Functionally independent AU-rich sequence motifs regulate KC (CXCL1) mRNA

Certain pro-inflammatory chemokine mRNAs containing adenine/uridine-rich sequence elements (AREs) in their 3′ untranslated regions (3′-UTRs) are known to exhibit constitutive instability and sensitivity to pro-inflammatory stimuli resulting in the stabilization of the message. Using tetR-regulated transcription we now show that the 3′-UTR of the mouse CXCL1 (KC) mRNA contains at least two ARE motifs that are structurally and functionally distinct. A fragment of 77 nucleotides containing 4 clustered AUUUA pentamers located at the 5′-end of the KC 3′-UTR is only modestly unstable yet promotes markedly enhanced, post-transcriptional protein production in response to either interleukin-1α (IL-1α) or lipopolysaccharide (LPS), suggesting translational regulation. In contrast, a fragment containing 3 isolated AUUUA pentamers corresponding to the residual 3′ 400 nucleotides of the KC 3′-UTR confers both instability and is stabilized in response to IL-1α. Although the clustered AUUUA pentamers in the upstream region are required for stimulus sensitivity, mutation of all three pentamers in the downstream region has little or no effect on either instability or stimulus sensitivity. The upstream region is comparably stabilized in response to either IL-1α or LPS, whereas the AUUUA-independent downstream determinant is differentially more sensitive to IL-1α. Finally, using UV-induced RNA cross-linking, these functionally independent sequences exhibit different patterns of interaction with RNA-binding proteins. Collectively, these findings document the presence of multiple independent determinants of KC mRNA function and demonstrate that these operate via distinct mechanisms.