Dendritic Targeted MRNA Expression Via A Cis-acting RNA UTR Element

Once thought to occur exclusively in the somatic space close to the nucleus,protein translation has been demonstrated far from the central perinuclear region in decentralized local domains—a process referred to as local translation.Local translation gives the growth cone autonomy to respond rapidly to signals without waiting for input from the cell body.In addition,axons may be limited by the size of large molecules and do not have enough space to store all the proteins they might need,so storing mRNA and synthesizing and degrading proteins as needed is more efficient.Local protein translation in the neurite is thought to be an important mechanism for the regulation of Synaptic plasticity.In Synaptic plasticity,the phenomenon is analogous to axonal guidance,long term enhancement,and a balance between protein synthesis and degradation during memory.Protein degradation mediates memory destruction and protein synthesis mediates memory reconstruction.These results suggest that the rapid regulation of local proteome is the basic principle of local translation.However,the mechanism of local protein translation has not been fully understood,and how to specifically express protein-coding genes in neurites is a challenge in the technical practice.Tick-borne encephalitis virus(TBEV)is a highly virulent neurotropic virus that mainly attacks human central nervous system and has a high mortality and disability rate.In recent years,the research on it is hot.It has been reported that TBEV UTR plays an important role in its invasion of cells and its replication and translation.Protein translation occurs only in the body of a cell near the nucleus or far from the central nucleus.This process is called local translation.Local translation can endow the growth cone with autonomy to respond to signals quickly without waiting for input from the cell body.In addition,axons may be limited by the size of large molecules and there are not adequate space to store all the proteins they might need,so storing mRNA and synthesizing and degrading proteins as needed is more efficient.Local translation in neurites is considered as an important mechanism to modulate synaptic plasticity of neurons.However,it is hard to specifically express a protein-coding gene in neurites.Recently,the 5?-UTR of TBEV is reported to be able to drive its RNA to the dendrites of infected neurons,as a cis-acting RNA element.To construct a neurite specific gene expression system,present study tested the ability of 5?-UTR of TBEV to bring mRNA(m Cherry CDS)to the neurites for targeted expression.First,we conducted in vitro cell experiments.The cell bodies and processes of neurons were separated by microfluidic platform.In the background,pm R-TBEV 5'-UTR m Cherry plasmid was successfully constructed and introduced into primary cultured neurons to detect the expression of m Cherry protein in dendrites in vitro.It was found that both TBEV 5'-UTR and Acbt 3'-UTR enhanced the fluorescence of m Cherry in the dendrites of the neurons,and more m Cherry signals were found in the dendrites of the neurons in the TBEV 5'-UTR group,which indicated that after fusing TBEV 5'-UTR into CDS mRNA,the reporter genes may be concentrated on the dendrites of neurons.Then,we measured the levels of m Cherry mRNA.We showed that both the 5?-UTR of TBEV and the 3?-UTR of Actb gene could bring the protein coding mRNA to neurites,and the TBEV 5?-UTR is more efficient.About the safety of the TBEV 5?-UTR,there was no obvious cytotoxicity to the neurons when adding either cis-acting RNA element to the protein-expressing plasmid vectors.Given the short length and high efficiency of the TBEV 5?-UTR,the 5?-UTR of TBEV were assemble into an AAV plasmid to produce virus particles for expressing protein-coding gene in vivo.After two weeks infection,the TBEV 5?-UTR infected neurons expressed more m Cherry protein in neurites.Based on the results,we can further explore the mechanism of TBEV 5'-UTR mediated RNA localization in dendrites and the relationship between UTR and mRNA and mRNA transport.The innovation of the paper is that we compared the different effects of UTR in vitro and in vivo,and proved that TBEV 5'-UTR is a short but efficient cis-acting RNA element,it can be used to accurately localize specific proteins into the neurites of the nervous system.In addition,whether the targeted expression of TBEV 5'-UTR as a cis-acting element used in mice can make up for some protein defects or deficiencies to achieve therapeutic effect remains to be studied.