In vivo studies of translational repression mediated by the granulocyte-macrophage colony-stimulating factor AU-rich element

The AU-rich element (ARE) controls the turnover of many unstable mRNAs and their translation. The granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE is known to be a destabilizing element, but its role in translation remains unclear. Here we studied in vivo the role of the GM-CSF ARE on the mRNA and protein expressions of an enhanced green fluorescent protein reporter gene. The GM-CSF ARE had a repressor effect on translation independently of its effect on mRNA levels. In the context of an internal ribosome entry site, the GM-CSF ARE still repressed translation but was no longer functional as a destabilizing element. Gel retardation assays showed that poly(A)-binding protein is displaced from the poly(A) tail when the ARE is present in the 3'-untranslated region. These data suggest that the GM-CSF ARE controls translation and mRNA decay by interfering with poly(A)-binding protein-mediated mRNA circularization.     

Inhibition of translation by UAUUUAU and UAUUUUUAU motifs of the AU-rich RNA instability element in the HPV-1 late 3' untranslated region

The human papillomavirus type 1 (HPV-1) late mRNAs contain a 57-nucleotide adenosine- and uridine-rich RNA instability element termed h1ARE in their late 3' untranslated regions. Here we show that five sequence motifs in the h1ARE (named I-V) affect the mRNA half-life in an additive manner. The minimal inhibitory sequence in motifs I and II was mapped to UAUUUAU, and the minimal inhibitory sequence in motifs III-V was mapped to UAUUUUUAU. We also provide evidence that the same motifs in the AU-RNA instability element inhibit mRNA translation, an effect that was entirely dependent on the presence of a poly(A) tail on the mRNA. Additional experiments demonstrated that the h1ARE interacted directly with the poly(A)-binding protein, suggesting that the h1ARE inhibits translation by interfering with the function of the poly(A)-binding protein.     

Rapid degradation of AU-rich element (ARE) mRNAs is activated by ribosome transit and blocked by secondary structure at any position 5' to the ARE.

The 3' noncoding region (NCR) AU-rich element (ARE) selectively confers rapid degradation on many mRNAs via a process requiring translation of the message. The role of cotranslation in destabilization of ARE mRNAs was examined by insertion of translation-blocking stable secondary structure at different sites in test mRNAs containing either the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE or a control sequence. A strong (-80 kcal/mol [1 kcal = 4.184 kJ]) but not a moderate (-30 kcal/mol) secondary structure prevented destabilization of mRNAs when inserted at any position upstream of the ARE, including in the 3' NCR. Surprisingly, a strong secondary structure did not block rapid mRNA decay when placed immediately downstream of the ARE. Studies are also presented showing that the turnover of mRNAs containing control or ARE sequences is not altered by insertion of long (1,000-nucleotide) intervening segments between the stop codon and the ARE or between the ARE and poly(A) tail. Characterization of ARE-containing mRNAs in polyadenylated and whole cytoplasmic RNA fractions failed to find evidence for decay intermediates degraded to the site of strong secondary structure from either the 5' or 3' end. From these and other data presented, this study demonstrates that complete translation of the coding region is essential for activation of rapid mRNA decay controlled by the GM-CSF ARE and that the structure of the 3' NCR can strongly influence activation. The results are consistent with activation of ARE-mediated decay by possible entry of translation-linked decay factors into the 3' NCR or translation-coupled changes in 3' NCR ribonucleoprotein structure or composition.     

Cutting edge: naturally occurring soluble form of mouse Toll-like receptor 4 inhibits lipopolysaccharide signaling

Toll-like receptors (TLRs) are a family of proteins playing important roles in host defense. Mice defective of functional TLR4 are hyporesponsive to LPS, suggesting that TLR4 is essential for LPS signaling. Here we report the cloning of an alternatively spliced mouse TLR4 (mTLR4) mRNA. The additional exon exists between the second and third exon of the reported mTLR4 gene and contains an in-frame stop codon. The alternatively spliced mRNA encodes 86 aa of the reported mTLR4 and an additional 36 aa. This alternatively spliced mTLR4 mRNA expressed a partially secretary 20-kDa protein, which we named soluble mTLR4 (smTLR4). In a mouse macrophage cell line, the exogenously expressed smTLR4 significantly inhibited LPS-mediated TNF-alpha production and NF-kappaB activation. Additionally, in mouse macrophages, LPS increased the mRNA for smTLR4. Taken together, our results indicate that smTLR4 may function as a feedback mechanism to inhibit the excessive LPS responses in mouse macrophages.