Laminar Shear Stress and 3′ Polyadenylation of eNOS mRNA

Abstract

The 3′ poly(A) tail is important in messenger RNA stability and translational efficiency. In somatic tissues, 3′ polyadenylation of mRNAs has been thought to largely be a constitutively active process. We have reported that laminar shear stress causes a brief increase in endothelial nitric oxide synthase (eNOS) transcription, followed by a prolonged increase in eNOS mRNA stability. We sought to determine whether shear stress and other stimuli affected eNOS 3′ polyadenylation in endothelial cells. Under basal (static) conditions, eNOS mRNA possessed short 3′ poly(A) tails of <25 nt. In contrast, laminar shear stress increased expression of eNOS transcripts with long poly(A) tails. ENOS transcripts with longer poly(A) tails had prolonged half-lives (6 hours in static cells versus 18 hours in sheared cells). Polysome analysis revealed that eNOS mRNA from sheared cells was shifted into more translationally active polysome fractions compared with eNOS mRNA from static cells. Shear-induced lengthening of the eNOS 3′ poly(A) tail was the result of increased nuclear polyadenylation. Furthermore, hydrogen peroxide and HMG Co-A reductase inhibitors, other stimuli known to modulate eNOS expression posttranscriptionally, also induced eNOS 3′ poly(A) tail lengthening. These results support the concept that shear stress modulates eNOS mRNA stability and translation via increased 3′ polyadenylation. We suggest that mRNA 3′ polyadenylation is a posttranscriptional mechanism used by endothelial cells to regulate gene expression.