From the
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 chaperone-type
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, 12, 13, 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, 16, 17, 18, 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 (20, 21, 22, 23, 24, 25, 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. 1and Table 1).
Figure 1:
RNA folding and the
effect of RNA chaperones. A, schematic free energy profiles
for folding of RNA in the absence (solid line) and presence (dashed line) of an RNA chaperone. Species with only secondary
structure are shown to emphasize the stability of RNA secondary
structure and the tendency to be trapped in incorrect secondary
structures, even though such species may not exist as discrete
intermediates. Only one alternative RNA secondary structure and only
one tertiary structure are shown for simplicity. The barrier for going
from unfolded to the correct secondary structure with and without
chaperone is shown as the same, although binding of a chaperone could
slow correct folding, as observed with protein chaperones. A protein
that lowers this barrier to speed the folding process can be referred
to as a ``guide.'' Chaperones work by decreasing the barrier
for escaping from the incorrectly folded structure. B, one of
several physical models of how an RNA chaperone could facilitate
refolding of misfolded RNAs. Imperfect charge complementarity between
the protein surface and RNA secondary structure (protRNA
). The protein speeds unfolding by favoring
conformational excursions that increase charge complementarity. The
protein binds more strongly to the unfolded RNA (prot
RNA
) relative to the folded RNA
because the unfolded RNA is free to rearrange to give charge
complementarity with the protein. Refolding can occur from this state
to either the correct or incorrect secondary structure and is driven by
the stabilization from base pair formation, which counters the
destabilization from loss of charge
complementarity.
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) .
Solutions to the RNA Folding Problems:
RNA Chaperones and Specific RNA-binding Proteins
This idea was apparently suggested for RNA over 20 years
ago(38, 39) . It was shown that the protein UP1, a
fragment of hnRNP ()A1 protein, could renature 5 S and tRNAs
that were kinetically trapped in alternative conformations and
suggested that such activities would be necessary in biology. DNA
annealing experiments appear to have provided the intellectual roots
for these ideas(40) . Long single strands of RNA or DNA
reassociate orders of magnitude slower than short oligonucleotides, in
part because the longer nucleic acids form intramolecular structures
that limit access by the complementary strand. Catalysis of
polynucleotide annealing by single-strand nucleic acid-binding
proteins, such as T4 gene 32 protein and Escherichia coli SSB,
would then arise from a disruption of intramolecular structure that
enhances access for intermolecular
base-pairing(40, 41) . The RNA chaperone proposal
brings molecular chaperones full circle, as early speculation about the
involvement of chaperones in protein folding, which is now
well established, was framed by analogy to the ability of single-strand
nucleic acid-binding proteins to catalyze nucleic acid duplex
formation: both facilitate correct folding by preventing
misfolding(42) . The energetics of RNA chaperone action are
depicted schematically in Fig. 1A, and one physical
model is portrayed in Fig. 1B.
Several recent experiments strongly support such an in vitro RNA chaperone activity of RNA-binding proteins. Slow physical steps in the reaction of a hammerhead ribozyme limit turnover and specificity (43) so this system provides an intermolecular model for the kinetic problems in RNA folding, i.e. dissociation of intermolecular duplexes is crucial for turnover and for discrimination against incorrect (mispaired) RNA substrates(44) . This can be likened to the unraveling of RNAs that have adopted incorrect secondary structures during folding. Proteins such as the NC protein from HIV-1 and the hnRNP A1 protein were shown to facilitate these physical steps and thereby enhance the ribozyme reaction. In addition, the NC protein resolved a kinetically trapped misfolded complex with HH16(4, 6, 46, 47) .
As mentioned above, the self-splicing of group I introns in vitro is often slow and inefficient, whereas splicing in vivo appears to be fast and efficient(48) . In some cases, proteins facilitate splicing in vivo by binding specifically to and stabilizing the catalytically active conformation of the intron (49, 50, 51) . In contrast, the E. coli S12 ribosomal protein facilitates proper folding of group I introns by nonspecific binding, suggesting a second mechanism for aiding group I intron splicing in vivo(5) . Characterization of the S12 protein facilitation further strengthened the analogy between RNA chaperones and protein chaperones. (i) The S12 protein shows no preferential binding to group I introns over exons or other RNAs, suggesting that S12 does not act by specifically stabilize the intron's catalytic conformation. (ii) The S12 protein is also able to facilitate a hammerhead ribozyme reaction, analogous to the NC and hnRNP A1 proteins, further suggesting a nonspecific rather than specific mode of action. (iii) The S12 protein promotes splicing of a population of kinetically trapped, unreactive precursor RNA, suggesting an ability to resolve misfolded RNAs. (iv) Protein chaperones function solely during a folding step and are not present in the final active species. The same stimulatory effect on group I self-splicing was observed whether or not S12 was removed by proteolysis prior to initiation of the self-splicing reaction. Thus, the S12 protein is required solely for folding and can act as a true chaperone.
