Transcriptome-Wide Analysis Of Hypoxic Cancer Cells Identify Alternative Splicing As A Mechanism To Inhibit Translation

Cellular adaptation to hypoxia involves downregulation of energy-consuming processes such as macromolecular synthesis, and leads to tumor aggressiveness and resistance to therapies for many solid cancers. To delineate mechanisms underlying this process, I carried out a transcriptome-wide study to measure hypoxia-mediated changes in gene expression and alternative splicing in in vivo and in vitro models of hypoxic head and neck carcinoma (HNC) cells. These data represent the first nucleotide-resolution study of the hypoxic transcriptome of HNC cells in vivo and in vitro. This investigation uncovered a global downregulation of genes known to regulate RNA processing, including a significant number of genes involved in splicing catalysis. Exon-level analyses classified >1,000 mRNAs to be affected by alternative splicing, and led to the discovery of a unique retained intron in the master regulator of translation initiation, EIF2B5. In this dissertation, I will describe a previously uncharacterized mechanism by which a hypoxia-mediated retained intron in EIF2B5 leads to a truncated isoform that opposes full-length eIF2Bε to inhibit translation. A functional investigation of this hypoxia-induced isoform confirmed that expression of the truncated 65kDa isoform of eIF2Bε confers a survival advantage to HNC cells under conditions of hypoxia. Moreover, expression of this isoform was observed in solid tumors of patients with HNC in a stage-dependent manner. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5: a weak splice site with an alternate splice site at the retained intron, hypoxia-induced expression of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II (RNAPII) specifically at the intron retained under hypoxia. Altogether, these data reveal differential splicing as a previously uncharacterized mode of translational control under hypoxia and are supported by a model in which hypoxia-induced changes to co-transcriptional processing lead to selective retention of an intron containing a premature-termination codon in EIF2B5.