Spinal Cord Injury regulates circular mRNA expression in axons

Injured axons in the CNS do not spontaneously regenerate leading to paralysis. Neurons can transport RNA and ribosomes to the site of injury where de novo translation has been known to occur, facilitating repair. Yet the tools available for studying axonal specific RNA are limited. We came up with a simple modification using Cold Active Proteases that enabled us to enrich for axonal RNA from rats with spinal cord injury (SCI) compared to non-injured. Our enriched axonal prep was then analyzed by bulk RNA-seq, where we looked at total RNA and we found differentially expressed genes (DEGs). Analyzing our bulk RNA-seq by Gene Ontology, we found the second most significant pathway for all DEGs was for axonogenesis, with markedly low glial cell contamination. From our DEGs we detected Rims2, which is reported to be predominately a circular RNA (circRNA) in the rodent CNS. Upon further annotation, we found over 200 putative circRNAs. circRNAs are abundant and well conserved in the brain, they are thought to be transcriptional regulators by functioning as microRNA (miR) sponges or binding to RNA-binding proteins (RBPs). By computational analysis using Circular RNA Interactome, we are able to predict which miRs or RBPs could bind to circRims2. Overall design: Our approach was to utilize the latest in detection of novel RNAs from spinal cord tissue that we enriched for axons, with advanced computational techniques, to determine novel Differential Expressed Genes (DEGs) expressed. Using spinal cords from rats with spinal cord contusions or controls with no injury, we utilized Cold Active Proteases to separate out the Acellular Fraction from the pelleted cells. We performed bulk-seq analysis on the Acellular Fraction and determined that it was enriched for known Axonal RNA, with limited glial cell contamination and detected novel DEGs. We used computational programs such as Gene Ontology and found axonogenesis to be a highly significant pathway for all DEGs. We confirmed for expression of some of the novel DEGs detected by RNA-scope in primary cortical cultures and adult rat spinal cord. Upon further annotation we detected novel circRNAs in our enriched axonal RNA. Using Ribonuclease R which digests linear RNA, but not circRNA, we validated that we could indeed detect circRNA by both RNA-scope of the spinal cord and RT-qPCR.