Abstract
The role of ribosomal protein S6 (rpS6) phosphorylation in mRNA translation remains poorly understood. Here, we reveal a potential role in modulating the translation rate of chemokine (C-X-C motif) ligand 8 (CXCL8 or Interleukin 8, IL8). We observed that more CXCL8 protein was being secreted from less CXCL8 mRNA in primary macrophages and macrophage-like HL-60 cells relative to other cell types. This correlated with an increase in CXCL8 polyribosome association, suggesting an increase in the rate of CXCL8 translation in macrophages. The cell type-specific expression levels were replicated by a CXCL8- UTR-reporter (Nanoluc reporter flanked by the 5’ and 3’ UTR of CXCL8). Mutations of the CXCL8-UTR-reporter revealed that cell type-specific expression required: 1) a 3’ UTR of at least three hundred bases; and 2) an AU base content that exceeds fifty percent in the first hundred bases of the 3’ UTR immediately after the stop codon, which we dub AU-rich proximal UTR sequences (APS). The 5’ UTR of CXCL8 enhanced expression at the protein level and conferred cell type-specific expression when paired with a 3’ UTR. A search for other APS-positive mRNAs uncovered TNF alpha induced protein 6 (TNFAIP6), another mRNA that was translationally upregulated in macrophages. The elevated translation of APS-positive mRNAs in macrophages coincided with elevated rpS6 S235/236 phosphorylation. Both were attenuated by the ERK1/2 signaling inhibitors, U0126 and AZD6244. In A549 cells, rpS6 S235/236 phosphorylation was induced by TAK1, Akt or PKA signaling. This enhanced the translation of the CXCL8-UTR-reporters. Thus, we propose that the induction of rpS6 S235/236 phosphorylation enhances the translation of mRNAs that contain APS motifs, such as CXCL8 and TNFAIP6. This may contribute to the role of macrophages as the primary producer of CXCL8, a cytokine that is essential for immune cell recruitment and activation.
Highlights
Translation is an essential step in protein synthesis
Translational control is mRNA-specific and the specificity is sometimes dependent on sequence motifs within the 5’ untranslated region (UTRs), such as 50 terminal oligopyrimidine (TOP) [4], which lead to selective protein synthesis during increased activity of eukaryotic translation initiation factor 4E
CXCL8 protein secretion and polysome association is increased in HL-60 macrophages
Summary
Translation is an essential step in protein synthesis. Mechanisms that regulate the rate of translation determine the expression levels of a large fraction of the genome. This was revealed by metabolic pulse labeling of global cellular mRNA and protein synthesis rates [1]. Multiple large-scale transcriptomic and proteomic studies have revealed a lack of correlation between mRNA and protein abundance across different mammalian cell-types and tissues [2,3]. Translational control is mRNA-specific and the specificity is sometimes dependent on sequence motifs within the 5’ untranslated region (UTRs), such as 50 terminal oligopyrimidine (TOP) [4], which lead to selective protein synthesis during increased activity of eukaryotic translation initiation factor 4E (eIF4E). Besides the 5’ UTR, the involvement of the 3’ UTR in conferring translational control has been hinted [6]
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