The Skaggs Institute
for Chemical Biology
Intracellular RNA Assembly
M.J. Fedor, J.W. Cottrell, L. Li, L. Liu, O. Tam, P. Watson, S. Zimmerman
Our goal is to
understand how RNAs fold into the correct 3-dimensional structures inside cells.
The mRNAs have long been recognized as key intermediates in the transmission of
information from the DNA sequence of genes to the amino acid sequence of proteins,
and noncoding RNAs are now known to perform several essential biological functions
previously attributed to proteins. Although RNAs must adopt precise 3-dimensional
structures to perform these functions, these nucleic acids tend to assemble into
a mixture of properly folded and misfolded structures during assembly reactions
carried out in vitro. We hope to learn how RNAs avoid misfolding and assemble into
functional structures during biogenesis in vivo.
have developed a way to use self-cleaving ribozymes embedded in chimeric mRNAs and
noncoding RNAs to probe RNA assembly in living cells. Ribozyme self-cleavage provides
a sensitive, quantitative signal that functional ribozyme structures have formed.
We began by engineering ribozyme variants with flanking inserts that have the potential
to form complementary base pairs with parts of the ribozyme sequence. The inserts
are located either upstream or downstream of the ribozyme sequence so they will
be transcribed either before or after the ribozyme. This arrangement allows us to
examine how the sequential nature of RNA synthesis affects folding outcomes (Fig.
1). Assembly of AltH1, the alternative helix, is incompatible with assembly of H1,
the ribozyme helix, and prevents self-cleavage. If RNA helices assemble sequentially
during transcription, an upstream insert would inhibit ribozyme assembly more than a downstream insert would. We
also designed AltH1 and H1 with different numbers of stabilizing base pairs to learn
how thermodynamic stability contributes to folding outcomes.
Chimeric RNAs contain sequences with the potential to form self-cleaving ribozymes
or nonfunctional structures, depending on the relative contributions of kinetics
and thermodynamics to RNA-folding outcomes. The secondary structure of this self-cleaving
ribozyme contains an essential base-paired helix, called H1, with 14 bp. A, A downstream
insert (green) is complementary to the adjacent ribozyme sequence and has the potential
to form an alternative helix with 12 bp, called 3′AltH1.
During cotranscriptional RNA assembly in vitro and in vivo, the H1 structure that
forms first resists competition from the downstream 3′AltH1
and allows assembly of a functional, self-cleaving ribozyme. B, In a second ribozyme
variant, an upstream insert (red) is complementary to the ribozyme sequence and
can form a helix with 12 bp, called 5′AltH1,
which is incompatible with H1 assembly. Although the 5′AltH1
helix has lower thermodynamic stability than does H1, 5′AltH1
can form first during transcription to create a kinetic trap that blocks subsequent
assembly of a functional ribozyme both in vitro and in vivo.
In the first set of ribozyme variants
we examined, upstream or downstream AltH1 sequences with 10 bp were able to compete
with assembly of H1 sequences with 8 bp. This result suggested that thermodynamic
stability, and not the sequential nature of RNA synthesis, was the major determinant
of folding outcomes in vivo. In vitro, an H1 helix with 8 bp dissociates slowly,
with a half-time on the order of days, so this evidence that a downstream AltH1
can block assembly of an upstream H1 implied that some feature of the intracellular
environment accelerates exchange between upstream and downstream structures.
In a second set of variants, H1 helices
with 14 bp were combined with AltH1 helices with 12 bp. These changes slowed helix
dissociation rates even further and reversed the relative thermodynamic stability
of H1 and AltH1 so that H1 helices were now more stable. Strikingly, assembly of
these variants did not always produce the most thermodynamically favored outcome.
As expected, significant cleavage did occur in the presence of a downstream insert,
evidence that stabilizing H1 by adding base pairs can prevent competition from a
shorter downstream 3′AltH1
that has lower kinetic and thermodynamic stability. However, the upstream 5′AltH1
also inhibits self-cleavage, even though H1 is longer and more stable, in contrast
to previous evidence that the most thermodynamically stable structure predominates
in vivo. Thus, ribozymes with 8 bp in H1 and 10 bp in AltH1 seem to reach thermodynamic
equilibrium during intracellular assembly, whereas ribozymes with 14 bp in H1 and
12 bp in 5′AltH1
seem to become trapped in an upstream 5′AltH1
with lower thermodynamic stability. Evidently, slow helix dissociation can trap
RNAs in thermodynamically less favored structures in vivo, but kinetic trapping
requires much longer helices with much slower dissociation kinetics than expected
from the behavior of similar RNA structures in vitro. Current efforts focus on understanding
the molecular basis of accelerated exchange between adjacent helices during RNA
biogenesis in living cells.
Fedor, M.J. Comparative enzymology and structural biology of RNA self-cleavage. Annu. Rev. Biophys.,