MYRF-1 And Its Paralog MYRF-2 Interact With Each Other And Cooperatively Regulate Synaptic Rewiring

The nervous system is a complex and sophisticated network constructed by neurons.During the development of the nervous system from the initial state to the mature state,it has a wide range of remodeling phenomena,including the pruning of neuronal axons;the generation of new synapses;the elimination of old synapses and so on.Different from the adult neural plasticity,the changes of neural connections are irreversible,during the developmental neural plasticity.At present,very little mechanism is known about the molecular events during neural plasticity.One prominent example showing developmental plasticity of neurons in C.elegans is an event that DD motor neurons completely reverse their synaptic connections made en passant without obvious morphological change.This system provides a powerful genetic model to explore the mechanism of synaptic remodeling.In our previous work,we obtained a novel myrf-1 mutant ju1121,which blocked the remodeling in DDs.Detailed analysis of the MYRF-1 protein,we found that the full length of MYRF-1 located to ER membrane.Its N-terminal part could be cleaved and enter the nucleus.Overexpressing the N terminal fragment of MYRF-1 could effectively promote early synaptic remodeling of DD neurons.However,previous work has shown that the synaptic refinement of the myrf-1 gene deletion mutant can occur normally.Therefore the nature of myrf-1(ju1121)mutation,and whether the normal function of MYRF-1 is to positively regulate synaptic rewiring remain to be clarified.We generated myrf-1 null mutations by introduction of indel mutations using CRISPR genome editing.To our surprise,DDs remodeling is normal in myrf-1 null mutants.We hypothesized that there may be other genes that are functionally redundant with myrf-1 to promote synaptic remodeling.MYRF-1 has a high similar paralog MYRF-2.We generated null mutants at early coding region of myrf-2 using CRISPR-Cas9 system.Such myrf-2 mutants also grow like wide type and show normal DD remodeling.However,in myrf-1 and myrf-2 double mutant,the synaptic remodeling is blocked,suggesting that myrf-1 and myrf-2 are located in the same genetic pathway,both of which together promote the formation of synaptic remodeling.In order to further explore the mechanism of myrf-1(fu1121),we over-expressed MYRF-1(G274R as ju1121)in DD neurons and found that the transgene expression inhibited the synaptic remodeling.The severity of the rewiring suppression depended on the expression of wild type MYRF-1.When MYRF-1 and MYRF-2 were co-expressed in cultured HEK293 cells,MYRF-1 and MYRF-2 can co-immunoprecipitate with each other,suggesting that they exist in a protein complex together.myrf-1 and myrf-2 are first genes identified as to promote the occurrence of synaptic remodeling in DD neurons,which provides a framework for further study of synaptic remodeling.