Identification of the RNA Cis-Elements that Interact with SRp30a to Regulate the Alternative Splicing of Caspase 9 Pre-mRNA

Studies have shown that the alternative splicing of caspase 9 and the phospho-status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and de novo ceramide via the action of protein phosphatase-1 (PP1). Two RNA splice variants are derived from the caspase 9 gene, pro-apoptotic caspase 9a and anti-apoptotic caspase 9b, via alternative splicing by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. In this study, the link between SR proteins and the alternative splicing of caspase 9 was established. Sequence analysis of the exon 3, 4, 5, and 6 cassette of the caspase 9 gene identified five possible high affinity sequences for interaction with the SR protein, SRp30a, a well-established regulator of exon inclusion/exclusion. Replacement mutagenesis identified purine-rich sequences between exons 4 and 5 and wthin exon 6 as important for binding SRp30a and required for expression of the caspase 9a splice variant. In vitro binding assays coupled with competitor studies demonstrated specific binding of RNA trans-acting proteins and SRp30a with these sequences. Furthermore, SDS-PAGE analysis of cross-linked RNA trans-acting factors with these possible RNA cis-elements revealed the specific binding of an approximate 66, 56, 45, and 38 kDa protein/protein complex to these sequences. A previous application of RNAi technology to downregulate SRp30a in A549 lung adenocarcinoma cells induced an approximately 75% decrease in SRp30a expression and induced a dramatic change in the ratio of caspase 9a/caspase 9b. Therefore, these studies have identified SRp30a as a major regulator of the alternative splicing of caspase 9 directly linking de novo ceramide generation, PP1, and SRp30a as the signal transduction pathway regulating the expression of caspase 9.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Biochemistry

Year dc:date.available

2005

Author dc:creator

• Mukerjee, Prabhat

Contributors dc:contributor

• Dr. Charles E. Chalfant

• PP1
• mutagenesis
• chemotherapy
• cancer
• CAPPs
• immunoblotting
• PP2A
• CAPK
• capcase
• splice
• ceramide
• apoptosis
• caspase 9
• cathepsin D
• Biochemistry, Biophysics, and Structural Biology
• Life Sciences

Statement dc:rights

• © The Author

Identifier

https://scholarscompass.vcu.edu/etd/1506

OAI identifier oai:identifier

oai:scholarscompass.vcu.edu:etd-2505