RNA Chaperones and RNA Folding Problem

• functional and structural property of RNA critical role in procecsses like RNA processing, splicing, translation
• folding of RNA influenced nature of RNA and protein interactions → origin of view outlined as follows
• RNA has two folding problems → 1) tendency to fold into and become kinetically trapped in alternative conformations 2) difficulty in specifying single tertiary structure that is thermodynamically strongly favored over competing structures (there is no fitness to folds, can always change so makes it hard to predict, thus meaning lack of variety of individual things)
• rna-binding proteins can help solve these folding problems
  • nonspecific RNA-binding proteins solve kinetic folding problem in vivo by acting as RNA chaperones that prevent RNA misfolding and fix misfolded RNA → fixes RNA
  • specific rna-binding proteins can solve thermodynamic folding problem by stabilizing a specific tertiary structure
  • emergence of nonspecific RNA-binding peptides with chaperone-type activities may have been early step in transition from RNA world to RNA-protein
  • specific RNA-binding proteins also have Chaperone activities that help prevent misfolding of their cogate 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 → similar concept to protein chaperones
• RNA chaperones = proteins that aid in process of RNA folding by preventing misfolding or by resolving misfolded species → not same as proteins that directly catalyze or work at the actual folding process/pathway or stabilize final folded protein or RNA structure

The Two Fundemantal Folding Problems of RNA

• inactive or alternative conformer is kinetically trapped to prevent activation
• several transfer RNAs were isolated in two conformations, only one of which could be charged by cognate aminoacyl-tRNA synthetase → inactive tRNA was found to be stable on hour time scale in presence or absence of Mg ion, but converted to active conformation upon heating in presenve of same Mg ion
• the same inactive tRNAs apparently adopt stable alternative secondary structures
• larger RNAs provide much additional evidence for kinetic folding problem → eg: in vitro self-splicing reactions of group 1 introns (group 1 indicates that they are >200 nucleotides) do not proceed to completion
  • no splicing indicates that these conformers are kinetically trapped, alternatively folded
• in vitro folding problem could be relevant to in vivo behaviour of RNA or might not be a good picture of all cellular interactions →
• primary, secondary, tertiary structuere comparison to proteins shows that kinetic folding problems described and additional thermodynamic folding problems are intrinsic to RNA

Primary:

• RNA's primary structure less diverse with smaller parameter space → only 4 nucelotides (ACGT) instead of 20 amino acids for proteins (nucleotides are either purines and pipyrimidinerimidines with minimal differences in structure [presence of Oxygen for example] and have complimentary pairing based on hydroxide bonds compared to protein amino acids made up of different sequence of nucleotides in certain order)
  • in primary stucture, constituents known as side chains
• RNA side chains come in purines/pyrimidines whereas proteins comprise of hydrophobic, hydrophilic, and charged groups of varying sizes and shapes
• dearth of primary structure diversity (essentially means that there is smaller parameter space, and thus low information content or parameter space) is expected to mean that RNA sequences for folding at the tertiary level will have a lower level of specificity or uniqueness

Secondary:

• high thermodynamic stability of RNA duplexes (?? comes in pairs I guess) expected to result in kinetic folding problems →
  • most stable part of the protein (alpha-helixes dissociate on sub-microsecond time scale)
  • in contrast, RNA duplex of 10 base pairs has half-time for dissociation of 30 minutes, and G/C-rich duplezes of 10 base pairs have disassociation half-times of 100 years at 30deg celsius → this means that RNA can get stuck in wrong conformation for longer periods of time
• this is a good indication of the kietic problem which can prevent structured RNA from adopting correct conformation and prevent access to mRNA, and even prevent turnover of an RNA subsequenct to correct folding (as per usual function)
• potential for alternative folds appear to be common property of RNAs → even random RNAs predicted to have structures with about half of residues base-paired, consistent with estimated helical content of randomly associated RNAs → (?? adds to diversity I guess)

Tertiary:

• probelm of stable alternative secondary folds is exacerbated by fortuitous tertiary interactions with 2' - hydroxlys, phosphoryl groups, and metal ions and by formatino of nonstandard base/base interactions that can further stabilitze incorrect RNA conformers
• low information content of RNA primary structure is further decreasesd by sequestering the base-pairing faces of residues in interior of duplexed regions, while side chains of proteins face outward in alpha-helixes and beta-sheets
• each RNA secondary strucutre elemnt thus has strong resemblance to others, so that RNA cna have difficult time sepcifying unique tertiary structure
  • eg. duplex of Tetrahymena group I ribozyme docks into tertiary interactions incorrectly approximately 1/1000 of the time + mutations increase this misdocking to about one-half
    • although there may be difficulty in ensuirng that correct tertiary structure of an RNA is formed, problem is not insurmountable → free energy preference of only 2 kcal/mol is sufficient to ensure >95% correct folding

RNA Chaperones as a Solution:

• RNA has fundamental folding problem, tendency to be kinetically trapped in misfolded forms (Nonspecific RNA-binding proteins can overcome problem in vitro)
• a protein UP1 → fragment of hnRNP^5 A1 protein can renature 5 S and tRNAs that were kinetically trapped in alternative conformations → long single strands of RNA or DNA reassociate orders of magnitude slower than short oligonucleotides becase the longer nucleic acids form intramolecular structures that limt access by complementary strand