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A genetic and biochemical analysis of pre-mRNA splicing in Saccharomyces cerevisiae

Citation

Vijayraghavan, Usha (1989) A genetic and biochemical analysis of pre-mRNA splicing in Saccharomyces cerevisiae. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:10172013-115606044

Abstract

Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.

Yeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.

To study trans -acting splicing factors we generated and screened a bank of temperature- sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP 18 gene has been cloned and its polyadenylated transcript identified.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biology
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Abelson, John N.
Thesis Committee:
  • Unknown, Unknown
Defense Date:1 October 1988
Record Number:CaltechTHESIS:10172013-115606044
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:10172013-115606044
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7997
Collection:CaltechTHESIS
Deposited By: John Wade
Deposited On:22 Oct 2013 16:15
Last Modified:22 Oct 2013 16:15

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