Translational control of apolipoprotein B mRNA: regulation via cis elements in the 5' and 3' untranslated regions.

Abstract

Translational control of apolipoprotein B (apoB) mRNA has been previously documented; however, the molecular mechanisms that govern translation of apoB mRNA are unknown. We investigated the role of the untranslated regions (UTR) in the regulation of apoB mRNA translation first by analyzing apoB UTR sequences using M-fold, a program used to predict RNA secondary structure. M-fold analysis revealed hairpin-like elements within the 5'UTR and 3'UTR of apoB mRNA with potential to form stable secondary structure. Luciferase (LUC) reporter assays were conducted to assess the biological activity of the putative RNA motifs within the UTR sequences by transiently transfecting HepG2 cells with chimeric mRNAs containing the 5' and/or 3' apoB UTRs linked to a LUC reporter gene. We observed statistically significant increases in LUC activity for the 5'UTRpGL3 and 5'/3'UTRpGL3 constructs. LUC mRNA levels remained constant for all constructs, suggesting that increased LUC activity was likely posttranscriptional in nature. When RNA isolated from transfected cells was translated in vitro, parallel increases in translatable LUC activity were observed. We also examined the role of UTR sequences within the context of the apoB coding sequence, using constructs containing the N-terminal 15% of apoB (apoB15). We observed a 40% and 25% increase in total protein mass with the 5'UTR-apoB15 construct and the 5'UTR-apoB15-3'UTR, respectively, over the control construct with no apoB UTR, with only a slight stimulation observed for apoB15 3'UTR. Radiolabeling analysis of apoB15 synthetic rate showed a more striking 4.5-fold stimulation of protein synthesis by 5'UTR while addition of both UTRs caused a 3.1-fold stimulation over the control construct. Deletion mutant analysis revealed that the stimulatory effect of the 5'UTR on apoB mRNA translation may be dependent on specific hairpin elements formed within the 5'UTR secondary structure. Overall, our data suggest that putative 5'UTR motifs are important for optimal translation of the apoB message whereas the presence of the 3'UTR appears to attenuate wild-type expression. Potential cis-trans interactions of these motifs with putative RNA binding proteins/translational factors are likely to govern apoB mRNA translation and protein synthesis and may play an important role in dysregulation of atherogenic lipoprotein production in dyslipidemic states.