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|Title:||Sequences and factors required for 5' splice site selection in Schizosaccharomyces pombe|
|Author(s):||Alvarez, Consuelo Jackeline|
|Doctoral Committee Chair(s):||Wise, Jo Ann|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||I first undertook a systematic study of the region of U1 snRNA complementary to the 5$\sp\prime$ splice site in S. pombe with the goal of expanding our knowledge of how this sequence functions in pre-catalytic events in pre-mRNA splicing. To determine the contribution to U1 function of each position complementary to the first six nucleotides of the intron, I mutagenized the relevant U1 nucleotides (1 through 6) and examined the ability of each mutant allele to support growth as the sole copy of U1 in S. pombe. Splicing assays performed on RNA from cells harboring viable alleles indicate that the 5$\sp\prime$ end of U1 snRNA plays an extremely critical role in S. pombe splicing.
The second goal of my project was to systematically mutagenize the 5$\sp\prime$ splice sites of the second intron of the S. pombe:cell division cycle gene (cdc2-I2). At least one transition and one transversion was constructed at each position and analyzed for effects on splicing. My data indicate that all intron positions are important for splicing in S. pombe, and in each case, splicing is blocked prior to the first transesterification reaction. I next proceeded to test the ability of U1 mutants to rescue the splicing defects conferred by 5$\sp\prime$ splice site mutations. The pattern of phenotypic rescue revealed that overall complementarity, rather than U1 pairing at specific positions, determines whether a given splice site is selected by this snRNA.
The third goal of my project was to explore the sequence and spacing requirements for activation of an unusual cryptic 5$\sp\prime$ splice site within cdc2-I2. Mutations that restore the 5$\sp\prime$ splice site consensus sequence to this cryptic junction result in its exclusive use, suggesting that it may lie in a more favorable context for splicing than the normal 5$\sp\prime$ splice site. Distance constraints as well as potential base pairing to U1, U6, and U5 snRNAs were analyzed. These studies revealed that a critical factor in the use of a given 5$\sp\prime$ splice site in S. pombe is its distance from the 3$\sp\prime$ splicing signals. Although U1 and U6 snRNAs participate in splicing at the cryptic site, they do not appear to play decisive roles in imposing this distance constraint unlike U5 snRNA.
|Rights Information:||Copyright 1996 Alvarez, Consuelo Jackeline|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9702443|