The Skaggs Institute
for Chemical Biology
Intracellular RNA Folding and Catalysis
M.J. Fedor, J.W. Cottrell, C.P. Da Costa, S.B. Daudenaurde, J.W. Harger, E.M. Mahen, M.R. Saha
RNAs play key
roles in normal growth and development, providing potential therapeutic targets
and reagents. RNAs are transcribed as linear polymers of 4 nucleotides, which must
adopt unique 3-dimensional structures in order to carry out their biological functions.
We focus on the pathways through which RNAs form specific functional structures
even though they can form many alternative nonfunctional structures that have similar
thermodynamic stabilities. Understanding how RNA assembly pathways produce unique,
functional outcomes will help explain the pathologic changes associated with RNA
misfolding and will guide the development of therapeutic approaches for disrupting
the RNA structures needed for proliferation of viral and microbial pathogens.
devised a way to evaluate RNA assembly during transcription in simple solutions
and in living cells by using catalytic RNA self-cleavage rates to quantify partitioning
between functional and nonfunctional RNA structures. We began by engineering ribozyme
variants with upstream and downstream inserts that potentially can form complementary
base pairs with either upstream or downstream parts of the ribozyme sequence (Fig.
1). Complementary base pairing between an insert and the ribozyme sequence creates
an AltH1 stem loop that is incompatible with assembly of the H1 stem loop that is
required for ribozyme function. We found that complementary inserts located upstream
of the ribozyme inhibited ribozyme assembly more than did downstream inserts during
transcription in vitro, consistent with a sequential folding model in which the
outcome of assembly is determined by the structure that forms first. In contrast,
both upstream and downstream inserts strongly inhibited assembly of the same ribozyme
variants when the variants were expressed as chimeric mRNAs in yeast.
|Fig. 1. Models
for intracellular RNA folding. A, In a rapid exchange model, secondary structures
that form during transcription can interconvert at a rate that might be accelerated
through the action of RNA chaperone proteins (lavender spheres). Structures with
the greatest thermodynamic stability are expected to dominate the folding outcome.
B, In a delayed folding model, secondary structure assembly does not occur during
RNA synthesis, perhaps because nascent transcripts associate with RNA binding proteins
(turquoise ellipses). Stable structures might form only after an entire folding
domain has been released from bound proteins. In a delayed folding model, stable
structures that form most rapidly are expected to dominate the folding outcome.
This evidence that an inhibitory stem loop can form downstream, after an entire ribozyme has been transcribed, indicates
that some element that is missing from simple in vitro assembly reactions can contribute
to RNA-folding reactions in living cells. Two models might account for this striking
result (Fig. 1).
a rapid exchange model, alternative structures interconvert rapidly,
allowing assembly to reach an equilibrium in which the most thermodynamically stable
structure predominates. In a delayed folding model, on the other hand,
assembly does not occur during transcription, perhaps because proteins that associate
with nascent transcripts prevent intramolecular base pairing. Once an entire domain
has been released from protein, perhaps concomitant with export of RNA from the
nucleus to the cytoplasm, partitioning among alternative structures might be governed
by differences in assembly rates.
In the first set of variants we examined,
AltH1 stem loops assemble from contiguous sequences while the H1 stem loops assemble
from separate ends of the RNA so that nonfunctional AltH1 stem loops might be expected
to assemble faster than H1 stem loops do. If so, the ability of AltH1 stem loops
to dominate the folding pathway could reflect the importance of fast folding kinetics,
consistent with the delayed folding model. To learn whether assembly kinetics affect
partitioning between alternative structures, we next examined circularly permuted
ribozymes in which H1 stem loops assemble from contiguous strands (Fig. 2). In these
circular permutants, H1 is expected to assemble faster than AltH1 stem loops do
that form between noncontiguous sequences.
|Fig. 2. Circular
permutants. Functional structures that include the essential H1 helix of the ribozyme
predominate over nonfunctional structures that include AltH1 stem loops when H1
forms from contiguous sequences even when AltH1 has greater thermodynamic stability
than H1 does.
mRNAs containing circularly permuted ribozymes were less abundant in yeast and had
the accelerated decay indicative of efficient intracellular self-cleavage of functional
ribozyme structures. The high self-cleavage activity of circular permutants suggests
that contiguous H1 stem loops are better able to resist competition from noncontiguous
AltH1 stem loops, even when upstream and downstream AltH1 stem loops have high thermodynamic
stability. Thus, contiguity, which would accelerate stem loop assembly, evidently
contributes to folding outcomes, consistent with the delayed folding model of secondary