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.