Identification And Anabolic Amphioxus MicroRNA Related Proteins, Identification And Comparative Genome Analysis

As one of the closest living invertebrate relatives of vertebrate, amphioxus is usually referred to as living fossil to research the evolution and origin of vertebrate. MicroRNAs (miRNAs) are endogenous noncoding RNAs with about21-25nucleotides in length and play an important role in the gene regulation and the process of growth and development of the biology. At present, amphioxus miRNAs are far less understood. In this study, we used the bioinformatical methods to systematically study amphioxus miRNAs and relative proteins involved in the synthesis and metabolize of miRNAs. The results are as follows:1.We systematically investigated the Branchiostoma floridae miRNAs using the bioinformatics pipeline combining RNAfold and Triplet-SVM algorithm. Totally, two hundred and forty five B. floridae miRNAs were identified, in which183miRNAs were newly identified. Genome location analysis showed that there were9clustered miRNAs distributed in the scaffold211and these miRNAs were proved to be expressed. Sequence similarity analysis showed that there were closer relationship between cephalochordates and vertebrates. The predicted52B. floridae miRNA families could be sorted into species/families-specific groups. By comparison of miRNA family type and copy number within different species, we found amphioxus has undergone the gene expansion transition stage from the miRNA family copies to miRNA family types. Most miRNA families were single-copy family types in invertebrate. We found that only19miRNAs were expressed in all developmental stages, most miRNAs were developmental stage-specific. In addition, these potential miRNA-targeting genes were mainly classified into cell adhension, defense/immunity protein activity, catalytic and enzyme regulator activity. Seventy-nine target genes regulated by amphioxus miRNAs were found to be related with immune function, suggesting that amphioxus miRNAs play important roles in immune regulation.2. Using the bioinformatics methods to identify miRNAs from the Solexa high throughput sequencing data of Branchiostoma belcheri,483miRNAs were obtained, in which287miRNAs were newly identified. Further we obtained238miRNA precursor sequences. Genome location analysis showed304miRNAs matched86scaffolds, and the miRNA numbers of each scaffold were ranged from1-25. There were25clustered miRNAs distributed in scaffold22. There were11miRNAs matched more than one genome location, indicated there were the genome duplication events happened.58.39%predicted miRNAs were conserved across1～89species (E-value≤10-5), and we found miRNAs in B. belcheri were highly similar to121(64.71%) miRNAs of B. floridae. The predicted B. belcheri miRNAs could be classified into70miRNA families, in which99%families were conserved in B. floridae. The70B. belcheri miRNA families were species/family specific, in which39(55.71%) B. belcheri miRNA families were specific in amphioxus. These lineage-specific miRNA families may contribute to developmental novelties during evolution. By comparison of miRNA family type and copy number between amphioxus and other species, we found amphioxus has undergone the gene expansion transition stage from the miRNA family copies to miRNA family types. The results of repetitive sequence predicted in miRNA precursor sequences showed that there were few repetitive sequence found in amphioxus miRNAs, indicted that repeat elements did not have much impact on the amphioxus miRNA evolution.3. We used homology search and bioinformatics methods to identify the proteins related in miRNA anabolic in amphioxus, sea squirt, lamprey and sea urchin. Full or partial cDNA squences were obtained by GENSCAN and Gene Wise software. The expression of the predicted genes were verified by RT-PCR and EST expression analysis. The analysis of conserved domain showed that the proteins in the same family have consistent or similar domains. We found two specific domain YppG and Drf_FH1in amphioxus and mouse Drosha proteins. By comparison of protein sequences within different species, we found that the proteins in closely related species were highly similar. And compared to sea squirt, the proteins found in amphioxus were highly similar to vertebrate. EST expression analysis showed that the proteins predicted in B. floridae and C. intestinalis were expressed in specific development stage. We found5conserved motifs in Drosha and Dicer proteins, and10motifs in Piwi and AGO proteins. There were catalytic triad "D-D/G-H/R" in AGO proteins, and "D-D-H/K/R" in Piwi proteins. We found that the exon numbers were totally different between vertebrate and invertebrate, approximately30exons in mammals Drosha proteins but only3-11in insects. Amphioxus has similar genome structure with vertebrate. The average length of exons of Drosha and Dicer were larger than the length of introns in insects and elegans, the phenomenon was inverse in human and zebrafish species. Phylogenetic tree inferred by Bayesian algorithm indicated that total Piwi proteins were classified into two clusters, one cluster contained human PIWIL1, PIWIL3, PIWIL4and other homology proteins, the other cluster contained human PIWIL2and homology proteins. Evolutionary pressure analysis results showed that there were more positive selection sites in Dicer genes, which would play important role in the change of the structure and function of Dicer genes. The above results would help to deeply understand the function of amphioxus miRNAs and anabolic related proteins, and supply important theoretical basis to uncover the amphioxus own life activities rule and discover the origin and evolution mechanism of vertebrate. And also will supply theoretical significance and practical value to further study of amphioxus functional genomics.