Bridging the gap between in vitro and in vivo RNA folding

Abstract

Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functions in vivo. Within the last few years, methods have been developed to probe RNA structures in vivo and genome-wide. These studies reveal that RNA often adopts very different structures in vivo and in vitro, and provide profound insights into RNA biology. Nonetheless, both in vitro and in vivo approaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studies in vitro often lack direct cellular relevance, leaving a gap in our knowledge of RNA folding in vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under such in vivo-like conditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA folding in vitro and in vivo and discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.

Figure 1

The Classical View (top) and the Modern View (bottom) of RNA’s role in biology. In the classical view of biology, RNA (top) serves as a messenger molecule between DNA and proteins and proteins have all the main functions in cells. Messenger RNA serves to translate information from DNA to proteins. The modern view of biology (bottom) has emerged in the last 25 years as the field learns more about the many functions of RNA. Noncoding RNA (ncRNA) has vast regulatory functions, some of which include immune responses (‘ppp’=5′-triphosphate, which activates PKR)(Nallagatla et al., 2007), thermosensors, ribozymes, riboswitches, and genome editing. In the modern view of biology, proteins still have most cellular functions, but RNA plays essential roles in the cell beyond its classical functions.

Figure 2

RNA interactions with RNA binding proteins (RBP, left), metal ions (central), and ligands (star, right) can result in structure changes. Unlike typical in vitro conditions, there are other molecules and complex solutions conditions in vivo that can interact with RNA and change its structure. These structure changes can result in an RNA with less structure (top left) more structure (top right), or an alternate conformation than the structure that is prevalent in vitro (bottom). Also shown (bottom) are the bacterial expression platforms of riboswitches that switch between two mutually exclusive structures that turn a gene ON (left) or OFF (right) by exposing or sequestering the Shine-Dalgarno sequence (blue).

Figure 3

Artist’s rendition of in vitro conditions (left), in vivo conditions (right) and in vivo-like conditions (center). Typical in vitro solutions are dilute with high monovalent ion concentrations that are very different from cellular conditions. The cellular environment is complex with monovalent and divalent salts, macromolecules, cosolutes, and organelles. In vivo-like conditions (center) bridge in vitro and in vivo conditions and are more complex than in vitro conditions with added synthetic crowding agents and proteins and physiological ion concentrations. However in vivo-like conditions are still much less complex than those prevailing in vivo.

Figure 4

Depiction of the hierarchical RNA folding pathway, and folding funnels for non-cooperative and cooperative folding. (A) RNA folds in a hierarchical manner in which secondary structures form followed by tertiary structure. Hierarchical folding can be (B) rugged and non-cooperative in which the pathway intermediates are populated and the RNA can form misfolds (M_i) before populating the native state (N), or folding can occur in a (C) cooperative manner in which the intermediates do not populate and the RNA folds in a single transition.

Figure 5

Different RNA structures can be populated under in vitro, in vivo, and in vivo-like conditions. RNA structures induced by the cellular environment, including proteins and crowding, are shown in the two outermost structures. The conditions in vitro favor the population of a structure that may not always be the functional RNA structure (center two structures). Depending on the in vivo-like conditions chosen, specific RNA structures will be populated.

Figure 6

RNA modifications by DMS (dimethyl sulfate), SHAPE reagents (Selective 2′-hydroxyl acylation analyzed by primer extension), and CMCT (1-cyclohexyl-(2-morpholinoehyl)carbodiimide metho-p-toluene). SHAPE reagents modify the 2′-hydroxyl on the sugar of all four nucleobases. SHAPE reagents include 1M7 (1-methyl-7-nitroisatoic anhydride), NMIA (N-methylisotoic anhydride), and NAI (2-methylnicotinic acid imidazolide). DMS modifies the N1 of A and the N3 of C as well as the N7 of G. CMCT modifies N3 of U and N1 of G. The chemical modifications (except N7 of G) can be detected immediately by RT followed by gel electrophoresis or high-throughput sequencing, and the enzymatic cleavages, which modify single- and double-stranded RNA, can be read out through gel electrophoresis or high-throughput sequencing.

Figure 7

Under both in vitro (right, grey) and in vivo-like conditions with molecular crowding (left, pink) RNA fold into their native state that is functional, indicated in this figure by catalysis. High concentrations of Mg²⁺ (10 mM or higher) is needed to achieve the folded state in vitro compared to in vivo-like crowded conditions where low physiological Mg²⁺ (0.5 mM) folds the RNA. Reprinted with permission from Strulson, C. A., Yennawar, N. H., Rambo, R. P. & Bevilacqua, P. C. (2013). Molecular crowding favors reactivity of a human ribozyme under physiological ionic conditions. Biochemistry, 52, 8187–8197. Copyright 2016 American Chemical Society.