Abstract
Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1) is an intestine-specific RNA-binding protein. However, inflammation or exposure to DNA-damaging agents can induce ectopic APOBEC1 expression, which can result in hepatocellular hyperplasia in animal models. To identify its RNA targets, FLAG-tagged APOBEC1 was immunoprecipitated from transfected HuH7.5 hepatocellular carcinoma cells and analyzed using DNA microarrays. The interleukin-8 (IL8) mRNA was the most abundant co-precipitated RNA. Exogenous APOBEC1 expression increased IL8 production by extending the half-life of the IL8 mRNA. A cluster of AU-rich elements in the 3'-UTR of IL8 was essential to the APOBEC1-mediated increase in IL8 production. Notably, IL8 mRNA did not co-immunoprecipitate with APOBEC1 from lysates of other cell types at appreciable levels; therefore, other factors may enhance the association between APOBEC1 and IL8 mRNA in a cell type-specific manner. A yeast two-hybrid analysis and siRNA screen were used to identify proteins that enhance the interaction between APOBEC1 and IL8 mRNA. Heterogeneous nuclear ribonucleoprotein Q (hnRNPQ) was essential to the APOBEC1/IL8 mRNA association in HuH7.5 cells. Of the seven hnRNPQ isoforms, only hnRNPQ6 enabled APOBEC1 to bind to IL8 mRNA when overexpressed in HEK293 cells, which expressed the lowest level of endogenous hnRNPQ6 among the cell types examined. The results of a reporter assay using a luciferase gene fused to the IL8 3'-UTR were consistent with the hypothesis that hnRNPQ6 is required for APOBEC1-enhanced IL8 production. Collectively, these data indicate that hnRNPQ6 promotes the interaction of APOBEC1 with IL8 mRNA and the subsequent increase in IL8 production.
Highlights
APOBEC1 stabilizes target mRNAs by suppressing nonsense- or AU-rich element-mediated decay; the mechanisms regulating target selection are unknown
The IL8 mRNA exhibited the highest signal ratio (58.31) among all transcripts examined, but the signal ratio of the MYC mRNA (1.04) was not elevated significantly, despite its robust expression in HuH7.5 cells. These findings indicate that APOBEC1 binds preferentially to IL8 mRNA in HuH7.5 cells
The Association between APOBEC1 and IL8 mRNA Is Promoted by heterogeneous nuclear ribonucleoprotein Q isoform 6 (hnRNPQ6) —As mentioned above, an interaction between APOBEC1 and IL8 mRNA was not observed in HEK293 cells; we investigated whether overexpression of Heterogeneous nuclear ribonucleoprotein Q (hnRNPQ) in this cell line could lead to the promotion of this association
Summary
APOBEC1 stabilizes target mRNAs by suppressing nonsense- or AU-rich element-mediated decay; the mechanisms regulating target selection are unknown. The results of a reporter assay using a luciferase gene fused to the IL8 3-UTR were consistent with the hypothesis that hnRNPQ6 is required for APOBEC1-enhanced IL8 production These data indicate that hnRNPQ6 promotes the. Apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1) is a cytidine deaminase that converts a specific cytidine residue in the apoB mRNA (APOB) to uridine This deamination generates an in-frame premature stop codon that results in the production of apoB-48, the short isoform of apoB. Greeve et al [8] have suggested that most types of carcinoma, including hepatocellular carcinoma, are not associated with aberrant editing of the mRNAs encoding APOB, novel APOBEC1 target 1, or neurofibromin 1. Further exploration of the mechanisms underlying the interaction between APOBEC1 and IL8 mRNA revealed that another factor, heterogeneous nuclear ribonucleoprotein Q isoform 6 (hnRNPQ6), enhanced this interaction
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