Novel AU-rich proximal UTR sequences (APS) enhance CXCL8 synthesis upon the induction of rpS6 phosphorylation

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. Author summary Ribosomal protein S6 (rpS6) is a component of the cell translation system. This system produces proteins based on the instructions found on messenger ribonucleic acids or mRNAs. The activity of rpS6 is modified via the attachment of phosphate groups. This rpS6 phosphorylation regulates cell size, cell proliferation, glucose homeostasis and cancer defense. However, the molecular mechanism remains poorly understood. Here, we propose that rpS6 phosphorylation may affect the rate of translation of certain mRNAs. These mRNAs contain a high frequency of adenine and uracil bases in the first hundred or so bases of their 3' UTR sequences, which we dub AU-rich proximal UTR sequences (APS). APS-modulated mRNAs include the immune signaling proteins, CXCL8 and TNFAIP6. This novel mechanism allows macrophages to secrete sufficient CXCL8 protein to attract and activate other immune cells such as neutrophils during injury or disease. It may also be aberrantly upregulated in a subset of tumors, leading to the overexpression of APS-positive genes such as CXCL8. Indeed, both rpS6 phosphorylation and CXCL8 expression are consistently found, by separate studies, to be tumor-promoting and upregulated in cancers. Modulating these APS-positive mRNAs may be a novel strategy to treat diseases.