Sciatic nerve injury-responsive differential CSDE1 association efficiency of ribosomal protein coding mRNAs
Gpr151 is an injury-responsive gene that promotes axon regeneration through its interaction with the 5'UTR region of its mRNA transcripts and the RNA-binding protein CSDE1. We found that knocking down or overexpressing the 5'UTR of Gpr151 changes the ribosomal protein mRNA regulon regulated by CSDE1. After the sciatic nerve injury, the association between the ribosomal protein mRNA regulon and CSDE1 was enhanced at dorsal root ganglions(DRGs), but this enhancement was not observed in the AAV-shGpr151 injected mice. Overexpression of 5'UTRmut, which has increased binding affinity to CSDE1, enhanced the ribosomal protein mRNA regulon and its association with CSDE1 in embryonic DRG neurons. Overall design: Wild type or AAV-shGpr151 injected mice were subjected to sciatic nerve injury in order to profile the injury-responsive changes in CSDE1 binding RNA pools at DRGs. The DRG tissues were collected 72 hours after the injury, and the RNAs were extracted from the anti-CSDE1 immunoprecipitated lysates. Primary DRG neurons were spot cultured and infected with LV-control or LV-5'UTRmut at DIV6. Neurons were collected at DIV6, and their RNAs were extracted from the anti-CSDE1 immunoprecipitated lysates. Adult DRG neurons from the wild type or RiboTag+/+;Advillin-Cre+ mice were cultured after giving the pre-conditioning effects induced by sciatic nerve injury 24 hours prior to DRG tissue dissection, and infected with AAV-control or AAV-shGpr151 four hours after cell culture. Neurons were collected at DIV6, and their RNAs were extracted from the anti-CSDE1 or anti-HA immunoprecipitated lysates. In vivo gene delivery of control, shGpr151 or 5'UTRmut, packaged in the AAV9 viral vectors, was conducted through the facial vein injection at P1. In vitro gene delivery of control, shGpr151 or 5'UTRmut, packaged in the AAV9 or lentiviral vectors, was conducted through the infection to cultured neurons.