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The Skaggs Institute
for Chemical Biology

Scientific Report 2007

Intracellular RNA Assembly

M.J. Fedor, J.W. Cottrell, C.P. Da Costa, S.B. Daudenaurde, E. Eren, L. Liu, M.R. Saha

Many RNA-mediated processes in growth and development require assembly and dissociation of specific RNA secondary structures (Fig. 1), yet the mechanisms that ensure assembly of functional structures and prevent the assembly of misfolded structures are poorly understood.

Fig. 1. RNAs must form specific base-paired secondary structures to carry out functions in growth and development, nd many RNAs must form different secondary structures at different steps along a reaction pathway.

We use self-cleaving ribozymes that can adopt defined nonfunctional structures to probe partitioning of RNAs among alternative structures in living cells. 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. 2). Base pairing between an insert and the ribozyme sequence creates an AltH1 stem loop that is incompatible with assembly of the H1 helix, an essential structural element of the ribozyme. We found that complementary inserts located upstream of the ribozyme inhibited ribozyme assembly more than did downstream inserts during RNA synthesis in vitro, consistent with a sequential folding model in which the upstream structure that forms first dominates the folding outcome. 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. 2. Hairpin ribozyme sequences with the potential to form either functional ribozymes or nonfunctional stem-loop structures. The secondary structure of an active ribozyme contains an essential base-paired helix called H1. The functional ribozyme that contains the H1 helix undergoes self-cleavage. The upstream Alt H1 ribozyme variant has a sequence added to the 5′ end (red) that can pair with 10 bases at the 5′ end of the ribozyme to form an Alt5′H1 stem loop that is incompatible with H1 assembly and prevents self-cleavage. If secondary structures assemble sequentially during RNA synthesis, Alt5′H1 would form before all of the sequences needed to form the essential H1 helix of the ribozyme have been synthesized, and the AltH1 insert would inhibit assembly of an active ribozyme. The downstream AltH1 variant has a sequence added to the 3′ end of the ribozyme (green) that can pair with 10 bases at the 3′ end to form an Alt3′H1 stem loop that is incompatible with assembly of a functional ribozyme. If secondary structures assemble sequentially, the complete ribozyme sequence could assemble into an active ribozyme with an H1 helix before the sequence needed to form Alt3′H1 has been synthesized, so the Alt3′H1 insert would not interfere with ribozyme self-cleavage activity.

This evidence that a downstream stem loop can form even after an entire ribozyme has been synthesized indicates that some feature of the intracellular environment alters the simple sequential folding pathway observed during RNA synthesis in vitro. Two models might account for this striking result. In a rapid exchange model, RNA chaperones might facilitate rapid interconversion among alternative structures, so that assembly reaches an equilibrium in which the most thermodynamically stable structure predominates. In the alternative delayed folding model, proteins that associate with nascent transcripts prevent assembly of RNA secondary structure during synthesis, and folding would occur only after an entire domain has been released from protein. In this model, the folding outcome is determined by differences among alternative structures in assembly and dissociation kinetics.

Recently, we have focused on distinguishing between these rapid conformational exchange and delayed folding models. In the first set of ribozyme variants we examined, AltH1 stem loops assembled from contiguous sequences while the H1 stem loops assembled from sequences at opposite ends of the ribozyme. Because AltH1 sequences were contiguous but H1 sequences were not, nonfunctional AltH1 stem loops would assemble faster than would H1 stem loops, and the ability of AltH1 stem loops to dominate the folding pathway would be consistent with the delayed folding model. To learn how contiguity and assembly kinetics affect assembly, we examined circularly permuted ribozymes in which H1 stem loops assemble from contiguous strands and would assemble faster than would AltH1 stem loops that form between noncontiguous sequences.

Chimeric 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 evidently affects partitioning among alternative structures, consistent with the delayed folding model.

To evaluate the importance of relative thermodynamic stability, we are investigating a series of variants with H1 and AltH1 structures that differ in relative thermodynamic stability. The pattern emerging from these studies is complex, suggesting that folding kinetics and thermodynamics both contribute to folding outcomes in vivo. Understanding how RNA assembly pathways produce functional RNA structures will help explain pathologic changes associated with RNA misfolding and will guide the development of therapeutic approaches for disrupting the RNA structures essential to proliferation of viral and microbial pathogens.


Da Costa, C.P., Fedor, M.J., Scott, L.G. 8-Azaguanine reporter of purine ionization states in structured RNAs. J. Am. Chem. Soc. 129:3426, 2007.

Cottrell, J.W., Kuzmin, Y.I., Fedor, M.J. Functional analysis of hairpin ribozyme active site architecture. J. Biol. Chem. 282:13498, 2007.


Martha J. Fedor, Ph.D.
Associate Professor

Fedor Web Site