Figure 2: Preassociation binding pathway in which a specific RNA-binding protein also has RNA chaperone activity, using nonspecific interactions to facilitate proper folding of its cognate RNA. The nonspecific and specific interactions are represented schematically by the absence and presence, respectively, of charge and shape complementarity within the complexes.
Protein chaperones appear to be a distinct class of molecules
designed to facilitate protein folding. In contrast, the significant
extent of nonspecific binding by RNA-binding proteins suggests that
many RNA-binding proteins may exhibit RNA chaperone activity in
vitro. Over 20 different proteins from E. coli extracts were able to facilitate group I intron splicing in
vitro(5) , but it is not known which proteins, if any, act
as cellular RNA chaperones. The hnRNP proteins, which coat pre-mRNA as
it is transcribed, represent the most obvious candidate class for
cellular RNA chaperones (see also (54) ).
Protein chaperones facilitate folding but do not remain bound to the final folded protein product, whereas RNA chaperones may facilitate the folding process and subsequently remain bound because of high levels of nonspecific binding affinity. This may represent a basic difference in the primary recognition element for the two classes of chaperones. Protein chaperones appear to recognize unfolded proteins because of exposed hydrophobic residues; when the protein folds and these residues are buried, the chaperone no longer binds strongly(52) . In contrast, the charged phosphodiester backbone and their bases are likely to be at least partially exposed in unfolded or misfolded RNA, allowing nonspecific binding, especially via electrostatic interactions.
Mechanistic studies of protein chaperones have suggested that they prevent misfolding by sequestering unfolded forms so that they cannot aggregate(52, 55, 56) . RNA chaperones may act similarly by binding to regions of an RNA and preventing or slowing formation of certain intramolecular structures. The RNA chaperones have also been shown to resolve RNAs that have already misfolded (see above), whereas the protein chaperones that have been best characterized can bind and sequester unfolded proteins but appear unable to bind efficiently to and resolve protein aggregates. The high nonspecific binding activity of RNA-binding proteins may account for this difference by allowing RNA chaperones to bind and subsequently to resolve misfolded RNA conformers (Fig. 1B). However, recent in vivo characterization of the Hsp104 protein has suggested that it actively resolubilizes protein aggregates(57, 58) , although the molecular basis for this is not known.
There are proteins other than the chaperones referred to above that aid proper protein folding such as prolyl isomerases and protein disulfide-isomerases(55) . Specific RNA-binding proteins can exhibit RNA chaperone activity by helping to prevent and resolve misfolding of both cognate and noncognate RNA (Fig. 2). Specific RNA-binding proteins could also aid the process of folding for the cognate RNA by acting as ``guides'' in the folding process, i.e. by trapping correctly folded domains or subdomains to help bias the RNA to follow along the folding path toward the final correctly folded structure. In addition, protein/protein interactions can bring together two RNAs (or two regions of one RNA), thereby increasing the probability of duplex formation or other interactions(2, 59, 60) . Proteins that do this might be referred to as matchmakers, rather than chaperones(7) . Such proteins may be involved in spliceosome assembly. There is evidence that the hnRNP A1 protein can act as both a chaperone and matchmaker (2, 6, 7) . RNA chaperones, matchmakers, and guides each can increase the observed rate of RNA/RNA assembly, so that it often may be difficult to distinguish these mechanistically.
RNAs could also act as RNA chaperones to assist in the folding of other RNAs. ``Facilitators'' are RNAs that base-pair to a ribozyme adjacent to the substrate(61) ; they presumably prevent the ribozyme from folding up upon itself, thereby increasing access for base-pairing to the substrate. This is analogous to the facilitation of duplex formation by single-strand binding proteins. There are several examples of intramolecular changes that either introduce or resolve problems in folding of an RNA (e.g.(62) and (63) ). This might be likened to the role of the prosequence in reducing a kinetic barrier in the folding of certain bacterial proteases(32) .
