Browsing by Author "Jarding, Ashley Marie"

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    Diverse Structures and Strategies of 5′ Exon Recognition in Group II Introns
    (2018-06-05) Jarding, Ashley Marie; Zimmerly, Steven; Muench, Douglas G.; Noskov, Sergei Yu; Van Marle, Guido; Cousineau, Benoit
    Group II introns are a family of mobile elements found in bacteria and bacteria-derived organelles, which are thought to have a direct evolutionary link to spliceosomal introns. These introns can self-splice in vitro thanks to their highly organized ribozyme structure. Studying the diverse structural features of group II introns illuminates the breadth of their structural diversity and resultant unique splicing and mobility capabilities. In the first of three projects presented in this dissertation, bioinformatic approaches were used to identify over 800 new introns in a large scale, systematic collection and analysis of group II introns. This allowed inference of class-specific mobility properties and the description of novel intron arrangements in genomes. Additionally, one new class and 15 unique, previously unclassified introns with novel structural features have been identified. The newly collected introns revealed a conserved structural feature for Class A introns: they appear to have two 5′ exon recognition motifs, whereas all other group II introns are reported to have one. The second project investigates the binary splicing hypothesis, which states that either of these two motifs can be used to recognize the exon. This was investigated and confirmed through both experimental and bioinformatic approaches. This strategy appears to expand the set of homing sites available to an intron by permitting splicing from one sequence and reverse-splicing into another. In the third project, the 5′ exon recognition mechanism for a IIC intron was investigated. IIC introns lack the canonical IBS2-EBS2 interactions found for other group II introns, but have a conserved transcriptional terminator in place of EBS2. In B.h.I1 a 5S rRNA upstream of this motif was shown to have an unexpected effect on the splicing reaction in vitro. Mutagenesis of 5S rRNA showed that the A-loop is critical for splicing. RNA protection experiments to characterize the interactions with the 5′ exon support the terminator stem interacting with Domain I while 5S has an auxiliary effect on exon ligation. In summary, this work has increased our understanding of the structural diversity of group II introns and corresponding specialized strategies used to recognize the 5′ exon during splicing.