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 controlled post-transcriptionally 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, polyadenyl-ated 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.
Jarzembowski
J A
JA
Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53792, USA.
Rajagopalan
L E
LE
Shin
H C
HC
Malter
J S
JS
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000241:Adenosine / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D020868:Gene Silencing; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011061:Poly A / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics* / Q000502:physiology; D013696:Temperature; D014529:Uridine / Q000235:genetics / Q000378:metabolism
D020121:5' Untranslated Regions; D012333:RNA, Messenger; D011061:Poly A; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000241:Adenosine; D014529:Uridine