Biology Ph.D. Dissertations

Title

Characterization of a Small Ribozyme with Self-Splicing Activity

Date of Award

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Scott Rogers, PhD

Second Advisor

Chris Keil, PhD (Committee Member)

Third Advisor

Carmen Fioravanti, PhD (Committee Member)

Fourth Advisor

Paul Morris, PhD (Committee Member)

Fifth Advisor

Vipaporn Phuntumart, PhD (Committee Member)

Abstract

Introns are common in the rRNA gene loci of fungal genomes, but biochemical studies to investigate splicing are rare. Here, self-splicing of a very small (67 nucleotide) group I intron is demonstrated. The PaSSU intron (located within the rRNA small subunit gene of Phialophora americana) splices in vitro under group I intron conditions. Most group I ribozymes contain pairing regions P1 - P10, with a conserved G•U pair at the 5' splice site, and a G at the 3' intron border. The PaSSU intron contains only P1, P7, and P10. While it contains the G•U pair at the 5' splice, a U is found at the 3' end of the intron instead of a G. Phylogenetic analysis places it within subgroup IC1, whose members are found in the nuclear rRNA genes of fungi. The structural elements are similar to those in the centermost regions of other group I introns. Its size can be explained by a single large deletion that removed P2 through much of P9. Part of the original P9region has assumed the function of P7. Its small size and genealogy makes it an excellent model to study RNA catalysis and evolution. Site-specific mutations have confirmed the essential character of 15% of the nucleotides that conform the intron. From the mutation done until today, around a third part of the intron may not be essential to achieve splicing characteristics. Ten nucleotides were mutated and inhibit intron removal. However, some induce alternative splicing. Nucleotides found in 5' exon-intron border, at P1, P7 and P10 that played essential roles are here demonstrated. Also, comparison among different Phialophora isolates that contain natural mutations have been studied to understand the evolutionary pattern involved in the gain, maintenance and loss of this intron in this species.

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