A novel protein factor is required for use of distal alternative 5' splice sites in vitro

Harper, J. E., Manley, J. L. (December 1991) A novel protein factor is required for use of distal alternative 5' splice sites in vitro. Mol Cell Biol, 11 (12). pp. 5945-53. ISSN 0270-7306 (Print)0270-7306 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/1658620
DOI: 10.1128/MCB.11.12.5945

Abstract

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

Item Type: Paper
Uncontrolled Keywords: Adenovirus Early Proteins Adenoviruses, Human/genetics Binding, Competitive Biological Factors/*metabolism HeLa Cells Humans Oncogene Proteins, Viral/genetics Potassium Chloride/pharmacology *RNA Splicing RNA, Messenger/metabolism RNA, Viral/metabolism Simian virus 40/genetics Viral Proteins/*metabolism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > Alternative Splicing
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date: December 1991
Date Deposited: 22 Dec 2015 20:08
Last Modified: 22 Dec 2015 20:08
PMCID: PMC361750
Related URLs:
URI: http://repository.cshl.edu/id/eprint/32116

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