Many functional RNAs are required to fold into specific three-dimensional structures. A fundamental property of RNA is that its secondary structure and even some tertiary contacts are highly stable, which gives rise to independent modular RNA motifs and makes RNAs prone to adopting misfolded intermediates. Consequently, in addition to stabilizing the native structure relative to the unfolded species (defined here as stability), RNAs are faced with the challenge of stabilizing the native structure relative to alternative structures (defined as structural specificity). How RNAs have evolved to overcome these challenges is incompletely understood. Self-splicing group I introns have been used to study RNA structure and folding for decades. Among them, the Tetrahymena intron was the first discovered and has been studied extensively. In this work, we found that a version of the intron that was generated by in vitro selection for enhanced stability also displayed enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective pressure to increase the energy gap between these structures. Further dissection suggests that the increased specificity against misfolding arises from two point mutations, which strengthen a local tertiary contact network that apparently cannot form in the misfolded conformation. Our results suggest that the structural rigidity and intricate networks of contacts inherent to structured RNAs can allow them to evolve exquisite structural specificity without explicit negative selection, even against closely-related alternative structures. To explore further how RNAs gain stability from intricate architectures, we examined a novel group I intron from red algae (Bangia). Biochemical methods and computational modeling suggest that this intron possesses general motifs of group IC1 introns but also forms an atypical tertiary contact, which has been reported previously in other subgroups and helps position the reactive helix at the active site. In the Bangia intron, the partners have been swapped relative to known group I RNAs that include this contact. This result underscores the modular nature of RNA motifs and provides insight into how structured RNAs can arrange helices and contacts in multiple ways to achieve and stabilize functional structures. / text

The oceans represent a wealth of biological diversity where many species remain to be discovered and described. Among seaweeds, a paucity of morphological features by which to differentiate species means that many genera harbour overlooked or cryptic species. Fucus, Porphyra and Ulva are three common genera of marine intertidal algae and all include species that are particularly difficult to distinguish morphologically. DNA barcoding has been championed as a revolutionary tool for species identification and discovery and applying this tool to algae was a logical step due to the difficulty of morphological identification of many algal species. This thesis is part of a significant initiative aimed at identification and discovery of all species of seaweeds in Canadian waters, using a DNA barcoding approach. The original concept of DNA barcoding relied on comparing the 5’ region of the mitochondrial cytochrome c oxidase 1 (COI-5P) gene among animal species. In this study, DNA barcoding with COI-5P was applied to the brown algal genus Fucus and worked as well as any other marker to assign morphologies to known species. The DNA barcoding results also uncovered substantial phenotypic diversity in Pacific F. distichus. Results were confirmed by comparison with sequences of the nuclear internal transcribed spacer region (ITS). For Porphyra, COI-5P DNA barcoding was compared with species identification using the chloroplast large rubisco subunit (rbcL) and the Universal Plastid Amplicon (UPA) in a floristic survey of Canadian Porphyra species. Two new species were discovered and described (Porphyra corallicola and Porphyra peggicovensis), and P. cuneiformis was synonymized with P. amplissima. The COI-5P emerged as the best marker for species discrimination despite difficulties with primer universality. To aid in choosing a marker for DNA barcoding for green algae, the universality and species discriminatory power of the rubisco large subunit (rbcL) (considering the 5’ and 3’ fragments independently), the UPA, the D2/D3 region of the nuclear large ribosomal subunit (LSU-D2/D3) and the ITS were evaluated. While the rbcL-3P highlighted several cryptic species, and worked well to distinguish Ulva species, more research is needed to recommend a marker for DNA barcoding generally in marine green macroalgae.