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

Abstract

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.

In Vivo Studies of Translational Repression Mediated by the Granulocyte-Macrophage Colony-stimulating Factor AU-rich Element

“…Blocking of ARE-mediated mRNA decay because translation initiates from an IRES suggests that the ARE-destabilizing function may be linked to events occurring at the 5Ј-end of the transcript, such as decapping. This is in accordance with recent data reported in mammalian cells, where the ARE stimulated decapping in HeLa cell extracts (7), and in vitro experiments, where the GM-CSF 5Ј-UTR was able to suppress the translational repression mediated by the GM-CSF ARE located in the 3Ј-UTR (9). Concerning this last point, we performed cell transfections with a plasmid containing both GM-CSF 5Ј and 3Ј-UTRs and repeated our analysis of EGFP expression by flow cytometry and Western and Northern blots.…”

“…However, in their report, Jarzembowski et al (9) show that the GM-CSF ARE could specifically inhibit translation of a polyadenylated transcript independently of its effect on the mRNA decay. Our in vivo results support these in vitro results since we showed that EGFP expression initiated by either cap-or IRES-dependent translation is dramatically inhibited by the GM-CSF ARE, whereas ARE-containing EGFP mRNAs persisted in the cells (Figs.…”

Expression of Creatine Kinase Isoenzyme Genes during Postnatal Development of Rat Brain Cerebrum: Evidence for Posttranscriptional Regulation

“…ment (nucleotides numbered 137-152 in figure 8), which is similar to AU-rich elements found to participate in translation repression of several other mRNAs (e.g. granulocyte-macrophage colony-stimulating factor [50], p53 [51],TNF-· [52], or Vg1 [53]). We used the m-fold program of Zuker and colleagues [54] to show that CKB 3) UTR can be folded into two classes of configuration each with considerable stability (approximately -60 kcal/mol; fig.…”

Regulated control by granulocyte-macrophage colony-stimulating factor AU-rich element during mouse embryogenesis

“…Because other regulatory sequences located within the mRNA could influence ARE activity, we kept in the constructs the ARE in its natural context. 15,16 Moreover, to analyze when and where the ARE controls the level of GM-CSF mRNA expression, the constructs were placed under the transcriptional control of the cytomegalovirus (CMV) early immediate promoter, which has been shown to be active early in development and in most tissues. 17,18 Because both constructs led to major lethality before birth, we analyzed transgene expression during late embryonic development.…”