Danielle Konings

Pattern analysis of RNA secondary structure: Similarity and consensus of minimal-energy folding

We describe an automated procedure to search for consensus structures or substructures in a set of homologous or related RNA molecules. The procedure is based on the calculation of optimal and sub-optimal secondary structures using thermodynamic rules for base-pairing by energy-minimization. A linear representation of the secondary structures of the related RNAs is used so that they can be compared and classified using standard alignment and clusterings programs. We illustrate the method by means of two sets of homologous small RNAs, U2 and U3, and a set of alpha-globin mRNAs and show that biologically interesting consensus structures are obtained.

Application of computational technologies to ribozyme biotechnology products

Abstract Ribozymes are RNA molecules that act enzymatically to cleave other RNA molecules. The cleavage reaction requires the binding of ribozyme to specific sites on the target RNA through (mostly) Watson-Crick base-pairing interactions. Association of ribozyme with target completes a three-dimensional ribozyme/target complex which results in cleavage of the target RNA. We are employing both computational and experimental approaches to identify sites on target RNA molecules that are open to ribozyme attack and to determine which ribozymes are most active against those sites. Two types of computational technologies are available for aiding in the identification of target sites and design of active ribozymes. First, DNA/RNA sequence analysis software is employed to identify sequence motifs necessary for ribozyme cleavage and to look for sequence conservation between different sources of the target organism so that ribozymes with the broadest possible target range can be designed. Second, RNA folding algorithms are employed to predict the secondary structure of both ribozyme and target RNA in an attempt to identify combinations of ribozyme and target site that will successfully associate prior to ribozyme cleavage. The RNA folding algorithms utilize a set of thermodynamic parameters obtained from measurements on short RNA duplexes; while these rules give reasonable predictions of secondary structure for a small set of highly structured RNAs, they remain largely untested for predicting the structure of messenger RNAs. This paper outlines the current status of designing ribozymes that fold correctly and of locating target sites that are sufficiently unfolded to allow ribozyme cleavage.

Equal G and C contents in histone genes indicate selection pressures on mRNA secondary structure

SummaryProtein-specific versus taxon-specific patterns of nucleotide frequencies were studied in histone genes. The third positions of codons have a (well-known) taxon-specific G+C level and a histone type-specific G/C ratio. This ratio counterbalances the G/C ratio in the first and second positions so that the overall G and C levels in the coding region become approximately equal. The compensation of the G/C ratio indicates a selection pressure at the mRNA level rather than a selection pressure or mutation bias at the DNA level or a selection pressure on codon usage. The structure of histone mRNAs is compatible with the hypothesis that the G/C compensation is due to selection pressures on mRNA secondary structure. Nevertheless, no specific motifs seem to have been selected, and the free energy of the secondary structures is only slightly lower than that expected on the basis of nucleotide frequencies.

Structural Analysis of a Group II Intron by Chemical Modifications and Minimal Energy Calculations

Folding of the yeast mitochondrial group II intron aI5c has been analysed by chemical modification of the in vitro synthesised RNA with dimethylsulfate and diethylpyrocarbonate. Computer calculations of the intron secondary structure through minimization of free energy were also performed in order to study thermodynamic properties of the intron and to relate these to data obtained from chemical modification. Comparison of the two sets of data with the current phylogenetic model structure of the intron aI5 reveals close agreement, thus lending strong support for the existence of a typical group II intron core structure comprising six neighbouring stem-loop domains. Local discrepancies between the experimental data and the model structures have been analyzed by reference to thermodynamic properties of the structure. This shows that use of the latest refined set of free energy values improves the structure calculation significantly.

Coexistence of multiple codes in messenger RNA molecules

Abstract The coexistence of multiple codes in messenger RNA molecules is investigated. A unique biological example of multiple coding is revealed by the rev responsive element (RRE) of lentiviruses. This element overlaps with the env gene and forms a specific, multibranched RNA conformation. An analysis of the similarity in the nucleotide sequences and the similarity in the secondary structures, as well as their relationship, is described. The data suggest a novel evolutionary pathway for the RRE structures: the shifting of base pairings in the secondary structure while preserving both a similar overall RNA conformation and, apparently, the activity of the protein encoded by the overlapping gene. This means that corresponding parts of the consensus secondary structure in different RRE species are formed by non-homologous nucleotides. This pattern has so far not been observed in comparative structural studies of other sets of functionally equivalent RNAs (like tRNAs and rRNAs). The multiple-coding problem is discussed with respect to the structure and function of the RRE and the high rate of viral evolution.

U1 snRNA: the evolution of its primary and secondary structure.

SummaryIn this paper we first show that the primary structure of U1 snRNA is homologous to that of tandem repeated pre-tRNA. Two sets of polymerase III promoter sites (the a and b boxes) are clearly recognisable at the appropriate positions in U1, although neither is functional; these sites occur in a degenerate form and their transcription is initiated by polymerase II. Moreover, several of the conserved subsequences of tRNAs that are not associated with transcription initiation (and supposedly are conserved because of their role in translation) are conserved in U1 as well, one of them being the pattern Py-Py-anticodon-Pu-Pu (for both anticodons of tandem tRNA).Second, we show that the secondary structure of U1 is apparently formed after fixation of the ‘B-hairpin loop’ by one of the associated proteins. If and only if this hairpin loop is fixed, a consensus secondary structure is produced by the minimisation-of-free-energy technique. Moreover, we show that this B-hairpin loop has been destabilised relatively recently in evolutionary time by deletions (e.g., in the polymerase III box). If we reinsert the deleted bases, the so constructed hypothetical “ancestral” molecule folds into the consensus secondary structure by unconstrained energy minimisation (i.e., without fixation of the B-loop).Some features of the secondary structure of tandem repeated pre-tRNA are conserved in U1, but the overall structure has changed dramatically. Like tRNA, U1 has a cloverleaf-like structure, but its overall size has doubled. By comparing their secondary structures and by alignment of the sequences, we trace the local events associated with the global change in secondary structure (and apparently in the function of the molecule).Finally, we discuss our results from the perspective of informatic prerequisites for heterarchical multilevel evolution.

Familial Alzheimer's mutation: mRNA secondary structure revisited

It has been suggested that the mutation at position 717 of the amyloid precursor protein (APP), found in several cases of familial Alzheimers disease, affects the secondary structure of the corresponding messenger RNA and the rate of its translation (10). Phylogenetic analysis based on comparison with other mammalian APP sequences does not support this possibility.