B52 And Dock Participate In Dscam Alternative Splicing And Expression Of Antimicrobial Peptides In Eriocheir Sinensis

Alternative splicing is ubiquitous in eukaryotes and is an important part of gene expression regulation.A series of differential transcripts can be produced through different combinations of exons,which greatly enriches the diversity of transcriptome and proteome of organisms.At the same time,the abnormal alternative splicing will directly lead to the disorder of physiological function.Therefore,alternative splicing events are crucial to the normal physiological function of organisms.Dscam is the representative gene of alternative splicing,which can potentially generate 38,016 alternative splicing isomers in D.melanogaster,and the phenomenon of alternative splicing of this gene only occurs in arthropods.It's exciting that a large number of isomers of arthropod Dscam gene have been proved to be immune-specific,and some scholars speculate that this gene may be related to immune priming or trained immunity.In order to clarify the regulation of Dscam on the expression of blood cell protein in Chinese mitten crab(Eriocheir sinensis),this paper conducted a quantitative analysis on the blood cell secretion protein that was silenced Dscam and stimulated by different bacteria based on isotope-labeled iTRAQ proteomics technology.The results showed that many cell physiological function-related proteins were influenced by Dscam,especially the expression of immune effector factors such as antibacterial peptides was significantly positive regulated by Dscam.ProPO and other immune factors were significantly up-regulated after Dscam silenced,indicating that the host may compensate for the decreased expression of antimicrobial peptides through the compensation mechanism.In addition,intracellular proteins such as spliceosomes have also been detected in cell culture media with significant changes,which may be related to the bacteria causing blood cells to rupture and release their contents.These results indicate that the extracellular part of Dscam,as a transmembrane protein,is highly likely to transmit the signal into the cell after receiving the dangerous information from bacteria,activate the relevant signal transduction pathway through the intracellular area,produce the immune effector factors such as antimicrobial peptides,and complete the immune defense process.Therefore,this paper will focus on the alternative splicing mechanism of transmembrane type Dscam of Chinese mitten crab and the signal transduction mechanism of cytoplasmic tail alternative splicing isomer to regulate the expression of antimicrobial peptides.Previous studies reported that RNA secondary structure and splicing factors may be key links that mediate Dscam alternative splicing.Therefore,we first compared the Dscam RNA secondary structure feature sequence of D.melanogaster based on the full length of Eriocheir sinensis Dscam gene(EsDscam),and it was found that the synergistic action of docking site,selector sequence and locus control region are the structural basis leading to splicing of Dscam gene pre-mRNA.We also isolated and characterised the B52 gene from E.sinensis(EsB52).The 876 bp open reading frame of EsB52 encodes a 291 amino acid residue polypeptide,and EsB52 has two RNA recognition motifs(RRMs)at the N-terminus and an arginine/serine-rich domain at the C-terminus.Each RRM contains two degenerate short submotifs,RNP-1 and RNP2.Analysis of tissue distribution revealed that EsB52 mRNA expression was widespread in all tested tissues,and especially high in brain and hemocytes.In hemocytes,Es B52 was upregulated significantly after stimulation with pathogenassociated molecular patterns and bacteria.Furthermore,EsB52 RNAi decreased the number of Ig7 inclusion in mRNA rather than Ig2 or Ig3.The above study indicated that docking site,selector sequence and locus control region were the structural basis of EsDscam alternative splicing,while splicing factors such as B52 were important regulatory elements that activated Dscam alternative splicing.Dscam is widely found in the immune system of arthropods and has been demonstrated to specifically bind to pathogens and phagocytize bacteria.However,how the immune-related signaling mechanism is activated after Dscam identification and combination with the pathogen remains unclear.Based on the previous work of our laboratory,we have confirmed that alternative splicing exons of EsDscam cytoplasmic tail and its isoforms in hemocyte of crab,expression of Es Dscam were acutely induced after immune challenge,significantly decreased AMPs expression were detected in EsDscam silenced hemocyte in vitro,which were coincidence with vulnerability to infection by Gram-positive bacterial and higher bacterial concentration in Dscam silenced crab in vivo.In this study,we found that the translation protein encoding SH3 binding motif of EsDscam cytoplasmic tail non-alternative splicing exon 32 interacted with the SH3 binding motif of Dock protein.Dock protein has functions similar to EsDscam regulation of ERK phosphorylation,Dorsal nuclear transport,and antimicrobial peptide expression.Further,through RNAi,Co-IP and other technical means,it was found that Dock protein regulates ERK phosphorylation through indirect binding.At the same time,this study also confirmed that alternative splicing exons do not have the ability to regulate ERK phosphorylation,Dorsal nuclear transport and antimicrobial peptide expression.The above studies indicated that the EsDscam cytoplasmic tail region regulates ERK phosphorylation through non-alternative splicing exon binding Dock protein,promotes Dorsal,a key molecule of the Toll signaling pathway,from the cytoplasm to the nucleus,and further up-regulates the expression of antimicrobial peptides to complete the process of antibacterial immune defense.