The use of energy by Rd-ATPases could also allow RNA folding and unfolding steps to be integrated and regulated within complex biological phenomena. For example, an Rd-ATPase may be used to dissociate the U4-U6 snRNP complex at just the right time in spliceosomal assembly, facilitating assembly of a catalytically active spliceosome and/or preventing inappropriate or premature splicing(68) . U4 may act as an RNA chaperone made of RNA that prevents misfolding of U6.) Rd-ATPases could also help select between alternative splice sites and prevent inaccurate splicing via a proofreading function that limits the time allotted for individual assembly and catalytic steps(69) . Rd-ATPases have also been implicated in ribosomal assembly and translational initiation.
The above ideas can be placed within a unifying but speculative evolutionary context in which an early step in the transition from the RNA world to the RNA/protein world was the emergence of nonspecific RNA-binding peptides with chaperone-type activities. These peptides could have provided a selective advantage in a primitive RNA-dominated world by rescuing RNAs from kinetic traps, aiding in the structural transition of a postreplicative duplex to a folded, functional single-stranded RNA, and helping RNAs more broadly explore structural alternatives. The appearance of a functional nonspecific RNA-binding peptide is expected to be more probable than the appearance of a specific RNA binder because there are more solutions to the problem of nonspecific binding and because a nonspecific binder would have many potential functional targets.
Later in evolution, the problems in folding RNA could have been parlayed into new opportunities for biological systems via cooperation between RNA and proteins, with nonspecific RNA-binding proteins with RNA chaperone functions developing binding preferences and ATP-dependent activities for use in control and regulation. For example, the hnRNP A1 protein has RNA chaperone activity (6, 7, 46) and also appears to be involved in splice site selection(70) , and the NC protein from HIV has chaperone activity and appears to bind viral RNA specifically during packaging(4) .
Orchestration of RNA Chaperone Activity in Vivo
The nonspecific RNA-binding proteins that enhance RNA function can also shut down RNA function at higher concentrations, so that there is a limited ``window of opportunity'' for each protein to be functional (1, 6, 7) . How then can a cell orchestrate the function of a large number of such proteins amidst a pool of near-random RNA without merely binding to and obscuring the function of a large subset of the RNAs? How are the chaperones removed to allow the RNA to function? How does a specific RNA-binding protein find its cognate RNA?
The answers to these questions are not known. Although the concentrations of the various RNA and protein components and their affinities can be regulated to influence RNA processing and function (e.g. Refs. 31, 33, 71, 72), it is not clear that affinities can be tuned and concentrations regulated precisely enough to fully avoid problems of inappropriate RNA/protein pairings and proteins obscuring RNA function. Higher order temporal and spatial cellular organization could be used to avoid these problems and to integrate RNA/protein interactions into other cellular processes. RNA could be ``handed off'' from one protein to another, with hnRNP proteins binding pre-mRNA as it is transcribed, perhaps being selectively replaced by proteins to set the stage for spliceosomal assembly and splicing(9, 34) . After splicing, the mRNA may be escorted to the cytoplasm by a subset of the hnRNPs and by other proteins (10) and then delivered to the ribosome for translation. Evidence for hand offs of proteins from one chaperone to another during folding (29, 56) provides a conceptual precedent for analogous action by RNA chaperones. The replacement of one protein by another could be spatially or temporally regulated by spatial segregation of specific RNAs and specific RNA-binding proteins within the nucleus (as for ribosomal assembly within the nucleolus) or by fast initial binding of more weakly bound proteins followed by slower binding of more strongly bound proteins and complexes.
A general advantage of keeping RNA molecules protein-bound is that proteins can dissociate faster than some RNA self-structures unravel on their own, allowing the RNA to change partners in a timely fashion throughout its processing odyssey. However, in some instances, the kinetic stability of RNA structures has been co-opted for cellular function; the classical example is attenuation of the trp and other bacterial operons that are regulated via a choice between alternative RNA secondary structures(45) .
It will be fascinating over the coming years to learn how RNA folding is controlled within the organization of RNA processing and RNA function. On the molecular level, it will be fascinating to unravel the mechanisms of RNA folding and RNA chaperone activity.