AU-rich mRNA binding proteins stabilize mRNA in human saliva

Objective: Human saliva contains thousands of mRNAs, some of which can be used as biomarkers for oral cancer. We have found that more than 60% of the mRNAs detected in human saliva contain AU-rich elements (ARE) in their 3' untranslated regions. As AREs are known to contribute to RNA turnover by forming complexes with ARE-binding proteins, we hypothesized that salivary mRNA stability is mediated by ARE-binding proteins. Thus, the goal of this study is to examine the role of ARE-binding proteins in stabilization of the mRNAs in human saliva. Methods: To test this hypothesis, we monitored the in vitro degradation of radiolabeled ARE-containing mRNAs (i.e., IL-8 and TNF-á) by salivary protein extracts. UV cross-linking and immunoprecipitation experiments were carried out to study the protein-RNA interactions in human saliva. Results: The degradation of IL-8 mRNA was accelerated by competition for saliva ARE-binding proteins through the addition of excess unlabeled IL-8 mRNA fragments containing four tandem AREs. UV cross-linking and immunoprecipitation experiments revealed the ARE-binding proteins AUF1 and HuR associated with IL-8 mRNA in the saliva. Immune-depletion of AUF1 and HuR from salivary protein extracts enhanced degradation of IL-8 mRNA, with the latter having a more pronounced effect. Moreover, the addition of purified HuR protein stabilized IL-8 mRNA in a dose-dependent manner. Conclusions: These studies indicate that ARE binding proteins AUF1 and HuR conferring stability to mRNAs in saliva. This analysis highlights the multitude of salivary mRNAs that contain ARE motifs (60% in saliva versus 5-8% in other transcriptomes) and their translational utility for saliva-based diagnostics. This work is supported by NIH grants to U01DE17790 (D.T.W) and K99DE018165-01A1 (V.P.)