Mechanism of DNA target site recognition by group II introns TeI3c and GsI-IIC and splicing activity of GsI-IIC reverse transcriptase

Abstract

Mobile group II introns are self-catalytic ribozymes found in bacteria and eukaryotic organelles. They can mobilize within the genomes by retrohoming, which involves RNA-catalyzed splicing followed by the excised intron reverse splicing into a target site. Both RNA splicing and retrohoming are facilitated by an intron-encoded reverse transcriptase (RT). Mobile group II introns are of interest as evolutionary ancestors of spliceosomal introns in higher organisms, for their use as bacterial gene targeting vectors known as targetrons, and as a source of thermostable group II intron reverse transcriptases (TGIRTs) for RNA-seq. The focus of this master’s thesis is on two thermophilic group II introns found in bacterial thermophiles: the subgroup IIB intron TeI3c and the subgroup IIC intron GsI-IIC. The TeI3c intron is known to rely on base pairing interaction between exon-binding site sequences 1/2 (EBS1/2), within the intron RNA, and intron-binding site sequences 1/2 (IBS1/2) in the 5’ exon of its target DNA, but it is not clear what targeting rules dictate one target sequence to be better or worse than others. I studied the targeting rules of TeI3c during retrohoming by using randomized libraries and next-generation sequencing followed by computational analysis of the sequence data. Understanding the targeting rules of TeI3c can be the important step in the development of thermostable targetron, which can be useful for metabolic engineering in the biofuel industry. Unlike TeI3c, which relies primarily on base pairing for DNA target recognition, the GsI-IIC intron recognizes a 5’-exon hairpin secondary structure of the target DNA. However, the secondary structure requirements of good targets have not been studied. I studied the secondary structure requirements during GsI-IIC retrohoming by using doped target libraries and next-generation sequencing to find conserved positions within a hairpin target site followed by mobility assays on different target sites with mutated conserved positions. Finally, I studied the forward splicing of GsI-IIC intron by comparing different hairpin target sites including the same mutated target sites tested for their mobility efficiency. These experiments address whether the 5’-exon hairpin structure is recognized similarly for RNA splicing and intron mobility.