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

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Abstract
Granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA levels are controlled post-transcrip-tionally by the 3′-untranslated region (UTR) adenosine-uridine-rich element (ARE). In untransformed, resting cells, the ARE targets GM-CSF mRNA for rapid degradation, thereby significantly suppressing protein expression. We used a rabbit reticulocyte lysate (RRL) cell-free system to examine translational regulation of GM-CSF expression. We uncoupled decay rates from rates of translation by programming the RRL with an excess of mRNAs. Capped, full-length, polyadenylated human GM-CSF mRNA (full-length 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 with an irrelevant synthetic leader sequence (syn 5′-UTR), translation of syn 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 GM-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 supported 50–60% of maximum translation. The 5′-UTR is highly conserved, suggesting similar regulation in multiple species and in these studies was the dominant element regulating GM-CSF mRNA translation, overriding the inhibitory effects of the ARE and the poly(A) tail.