Soft Interactions with Model Crowders and Non-canonical Interactions with Cellular Proteins Stabilize RNA Folding

Abstract

Living cells contain diverse biopolymers, creating a heterogeneous crowding environment, the impact of which on RNA folding is poorly understood. Here, we have used single-molecule fluorescence resonance energy transfer to monitor tertiary structure formation of the hairpin ribozyme as a model to probe the effects of polyethylene glycol and yeast cell extract as crowding agents. As expected, polyethylene glycol stabilizes the docked, catalytically active state of the ribozyme, in part through excluded volume effects; unexpectedly, we found evidence that it additionally displays soft, non-specific interactions with the ribozyme. Yeast extract has a profound effect on folding at protein concentrations 1000-fold lower than found intracellularly, suggesting the dominance of specific interactions over volume exclusion. Gel shift assays and affinity pull-down followed by mass spectrometry identified numerous non-canonical RNA-binding proteins that stabilize ribozyme folding; the apparent chaperoning activity of these ubiquitous proteins significantly compensates for the low-counterion environment of the cell.

Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change

“…It is becoming increasingly clear that excluded volume effects mediated by molecular crowding affect macromolecular structure, protein stability, enzyme activity and nucleo-cytoplasmic organization (Daher et al, 2018;Delarue et al, 2018;Hancock, 2004;Minton, 2001;Sukenik et al, 2018). Previous work has noted the potential for phase separation to dynamically buffer the intracellular protein concentration (Alberti et al, 2019).…”

“…Yet, the significance of the propensity of these biomolecules to phase-separate under the physiological conditions of their native intracellular environment, where molecular crowding is dominant, is poorly understood (Alberti et al, 2019). While it is possible to alter crowding within the test tube via the addition of synthetic macromolecules (Alberti et al, 2019), the nature and extent of crowding in the cellular context is quite different (Daher et al, 2018;Walter, 2019) and dynamically changes with the cellular state. For example, cell volume adjustments occur during processes critical to both cellular homeostasis and pathology, including the cell cycle (Tzur et al, 2009;Zlotek-Zlotkiewicz et al, 2015) as well as upon cell adhesion and migration (Guo et al, 2017;Watkins and Sontheimer, 2011).…”

Biological Pathway Specificity in the Cell—Does Molecular Diversity Matter?

“…The predicted significant impact of a large number of low‐affinity competitors on specific binding interactions is the reality of the cell's crowded molecular environment (Figure C). In fact, increasing evidence is emerging that, for example, many cryptic RNA‐binding sites exist on proteins of all trades, including many metabolic housekeeping enzymes . Similarly, such dynamic equilibrium considerations are the essence of recent modeling efforts of the regulatory RNA silencing machinery, whereby each RNA target competes both for protein partners of the RNA‐induced silencing complex (RISC) and microRNAs (miRNAs; Figure ) .…”

“…Yet, even well‐studied metabolic pathways have to be amended and expanded regularly, with important implications for normal physiology and diseases such as cancer . Similarly, recent studies have found that hundreds of proteins and RNAs “moonlight,” in that they each participate in multiple distinct biochemical pathways by using different parts of their surfaces to interact with diverse binding partners . When even metabolic enzymes that are thought to “normally” phosphorylate small metabolites equally function as protein kinases and phosphorylate a variety of protein substrates to regulate fundamental cellular functions, it is no wonder that cellular networks show much more redundancy and plasticity, and are harder to therapeutically target, than expected from simple linear cause‐and‐effect relationships.…”

Editorial Overview: Single-Molecule Approaches to Difficult Challenges in Folding and Dynamics

“…Daher and coworkers investigated the effect of crowding agents on the docking-undocking equilibrium of the hairpin ribozyme [24]. Two crowding agents are examined, polyethylene glycol and yeast extract.…”