The 5 '-untranslated region of GM-CSF mRNA suppresses translational repression mediated by the 3 ' adenosine-uridine-rich element and the poly(A) tail

Granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA levels are c ontrolled post-transcriptionally by the 3'-untranslated region (UTR) adenos ine-uridine-rich element (ARE), In untransformed, resting cells, the ARE ta rgets GM-CSF mRNA for rapid degradation, thereby significantly suppressing protein expression. We used a rabbit reticulocyte lysate (RRL) cell-free sy stem to examine translational regulation of GM-CSF expression. We uncoupled decay rates from rates of translation by programming the RRL with an exces s of mRNAs. Capped, full-length, polyadenylated human GM-CSF mRNA (full-len gth 5'-UTR AUUUA+A90) and an ARE-modified version (full-length 5'-UTR AUGUA +A90) produced identical amounts of protein. When the 5'-UTR was replaced w ith an irrelevant synthetic leader sequence (syn 5'-UTR), translation of sy n 5'-UTR AUUUA+A90 mRNA was suppressed by >20-fold, Mutation of the ARE or removal of the poly(A) tail relieved this inhibition. Thus, in the absence of a native 5'-UTR, the ARE and poly(A) tail act in concert to block QM-CSF mRNA translation. Substitutions of different regions of the native 5'-UTR revealed that the entire sequence was essential in maintaining the highest rates of translation. However, shorter 10-12 nt contiguous 5'-UTR regions s upported 50-60% of maximum translation. The 5'-UTR is highly conserved, sug gesting similar regulation in multiple species and in these studies was the dominant element regulating GM-CSF mRNA translation, overriding the inhibi tory effects of the ARE and the poly(A) tail.