In vitro selections with RNAs of variable length converge on a robust catalytic core.

In vitro selection is a powerful tool that can be used to understand basic principles of molecular evolution. We used in vitro selection to understand how changes in length and the accumulation of point mutations enable the evolution of functional RNAs. Using RNA populations of various lengths, we performed a series of in vitro experiments to select for ribozymes with RNA ligase activity. We identified a core ribozyme structure that was robust to changes in RNA length, high levels of mutagenesis, and increased selection pressure. Elaboration on this core structure resulted in improved activity which we show is consistent with a larger trend among functional RNAs in which increasing motif size can lead to an exponential improvement in fitness. We conclude that elaboration on conserved core structures is a preferred mechanism in RNA evolution. This conclusion, drawn from selections of RNAs from random sequences, is consistent with proposed evolutionary histories of specific biological RNAs. More generally, our results indicate that modern RNA structures can be used to infer ancestral structures. Our observations also suggest a mechanism by which structural outcomes of early RNA evolution would be largely reproducible even though RNA fitness landscapes consist of disconnected clusters of functional sequences.

Big on Change, Small on Innovation: Evolutionary Consequences of RNA Sequence Duplication.

The potential for biopolymers to evolve new structures has important consequences for their ability to optimize function and our attempts to reconstruct their evolutionary histories. Prior work with in vitro systems suggests that structural remodeling of RNA is difficult to achieve through the accumulation of point mutations or through recombination events. Sequence duplication may represent an alternative mechanism that can more readily lead to the evolution of new structures. Structural and sequence elements in many RNAs and proteins appear to be the products of duplication events, indicating that this mechanism plays a major role in molecular evolution. Despite the potential significance of this mechanism, little experimental data is available concerning the structural and evolutionary consequences of duplicating biopolymer sequences. To assess the structural consequences of sequence duplication on the evolution of RNA, we mutagenized an RNA sequence containing two copies of an ATP aptamer and subjected the resulting population to multiple in vitro evolution experiments. We identified multiple routes by which duplication, followed by the accumulation of functional point mutations, allowed our populations to sample two entirely different secondary structures. The two structures have no base pairs in common, but both structures contain two copies of the same ATP-binding motif. We do not observe the emergence of any other functional secondary structures beyond these two. Although this result suggests a limited capacity for duplication to support short-term functional innovation, major changes in secondary structure, like the one observed here, should be given careful consideration as they are likely to frustrate attempts to infer deep evolutionary histories of functional RNAs.

Evolution of ribozymes in the presence of a mineral surface.

Mineral surfaces are often proposed as the sites of critical processes in the emergence of life. Clay minerals in particular are thought to play significant roles in the origin of life including polymerizing, concentrating, organizing, and protecting biopolymers. In these scenarios, the impact of minerals on biopolymer folding is expected to influence evolutionary processes. These processes include both the initial emergence of functional structures in the presence of the mineral and the subsequent transition away from the mineral-associated niche. The initial evolution of function depends upon the number and distribution of sequences capable of functioning in the presence of the mineral, and the transition to new environments depends upon the overlap between sequences that evolve on the mineral surface and sequences that can perform the same functions in the mineral's absence. To examine these processes, we evolved self-cleaving ribozymes in vitro in the presence or absence of Na-saturated montmorillonite clay mineral particles. Starting from a shared population of random sequences, RNA populations were evolved in parallel, along separate evolutionary trajectories. Comparative sequence analysis and activity assays show that the impact of this clay mineral on functional structure selection was minimal; it neither prevented common structures from emerging, nor did it promote the emergence of new structures. This suggests that montmorillonite does not improve RNA's ability to evolve functional structures; however, it also suggests that RNAs that do evolve in contact with montmorillonite retain the same structures in mineral-free environments, potentially facilitating an evolutionary transition away from a mineral-associated niche.

In vitro evolution of distinct self-cleaving ribozymes in diverse environments.

In vitro evolution experiments have long been used to evaluate the roles of RNA in both modern and ancient biology, and as a tool for biotechnology applications. The conditions under which these experiments have been conducted, however, do not reflect the range of cellular environments in modern biology or our understanding of chemical environments on the early earth, when the atmosphere and oceans were largely anoxic and soluble Fe(2+) was abundant. To test the impact of environmental factors relevant to RNA's potential role in the earliest forms of life, we evolved populations of self-cleaving ribozymes in an anoxic atmosphere with varying pH in the presence of either Fe(2+) or Mg(2+). Populations evolved under these different conditions are dominated by different sequences and secondary structures, demonstrating global differences in the underlying fitness landscapes. Comparisons between evolutionary outcomes and catalytic activities also indicate that Mg(2+) can readily take the place of Fe(2+) in supporting the catalysis of RNA cleavage at neutral pH, but not at lower pH. These results highlight the importance of considering the specific environments in which functional biopolymers evolve when evaluating their potential roles in the origin of life, extant biology, or biotechnology.

Role of helical constraints of the EBS1-IBS1 duplex of a group II intron on demarcation of the 5' splice site.

