The Regulation of the Alternative Splicing of Caspase 9

The pro-apoptotic, caspase 9a, and the anti-apoptotic, caspase 9b, are derived from the caspase 9 gene by alternative splicing. This study demonstrates that the alternative splicing of caspase 9 is dysregulated in a large percentage of non-small cell lung cancer (NSCLC) tumors of the adenocarcinoma type. Furthermore, modulation of the levels of splice variants of caspase 9 had dramatic effects on the anchorage-independent growth and tumorigenic capacity of NSCLC cells. Due to these findings, the molecular mechanisms regulating the post-transcriptional processing of caspase 9 were therefore examined and an exonic splicing silencer (ESS) regulating the pre-mRNA processing of caspase 9 was identified. To study the possible RNA trans-factors interacting with this RNA sequence, we utilized an electromobility shift assay (EMSA) coupled with competitor studies and demonstrated three specific protein:RNA complexes for this ESS. Affinity purification and mass spectrometry analysis identified hnRNP L as part of these protein:RNA complexes. Downregulation of hnRNP L induced a significant increase in caspase 9a/caspase 9b mRNA ratio, which translated to the protein level. Expression of hnRNP L verified the siRNA specificity lowering the caspase 9a/9b ratio, but expression of hnRNP L produced the contrasting effect in non-transformed cells suggesting a post-translational modification specific for NSCLC cells. Indeed, the phospho-status of hnRNP L was significantly increased in NSCLC cells, and mutagenesis studies identified Ser52 as a critical residue regulating the ability of hnRNP L to repress the inclusion of the exon 3,4,5,6 cassette into the mature caspase 9 mRNA. The biological relevance of this mechanism was demonstrated by stable downregulation of hnRNP L in NSCLC cells, which induced a complete loss of both anchorage-independent growth and tumorigenic capacity. This effect of hnRNP L downregulation was due to distal modulation of the alternative splicing of caspase 9 as the loss of both phenotypes was “rescued” by ectopic expression of caspase 9b. Therefore, this study identifies cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase 9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Year dc:date.available

2010

Author dc:creator

• Goehe, Rachel

Contributors dc:contributor

• Charles Chalfant

• caspase 9
• hnRNP L
• Biochemistry, Biophysics, and Structural Biology
• Life Sciences

Statement dc:rights

• © The Author

Identifier

https://scholarscompass.vcu.edu/etd/144

OAI identifier oai:identifier

oai:scholarscompass.vcu.edu:etd-1143