AU-rich elements associated with HuR are essential for lncRNAs turnover in murine neural stem cells differentiation

A Growing number of studies are deeply investigating the functional role that lncRNAs play in gene expression regulation at multiple levels: transcriptional, translational and post-translational processes. Interestingly, a key and essential role for these molecules has been identified in brain development, where genetic ablation of specific lncRNAs (linc-ENC1, linc-BRN1a/b, FABL, TUG1, FENDRR, HOTTIP, PERIL and HAUNT) was shown to cause strong modifications in mouse brain’s development. To this day, it is unknown how these lncRNAs are regulated in the brain and in neural stem cells. Here, we report the identification of a new lncRNA turnover regulatory mechanism involving the binding of the RNA-binding-protein ELAV1/HuR to conserved AU-rich regions. We firstly investigated the lncRNAs expression in neural stem cells and we observed that their expression patterns change during the differentiation process. We demonstrated by RIP (RNA-immunoprecipitation) analysis that these lncRNAs are bound to HuR. We then suggested that HuR interaction with these lncRNAs plays a regulatory function necessary for the neural stem cells differentiation process, and we demonstrated this by pharmacologic (Di-Hydrotanshinone) and genetic (silencing) means. The treatment with actinomycin D, a transcriptional inhibitor, allowed to evaluate the half-life of these RNA molecules, and we found that HuR’s inhibition lead to a modification in their decay rates both in neural stem cells and in differentiated ones. We then investigated the presence of AU-rich regions in the lncRNAs sequences and found that these are indeed present. Moreover, by means of luciferase reporter assay we demonstrated that mutations of the AU-rich like regions, within the different lncRNA sequences, result in a modification of the molecule’s half-life. By means of immunofluorescence analysis, we demonstrated that HuR’s inhibition also leads to a powerful induction of the differentiation process towards their neuronal fate. In conclusion, we show that these lncRNAs play a role in neural stem cells biology influencing their differentiation capabilities as the alteration of their levels may have an effect on this process. Moreover, we report that the inhibition of HuR’s interaction with the analyzed lncRNAs leads to neural differentiation, suggesting a complementary role for the lncRNAs and the RNA-binding protein (HuR) in this process. Furthermore, we demonstrated that the turn-over regulation mechanism is similar to the one used by mRNAs, involving HuR interaction with AU-rich like regions.