Unraveling Intron Retention: Differential Expression Pattern in Brain and Kidney Cells After Hypoxia or Ischemia

Introduction The inclusion of introns in the mature mRNA (intron retention), has been recently uncovered as a mechanism for the regulation of genetic expression in vertebrates. Intron retention is implicated in tissue specific protein diversity, splicing regulation and control of gene expression. We have recently identified intron retention as a mechanism involved in the regulation of erythropoietin (EPO) . EPO is the key regulator of erythropoiesis and is transcriptionally regulated by hypoxia inducible factor 2 (HIF2). The ability to easily detect intron retention could add to our ability to better understand genetic regulation of our genomes in stress conditions, such as ischemic diseases (ex/ myocardial infarction (MI) or stroke). Objective To evaluate the role of intron retention mechanism in EPO expression level to gain a better understanding of its regulation in response to hypoxia and ischemia, conditions which result in EPO expression. Methods HTB16 (neuroblastoma), CHP212 (glioblastoma), hCMEC/D3 (blood brain barrier) and HEK293 (kidney) were placed in hypoxic (1% oxygen), ischemic (1% oxygen, no serum or glucose) or normoxic conditions (20% oxygen) for 24 hours prior to RNA extraction. Quantitative polymerase chain reaction (qPCR) was performed with two sets of primers for EPO, the first set was targeting intron inclusion with each primer in a separate exon (intron primers) and the second set was targeting a region that spans two exons, which would not amplify if introns were present (non‐introns). Relative quantification for the level of expression was determined by qPCR using β‐actin and YWHAZ as reference genes for normalization purposes. Statistical analysis of transcript expression and the ratio between intron and non‐intron detecting primer sets were determined using analysis of variance (ANOVA). Results EPO mRNA level was increased in HTB16, CHP212 and HEK293, but not in hCMEC/D3 cell lines in response to ischemia, but not hypoxia. However, when the ration of intron vs. non‐intron expression level was determined a trend towards more intron retaining transcripts in HTB16, CHP212 and hCMEC/D3 but not in HEK293 was observed, suggesting an induction for the expression of this gene under this ischemic condition. Conclusions Intron retention mechanism in EPO is taking place in brain‐derived cell lines, but not in kidney‐derived cells. These results indicate a tissue specificity of intron retention mechanism that could regulate differential expression level of EPO under different physiological and stressful conditions. These findings highlight the complexity of EPO regulation. Hence, intron retention must be considered when examining the expression of a gene to better understand expression in ischemic disease models, such as MI or stroke. Support or Funding Information Natural Sciences and Engineering Research Council of Canada (NSERC)

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