Functional Study Of A Short Open Reading Frame Encoding Ribosomal Protein S27 In Innate Immunity Of Marsupenaeus Japonicus

Analysis of the genome,transcriptome and proteome revealed the presence of hundreds to thousands of translated but unannotated small or short open reading frames(smORF or sORF).SmORF or sORF encodes a polypeptide of less than 100 codons.Studies have found that many sORFs play important roles in the life of the body,such as growth and development,apoptosis and innate immunity.We found a variety of different sORFs in the transcriptomes of virus-stimulated Marsupenaeus japonicus hemocytes and digestive tracts.Through bioinformatics analysis of these sORFs,we found that many sORFs belong to ribosomal proteins.More and more evidences show that many kinds of ribosomal proteins play other roles besides constituting ribosomes and participating in the biosynthesis of proteins,such as DNA repair,cell development regulation,and cell differentiation.In addition,some ribosomal protein genes were highly expressed in gastric cancer,colorectal cancer,esophageal cancer,liver cancer and other tumor tissues.What are the functions of these sORFs and their encoded peptides in the immune defense of shrimp?In order to answer this question,this paper carried out the functional study of sORFs in the immune response of shrimp.First,the tissue distribution and expression patterns of these sORFs in hemocytes were analyzed.They were found to be distributed in all the shrimp tissues tested and showed different degrees of up-regulated expression after WSSV challenged.We conducted further functional studies on the ribosomal protein MjRPS27.We found that MjRPS27 expression was significantly up-regulated in white spot syndrome virus(WSSV)infected shrimp.After knockdown of MjRPS27 by RNA interference,qPCR detected a significant increase in the expression of WSSV envelope protein VP28.We constructed an MjRPS27 a vector for expressing recombinant protein with a penetrating peptide.After injection of TAT-MjRPS27 into shrimp,the expression level of VP28 in shrimp was reduced.Analysis of the results of these experiments indicated that MjRPS27 has a function of inhibiting viral replication when WSSV infects Marsupenaeus japonicus.Further experiments showed that when we knocked down MjRPS27,the mRNA expression level of MjDorsal,MjRelish and AMPs were decreased,and thenuclear translocation of MjDorsal,MjRelish were also decreased by immunocytochemistry.This suggests that MjRPS27 may induce NF-κB pathyway and regulate the expression of AMPs after WSSV challenged,thereby inhibiting viral replication.At the same time,we also found that MjRPS27 interacts with the envelope proteins VP19,VP24 and VP28 of WSSV.It is suggested that MjRPS27 may also bind to WSSV envelope protein to inhibit the assembly into virions in shrimp.This study was the first to elucidate the function of the ribosomal protein MjRPS27 in antiviral immunity of shrimp.