of these folding intermediates are on-path and exhibit more native interactions. Thus, one of the most exciting challenges of an RNA folding pathway is recognizing folding phases and defining their structural composition. Categorizing intermediate folding states in vitro has advanced significantly up to this point, sometimes even at the atomic level.

A thorough evaluation of RNA folding in vitro under non-physiological conditions and in vivo is of great interest given the different settings involved in vitro refolding and the development of intracellular RNA structures. According to our knowledge, just one study has explored this fascinating topic. The P4-P6 and P3-P9 structural domains, which make up Group I introns, come together to form a gap for binding stem P1, which has the 5′ splice-site (5′SS). In vitro folding of the group, I introns, specifically of the Tetrahymena ribozyme, has been meticulously examined during the past few decades.

In conclusion, the Tetrahymena ribozyme travels across a challenging free-energy landscape, yet along the direct pathway, the P4-P6 domain folds quickly, followed by the slowly assembled P3-P9 domain. 43 Other group I introns also share these key features. 4,9,10 Using DMS chemical probing in E. coli, the structure of td wild-type and mutant introns was observed to evaluate group I intron folding in vivo. The tertiary structure of the td ribozyme is affected differently by intron mutations, indicating that they interfere with folding at various stages. For example, destabilizing stem P6 caused structural changes in both major domains, whereas weakening stem P7 only affects the folding of the P3-P9 domain. Analyzing the intracellular structure of these folding transitions allowed the key folding steps seen in vitro to be compared to a hypothesized order of events in a hierarchical in vivo folding process.


Current Techniques for Investigating RNA Structure in Vivo

To examine RNA/RNP structures in vitro, a variety of experimental techniques, including physical and chemical methods, are used. However, its application in examining the RNA structure and function in living cells is constrained by the complexity of cells. In vivo RNA structure has been investigated using a variety of chemical reagents that are sensitive to secondary and/or tertiary structures.

The most effective reagent for RNA structural probing in a wide range of organisms, including eukaryotic and bacteria, is DMS. The speed with which DMS penetrates all cell compartments without first permeabilizing the cells is a significant convenience. DMS methylates the N7 of guanines, the N1 of adenines, and the N3 of cytosines after absorption if they are not involved in H-bonding or are not covered by proteins. Likewise, lead-(II)-acetate was employed to examine the RNA structure of bacteria.

It is simple for this ion to enter bacterial cells, where it predominantly causes cleavages at sites of strong metal ion binding. Last but not the least, intermolecular interactions and RNA tertiary structure can be investigated using hydroxyl radical footprinting. 118 In a nutshell, X-rays from a high flux synchrotron produce hydroxyl radicals inside of cells that can remove a hydrogen atom from ribose and start the breakage of the RNA backbone. 118 By doing so, hydroxyl radical cleavage correlates with the backbone's solvent accessibility, giving nucleotide-level information. Notably, the modification or cleavage sites are then mapped via primer extension of total RNA extracts, providing details on the structure of RNA and the interactions between it and proteins in vivo.

Another effective method for discovering new RNA-protein interaction partners or defining RNA-protein interactions in vivo is UV-crosslinking. 134–139 This technique offers crucial spatial restraints and details on how RNP complexes are structured. Recently, Xenopus Leavis oocytes were employed for the first time to explore RNA-protein interactions at the atomic level using the chemo genetic methodology NAIM. 140,141 As nucleotide analog phosphorothioates, random alterations of base or backbone moieties are introduced into the transcript before being microinjected in oocytes and subsequently identified as functional groups necessary for RNP assembly in vivo. It is possible to alter UV-crosslinking and NAIM to investigate intra- or intermolecular RNA interactions.



Despite tremendous advancements in our understanding of RNA folding in vivo, there is still much to learn about the development of intracellular RNA structures.

The creation of novel methods to track the production of RNA structures in living cells is necessary for any advancement in our knowledge of the mechanisms that cause RNA folding in living cells. Particularly of great interest are methods like NMR that would make it possible to analyze folding kinetics in vivo.

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