Alternative Splicing And Immunoregulation Mechanism Of Membrane-bound Dscam In Eriocheir Sinensis

Classically,the two immune systems against invaders are innate immunity and adaptive immunity.Innate immunity is found in all animals,while adaptive immunity that has the defining characteristics of antigen specificity and immunological memory was believed to exist only in vertebrates.In the invertebrate innate immunity system,pattern recognition receptors(PRRs)either directly or indirectly activate Toll pathways upon interaction with pathogen associated molecular patterns(PAMP),lead to the nuclear translocation of Dorsal transcription factors that control transcription of distinct sets of immune effectors genes,such as antibacterial peptides(AMPs).Alternative splicing is an ancient and widespread mechanism used by eukaryotes to expand protein diversity and regulate gene expression.The most intriguing example of alternative splicing is that the hypervariable Dscam(Dscam-hv)gene is only found in arthropod,a member of the Immunoglobulin(Ig)superfamily.The domain architecture of Dscam comprises the typical pattern including 9 Ig domains(Ig)followed by 4 fibronectin III(FNIII)domains and 1 Ig domain-4 FNIII domains-transmembrane(TM)domain-cytoplasmic tail.Both transmembrane and secreted Dscam type(m-Dscam and s-Dscam)were found in arthropod,which can generate more than 10,000 different isoforms through mutually exclusive splicing of N terminal of Ig2 and Ig3,the whole Ig7 and the transmembrane domain.In addition,diversity was also identified in the cytoplasmic tail induced by exon inclusion or exclusion.Recently,Dscam was found to specifically recognize and bind invading pathogen because of its hypervariable diversity in arthropod innate immunity system.However,the mechanism of how pathogen-induced m-Dscam regulates the innate immune response in arthropods remains unclear.Here we identified m-Dscam gene in Eriocheir sinensis(Esm-Dscam)via genomic sequencing.The m-Dscam gene potentially has 30,600 isoforms due to three alternatively spliced Ig domains(Ig2,Ig3 and Ig7)and a transmembrane domain.We also found six different isoforms in Esm-Dscam cytoplasmic tail induced by exon splicing.The analysis of phylogeny shows that the evolutionary histories of exons encoding the Ig2,Ig3 and Ig7 regions of Esm-Dscam are independent,whereas the rapid evolution of exons encoding Ig3 region indicates that Ig3 domain might be play an important role in organism.Esm-Dscam was significantly upregulated after LPS,PGN,Gram-positive(G~+)bacteria and Gram-negative(G~-)bacterial challenge at both mRNA and protein levels.We demonstrated that Esm-Dscam receptor and Toll signaling pathway play important roles in host defense of S.aureus by regulating expression of antimicrobial peptides in carb and Dorsal translocation into nucleus using knockdown and immunofluroscence approaches.We then found that Esm-Dscam positively regulate phosphorylation of Dorsal and its translocation into the nucleus.We also found that Esm-Dscam receptor can regulate the phosphorylation level of ERK signaling molecules to regulate the expression of antimicrobial peptides,and the phosphorylation of ERK regulate the activation of Dorsal.Overall,our study suggest that Esm-Dscam protein has ability to positively regulate activation of MAPK/ERK molecule,actively regulates Toll signaling pathway by promoting Dorsal translocation to induce AMPs transcription upon S.aureus challenge.