Recognition of the 5' splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem-loop of domain 1 and a complementary sequence at the 3' end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5γ intron, we probed the solution structure of the ID3 stem-loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5γ and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5' splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1-IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5γ and O.i. introns and may help to fine-tune elements of recognition in group II introns.

Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex.

The complex formed between the U2 and U6 small nuclear (sn)RNA molecules of the eukaryotic spliceosome plays a critical role in the catalysis of precursor mRNA splicing. Here, we have used enzymatic structure probing, (19)F NMR, and analytical ultracentrifugation techniques to characterize the fold of a protein-free biophysically tractable paired construct representing the human U2-U6 snRNA complex. Results from enzymatic probing and (19)F NMR for the complex in the absence of Mg(2+) are consistent with formation of a four-helix junction structure as a predominant conformation. However, (19)F NMR data also identify a lesser fraction (up to 14% at 25°C) of a three-helix conformation. Based upon this distribution, the calculated ΔG for inter-conversion to the four-helix structure from the three-helix structure is approximately -4.6 kJ/mol. In the presence of 5 mM Mg(2+), the fraction of the three-helix conformation increased to ∼17% and the Stokes radius, measured by analytical ultracentrifugation, decreased by 2%, suggesting a slight shift to an alternative conformation. NMR measurements demonstrated that addition of an intron fragment to the U2-U6 snRNA complex results in displacement of U6 snRNA from the region of Helix III immediately 5' of the ACAGAGA sequence of U6 snRNA, which may facilitate binding of the segment of the intron adjacent to the 5' splice site to the ACAGAGA sequence. Taken together, these observations indicate conformational heterogeneity in the protein-free human U2-U6 snRNA complex consistent with a model in which the RNA has sufficient conformational flexibility to facilitate inter-conversion between steps of splicing in situ.

Impact of base pair identity 5' to the spliceosomal branch site adenosine on branch site conformation.

The branch site helix from Saccharomyces cerevisiae with pseudouridine (ψ) incorporated in a phylogenetically conserved position of U2 snRNA features an extrahelical branch site adenosine (A) that forms a base triple interaction with the minor groove edge of a widely conserved purine(U2 strand)-pyrimidine(intron strand) (R(U2)-Y(intron)) base pair two positions upstream. In these studies, NMR spectra of a duplex in which 2-aminopurine (2ap), a fluorescent analog of adenine lacking the proposed hydrogen bond donor, was substituted for the branch site A, indicated that the substitution does not alter the extrahelical position of the branch site residue; thus, it appears that a hydrogen bond between the adenine amino group and the R-Y pair is not obligatory for stabilization of the extrahelical conformation. In contrast, reversal of the orientation of A(U2)-U(intron) to U(U2)-A(intron) resulted in an intrahelical position for the branch site A or 2ap. Fluorescence intensity of 2ap substituted for the branch site A with the original R(U2)-Y(intron) orientation (AU or GC) was high, consistent with an extrahelical position, whereas fluorescence in helices with the reversed R-Y orientation, or with a mismatched pair (A-U → G•A or U•C), was markedly quenched, implying that the residue was stacked in the helix. The A 5' to the branch site residue was not extrahelical in any of the duplexes. These findings suggest that the R(U2)-Y(intron) base pair orientation in the ψ-dependent branch site helix plays an important role in positioning the branch site A for recognition and/or function.

[Diabetic ketoacidosis in children with newly detected type 1 diabetes in Montenegro from 1999 to 2008].

INTRODUCTION: The aim of the study was to analyze the prevalence of diabetic ketoacidosis during the period of 10 years (1999-2008) among children diagnosed with type 1 diabetes in Montenegro. MATERIAL AND METHODS: A retrospective population-based incidence study was performed. The study participants were selected from two independent sources: the diabetes register and hospital records. The following parameters were measured before the first insulin injection: plasma glucose, blood gas analysis, electrolytes, creatinine, insulin, c-peptide, and HbA1c. Diabetic ketoacidosis was defined as pH <7.3 and severe diabetic ketoacidosis as pH <7.1. The obtained data were analysed using SPSS for Windows (version 17). RESULTS: During the study period, 208 children <15 years of age (107 boys and 101 girls) were found to have newly diagnosed type 1 diabetes. The age- and sex-standardized incidence rate was 15.8/100,000 children/yr. Of these, 51 (24.5%) presented with diabetic ketoacidosis at the time of diagnosis and 8 (3.8%) had a severe form. and no one died. In children <5 years the prevalence was 30.4%. We found no statistically important correlation between diabetic ketoacidosis incidence, pH value and the age of children (p>0.05). There was also no significant difference in diabetic ketoacidosis incidence between the boys and girls (p>0.05). CONCLUSION: The frequency of diabetic ketoacidosis in children with newly diagnosed type 1 diabetes in Montenegro is still high with a trend to decrease in the last ten years. In particular, children under 5 years of age are at a high risk of developing diabetic ketoacidosis at the onset.