Juan Lin

Abstract

Experimentally introduced secondary structure, formed by a cis-targeted hammerhead ribozyme in exon 2 two nucleotides dowstream of the 3' splice site of a yeast ACT-E. coli lacZ fusion gene, virtually abolishes splicing in vivo and inhibits B-galactosidase production. This is the first demonstration of the splicing inhibitory effect of secondary structures occurring in exons in the absence of splice site sequestration. Our goal is to understand the mechanism by which utilization of this splice site is impeded. Two cis - and one trans-acting mutations that alleviate the splicing blockage were obtained by screening for restoration of B-galactosidase activity. Both cis-acting mutations potentially destabilize the stem in the region close to the 3' splice site. Combined with the results from other artificially constructed cis-acting mutants, this finding suggests that (1) splicing is inhibited by formation of the RNA secondary structures and (2) the extent of inhibition is directly related to the stablility of the secondary structures. Our results also indicate that there may be a general phenomenon of exon 2 secondary structure-specific splicing regulation.

The trans-acting mutation, designated rss1-1, restores B-galactosidase expression by both increasing the splicing efficiency and stabilizing the precursor and lariat intermediate. The gene encoding the trans-acting mutant protein has been cloned. The RSS1 gene is located on Saccharomyces cerevisiaechromosome V; and is a single copy, essential gene. The predicted RSS1 amino acid sequence has marked similarity to members of the putative ATP-dependent RNA helicase family. The trans-acting suppression activity of rss1-1 is highly allele-specific for the secondary structure of the hammerhead ribozyme. Different secondary structures, which also block splicing, are not suppressed by the rss1-1 allele. The single nucleotide change that confers the rss 1-1 suppresssor phenotype produces a Gly to Arg substitution near the GRAGR consensus motif. Thus, the GRAGR region might be involved in RNA substrate interaction. Two alternative models for suppression of the splicing blockage by rss 1-1 are proposed.

LLU Discipline

Microbiology and Molecular Genetics

Department

Microbiology, Molecular Biology and Biochemistry

School

Graduate School

First Advisor

John J. Rossi

Second Advisor

Adam M. Bailis

Third Advisor

Ren-Jang Lin

Fourth Advisor

Berry L. Taylor

Fifth Advisor

Anthony J. Zuccarelli

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1996

Date (Title Page)

12-1996

Language

English

Library of Congress/MESH Subject Headings

Gene Expression Regulation; RNA, Messenger; RNA Splicing.

Type

Dissertation

Page Count

xii; 147

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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Microbiology Commons

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