SRSF1 Is a Mediator of Radiation-Induced Alternative Splicing in B-Lymphocytes

Ionizing radiation is used in the treatment of Hodgkin and non-Hodgkin lymphomas (Spetch et al. 2014; Illidge et al. 2014). Despite its effectiveness, radiation is a "blunt" therapy that damages indiscriminately both cancer and normal cells, and can result in secondary malignancies (Dores et al. 2002). To better understand cellular response to radiation, we examined alternative splicing, and its regulation, in irradiated human cells.

To accomplish this, we exposed cultured B-lymphocytes from 10 individuals to 10 Gy of ionizing radiation, and performed RNA sequencing before, and two and six hours after radiation treatment. With these data, we first identified alternative splicing events. From about 60 million reads per sample, we detected over 20,000 alternatively spliced events. In total, 1,600 events were radiation responsive (ANOVA, FDR<5%). The splicing events in irradiated cells belong mainly to three categories, each of which occurred in genes with distinct biological functions. Cassette exons, which were the most numerous splicing event, were found primarily in DNA damage response and apoptotic genes, while alternative first or last exons were found in chromatin assembly genes; retained introns occurred in genes involved in RNA processing and translation. Using these radiation-responsive events, we then examined the kinetics of this response. We found that changes in alternative splicing were acute, occurring within two hours of radiation treatment. Overall, the splicing events produced shorter transcripts in irradiated cells.

Next, we looked for how alternative splicing is regulated in response to radiation. To identify putative mediators of this response, we determined the expression levels of over 60 trans-acting splicing factors. We found that the expression level of 26 splicing factors changed significantly in irradiated cells (ANOVA, FDR<5%). Of these putative mediators, we further examined serine/arginine-rich splicing factor (SRSF1)'s role in radiation response. First, we assessed SRSF1 expression in irradiated cells. Following radiation exposure, the transcript and protein expressions of SRSF1 decreased. Next, we performed motif enrichment analysis to identify target genes. SRSF1 RNA-binding sites were enriched in skipped cassette exons: specifically, of the 362 skipped cassette exons in irradiated cells, 93 (26%) contained SRSF1 binding motifs.

Finally, we examined the link between SRSF1 and exon skipping in irradiated cells more closely. For example, in the transcripts for RNA-binding protein 3 (RBM3), SRSF1 binds an RBM3 exon that contains a premature termination codon (Sanford et al. 2009), and mediates exclusion of this exon. We confirmed experimentally that this results in an increase in RBM3 protein expression. Together, our data demonstrate that alternative splicing is a key part of cellular response to radiation, and SRSF1 plays a role in mediating exon skipping. In this presentation, we will describe radiation-induced alternative splicing by discussing the genes that are alternatively spliced, and its regulation by the splicing factor SRSF1.