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.
Microbiology and Molecular Genetics
Microbiology, Molecular Biology and Biochemistry
John J. Rossi
Adam M. Bailis
Berry L. Taylor
Anthony J. Zuccarelli
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Gene Expression Regulation; RNA, Messenger; RNA Splicing.
Loma Linda University Libraries
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.
Lin, Juan, "Structural and Functional Analysis of Secondary Structure Block to mRNA Utilization" (1996). Loma Linda University Electronic Theses, Dissertations & Projects. 729.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives