Transcriptome analysis of hypoxic cancer cells uncovers intron retention in EIF2B5 as a mechanism to inhibit translation

Abstract

Cells adjust to hypoxic stress within the tumor microenvironment by downregulating energy-consuming processes including translation. To delineate mechanisms of cellular adaptation to hypoxia, we performed RNA-Seq of normoxic and hypoxic head and neck cancer cells. These data revealed a significant down regulation of genes known to regulate RNA processing and splicing. Exon-level analyses classified > 1,000 mRNAs as alternatively spliced under hypoxia and uncovered a unique retained intron (RI) in the master regulator of translation initiation, EIF2B5. Notably, this intron was expressed in solid tumors in a stage-dependent manner. We investigated the biological consequence of this RI and demonstrate that its inclusion creates a premature termination codon (PTC), that leads to a 65kDa truncated protein isoform that opposes full-length eIF2Bε to inhibit global translation. Furthermore, expression of 65kDa eIF2Bε led to increased survival of head and neck cancer cells under hypoxia, providing evidence that this isoform enables cells to adapt to conditions of low oxygen. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5: a weak splicing potential at the RI, hypoxia-induced expression and binding of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II 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 cotranscriptional processing lead to selective retention of a PTC-containing intron in EIF2B5.

Gallop Racing Shifts Mature mRNA towards Introns: Does Exercise-Induced Stress Enhance Genome Plasticity?

“…In literature a general increase of intron retention events has been observed in response to some types of stress like thermal stress exposure [47,48], chemotherapy-induced stress in cancer cells [49] and hypoxia [50]. The intron retention phenomenon could be triggered by the transcriptional activation of repeated elements contained within the intron sequence [36,37].…”

Increased intron retention is a post‐transcriptional signature associated with progressive aging and Alzheimer’s disease

“…Accumulated evidence indicated that IR may also play an important regulatory role during normal development, including translational inhibition in response to hypoxic stress (Brady et al, ), regulation of mRNA expression patterns during hematopoiesis (Cho et al, ; Wong et al, ) and neurogenesis (Braunschweig et al, ; Buckley et al, ). Therefore, defining age‐associated changes to IR may allow a far better understanding into how IR may regulate the transition from healthy to the pathological state during aging.…”

Splice variant of growth hormone-releasing hormone receptor drives esophageal squamous cell carcinoma conferring a therapeutic target

“…Alternative splicing in tumor cells is one of the cellular adaptations that are largely promoted by the hypoxic microenvironment in solid tumors. Given that intron-containing mRNAs are the most frequent splicing products that result from hypoxia (34)(35)(36), it is reasonable to predict that the enhanced expression of SV1 in ESCC is induced by hypoxia. We also demonstrated that SV1 in ESCC cells possesses both ligand-dependent and ligand-independent functions.…”