Antisense Boosts EPO Receptors

Abstract

Erythropoetin (EPO) signaling contributes to organ development, as well as to differentiation of erythrocytes. Previously, the abundance of EPO receptor was reported to increase following surgical removal of one lung (pneumonectomy) in postnatal dogs. Zhang et al . now report that the abundance of EPO-R appears to be regulated by both transcriptional mechanisms from the sense EPO-R transcript ( sEPO-R ), as well as by either the antisense transcript ( asEPO-R ) itself or proteins encoded within asEPO-R . Both sense and antisense transcripts of EPO-R were detected by Northern blot in normal canine lung, as well as other canine tissues and several human tissues. The abundance of sEPO-R increased modestly after pneumonectomy (23% above control), whereas the abundance of asEPO-R increased substantially (119% above control) as did the abundance of EPO-R (169%). Cloning and characterization of canine lung asEPO-R revealed two putative open reading frames (ORF1 and ORF2). Both EPO-R transcripts, as well as the EPO-R protein, were detected in the same bronchiolar epithelial cells by in situ hybridization and immunofluorescence, whereas antisense transcripts for surfactant-associated protein-A were not detected. Coexpression of sEPO-R and asEPO-R increased the abundance of the EPO-R produced by transfected human embryonic kidney (HEK) 293 cells compared with those transfected with only sEPO-R ; the effect of asEPO-R increased with increasing dose. When the start codon of ORF1 was mutated, EPO-R abundance was still elevated in the cotransfected cells, which suggests that the putative encoded protein is not important for the enhancing effect of asEPO-R . In contrast, the region near ORF2 appeared to play a more complex role in EPO-R regulation. When a long construct that included ORF2 and 300 base pairs of 5′ of the ORF2 start site were cotransfected with sEPO-R , then EPO-R production was increased compared with cells only transfected with sEPO-R . However, when the 300 extra base pairs were not included, then EPO-R abundance was not enhanced. Further complicating the effect of the ORF2 region, when the start codon of the ORF2 was mutated in the construct lacking the extra 300 base pairs, enhancement of EPO-R abundance was restored. The authors propose that the asEPO-R has several regulatory elements: The RNA itself has stimulatory effects, and the protein encoded by ORF2 is a negative regulator of EPO-R synthesis. Thus, when the ORF2 start codon is eliminated, the positive effects of the antisense RNA dominate and EPO-R production is enhanced. Q. Zhang, J. Zhang, O. W. Moe, C. C. W. Hsia, Synergistic upregulation of erythropoietin receptor (EPO-R) expression by sense and antisense EPO-R transcripts in the canine lung. Proc. Natl. Acad. Sci. U.S.A. 105 , 7612-7617 (2008). [Abstract] [Full Text]