Abstract 2895: Radiation-induced alternative splicing as detected in total and polysome-bound mRNA in glioblastoma stem-like cells
Authors: Amy Wahba, Michael C. Ryan, Uma Shankavaram, Kevin Camphausen, Philip J. Tofilon
Journal: Cancer Research (2016)

Abstract The vast majority of human genes undergo alternative splicing, which is dependent on tissue type, developmental stage, and environmental context. Given that alternative splicing regulates gene expression and is a major source of diversity within the proteome, we used RNAseq to determine the effects of ionizing radiation on the generation of alternative transcripts in the human glioblastoma stem-like cell line NSC11. For these studies, total cellular mRNA, reflecting the transcriptome, and polysome-bound mRNA, reflecting the translatome, were collected from control and irradiated (2Gy, 1h) NSC11 cells and subjected to RNAseq to identify all transcripts; SpliceSeq was then used to visualize and quantitate splice variations induced by radiation. We found that after irradiation 122 genes were differentially spliced in the transcriptome, which indicates that radiation induces alternative splicing, while 357 were differentially spliced in the translatome suggesting that radiation also increases the translation of specific alternative transcripts. However, 33 genes were in common between the transcriptome and translatome suggesting that radiation increases the level and translation of these alternative transcripts. IPA analyses of the radiation-induced alternative transcripts in the translatome identified DNA Double-Strand Break Repair by Homologous Recombination, Ceramide Degradation, and Sphingosine and Sphingosine-1-phospahate Metabolism as the top 3 canonical pathways enriched in this dataset. The top 3 molecular and cellular functions in this analysis were Cellular Assembly and Organization, DNA Replication, Recombination, and Repair, and Cell Cycle. The same analysis of alternative transcripts in the transcriptome did not show an enhancement of DNA damage and repair networks after radiation, suggesting that it is the polysome loading of these transcripts that is changed after irradiation. Further analyses of alternative transcripts in the translatome using EnrichR demonstrated that the top 3 pathways, analyzed using the Reactome 2015 curated pathway database, were Homologous Recombination Repair of Replication-Independent Double-Strand Breaks, Homologous Recombination Repair, and Double-Strand Break Repair. Finally, GeneCodis was used to analyze the biological processes enhanced in the radiation-induced translatome. The top 3 biological processes were Regulation of Transcription, DNA-Dependent, Negative Regulation of Transcription, DNA-Dependent, and Double-Strand Break Repair. These results indicate that radiation affects alternative splicing at both the level of mRNA abundance and translation and suggest that these processes may play a role in radiation-induced translational control of gene expression. Citation Format: Amy Wahba, Michael C. Ryan, Uma Shankavaram, Kevin Camphausen, Philip J. Tofilon. Radiation-induced alternative splicing as detected in total and polysome-bound mRNA in glioblastoma stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2895.