RNA Chaperones and the RNA Folding Problem

Abstract

Functional and structural inter-relationships of RNA and proteins in the execution and control of biological processes such as RNA processing, RNA splicing, and translation are increasingly apparent. In this minireview, I present an RNA chaperone hypothesis, which fosters the view that constraints imposed by fundamental problems in the folding of RNA have profoundly influenced the nature of RNA/protein interactions in biology. The origin of this view is outlined as follows. RNA has two fundamental folding problems: a tendency to fold into and become kinetically trapped in alternative conformations and a difficulty in specifying a single tertiary structure that is thermodynamically strongly favored over competing structures. RNA-binding proteins can help solve both RNA folding problems. Nonspecific RNA-binding proteins solve the kinetic folding problem in vivo by acting as RNA chaperones that prevent RNA misfolding and resolve misfolded RNAs, thereby ensuring that RNA is accessible for its biological function. In addition, specific RNA-binding proteins can solve the thermodynamic folding problem by stabilizing a specific tertiary structure. The emergence of nonspecific RNA-binding peptides with chaperonetype activities may have been an early step in the transition from the RNA world to the RNA/protein world. Specific RNA-binding proteins may also have RNA chaperone activities that help prevent misfolding of their cognate RNAs. RNA-dependent ATPases may act as RNA chaperones that spatially and temporally control RNA conformational rearrangements. “RNA chaperone” refers to proteins that aid in RNA folding and is not meant to refer to chaperones made of RNA. For clarity, the classical chaperones that aid protein folding are referred to as “protein chaperones.” In keeping with the accepted definition of protein chaperones, RNA chaperones are defined as proteins that aid in the process of RNA folding by preventing misfolding or by resolving misfolded species. This is in contrast to proteins that help protein or RNA folding by catalyzing steps along the folding pathway or by stabilizing the final folded protein or RNA structure. There are no established examples of RNA chaperones that act in vivo. This hypothesis is presented because the in vitro data reviewed herein provide support for the hypothesis and this view provides a conceptual framework for RNA folding and RNA/protein interactions. The kinetic problem in RNA folding is emphasized, while space constraints have greatly limited discussion of the thermodynamic problem. The Two Fundamental Folding Problems of RNA Many of the examples of RNA misfolding in vitro suggest that the inactive or alternative conformer is kinetically trapped such that it does not revert to the active conformation even after long periods of time. Early work showed that several tRNAs were isolated in two conformations, only one of which could be charged by the cognate aminoacyl-tRNA synthetase (11–14). An inactive tRNA was stable on the hour time scale in the presence or absence of Mg, but was converted to an active conformation upon heating in the presence of Mg (12). These inactive tRNAs apparently adopt stable alternative secondary structures (15–19). Larger RNAs provide much additional evidence for a kinetic folding problem. For example, in vitro self-splicing reactions of group I introns, which are 200 nucleotides, typically do not proceed to completion. This suggests the presence of kinetically trapped, alternatively folded conformers (see also Refs. 20–26). The RNA folding problems observed in vitro could be relevant to the in vivo behavior of RNA or could instead arise as an artifact of in vitro handling of RNA, as RNA is typically purified under denaturing conditions and then renatured. A comparison of the primary, secondary, and tertiary structure of RNA and proteins, based in part on an insightful analysis of tRNA structure (27), suggests that the kinetic folding problems described above and additional thermodynamic folding problems are intrinsic to RNA (summarized in Fig. 1 and Table I). Primary Structure—RNA has a paucity of primary structure diversity compared with proteins, with just 4 side chains instead of 20. Furthermore, the 4 RNA side chains are more similar to one another than the protein side chains. The RNA side chains come in only two “sizes,” purine and pyrimidine, and each is a planar group decorated with hydrogen bond donors and acceptors, whereas the protein side chains comprise hydrophobic, hydrophilic, and charged groups of varying sizes and shapes. The dearth of primary structure diversity, or low “information content,” of an RNA polymer (relative to a protein polymer) would be expected to render it more difficult for an RNA sequence to specify a unique tertiary structure. Secondary Structure—The high thermodynamic stability of RNA duplexes is expected to result in kinetic folding problems. The most stable protein !-helices dissociate on the sub-microsecond time scale (28). In contrast, an RNA duplex of 10 base pairs has a half-time for dissociation of #30 min, and G/C-rich duplexes of 10 base pairs have dissociation half-times up to #100 years at 30 °C (30). Thus, RNA can get stuck in the wrong conformation (Fig. 1). This kinetic problem could prevent a structured RNA from adopting the correct conformation, could prevent access to mRNA, and could even prevent turnover of an RNA subsequent to correct folding. The potential for alternative folds appears to be a common property of RNAs. Even random RNAs are predicted to have structures with about half of the residues base-paired, consistent with the estimated helical content of randomly associated RNAs (35, 36). Tertiary Structure—The problem of stable alternative secondary folds is exacerbated by fortuitous tertiary interactions with 2$hydroxyls, phosphoryl groups, and metal ions and by the formation of nonstandard base/base interactions that can further stabilize incorrect RNA conformers. Even after RNA adopts the correct secondary structure, it is not yet “out of the woods.” The low information content of RNA primary structure is further decreased by sequestering the base-pairing faces of residues in the interior of duplexed regions, while the side chains of proteins face outward in !-helices and -sheets. Each RNA secondary structure element thus has a strong resemblance to others, so that RNA can have a difficult time specifying a unique tertiary structure. For example, a duplex of the Tetrahymena group I ribozyme docks into tertiary interactions incorrectly approximately 1/1000 of the time, and mu* This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995. This work was supported by National Institutes of Health Grant GM49243. ‡ Lucille P. Markey Scholar in Biomedical Sciences and a Searle Scholar. 1 “Nonspecific” is used for simplicity, even though there is presumably no truly nonspecific RNA-binding protein. It refers to RNA-binding proteins with low or wide binding specificities. However, RNA-binding proteins, even those that bind a particular target RNA in vivo, bind other RNAs with reasonably high affinity. The difference between specific and nonspecific or widely specific proteins is quantitative rather than qualitative, so that an absolute distinction is not possible. 2 The term RNA chaperone is already in use by some in the field (1–8). I suggest that only proteins with demonstrated biological roles as chaperones in RNA folding be referred to as RNA chaperones, while the ability to facilitate folding in vitro be referred as “RNA chaperone activity.” The definition of RNA chaperones is further honed in the text, and some possible ambiguities are addressed. For example, specific RNA-binding proteins can exhibit biological or nonbiological RNA chaperone activity in the folding of cognate or noncognate RNAs (see Fig. 2 and text). I suggest that these proteins not be referred to as RNA chaperones, in deference to their other functions. Minireview THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 36, Issue of September 8, pp. 20871–20874, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.