Identification Of The Silkworm Anillin Gene And Aurora B-mediated Phosphorylation Of Anillin

Anillin is an actin binding protein and plays crucial roles during mitotic cell cycle progression in metazoan.However,the sequence and functions of the Anillin gene have not been yet characterized in the silkworm,Bombyx mori.In addition,during mitosis,Anillin undergoes significant phosphorylation,but the kinase,modification site,and corresponding function during mitosis remain unclear.In this study,we cloned the full-length cDNA sequence of the silkworm Anillin(BmAurB)gene,analyzed spatio-temporal expression profiling and BmAnillin function in the silkworm cell cycle using RNAi.At the same time,we also investigated the role of Aurora B kinase in the phosphorylation regulation of Anillin during cell cycle progression.The main results are as follows: 1.Full-length cDNA cloning and spatio-temporal expression profiling of the silkworm BmAnillin geneWe used the amino acid sequence of Drosophila Anillin gene to search the silkworm genome database and found that a predicted gene,namely BMgn004275,shares high sequence similarity with the query.Based on rapid amplification of cDNA ends(RACE)approach,we isolated the full-length cDNA of silkworm Anillin(BmAnillin)gene from wing disc,which comprises of a total of 6,552bp(GenBank accession no.MK327369).Further sequence analysis showed that the BmAnillin gene contains a 5? untranslated region(UTR)with 167 bp,a 3? UTR with 2,374 bp,and an open reading frame(ORF)with 4,011 bp,which consists of 21 exons and encodes 1,336 amino acid residues.The deduced amino acid sequence for the BmAnillin ORF has a specific region of Anillin family,namely AHR in the C-terminus,which covers an AH domain and a PH domain.We identified the Anillin genes in several organisms by a BLAST search,and evaluated the molecular evolutionary relationships among the Anillin genes.The Anillin gene exists as a single copy in silkworm and other surveyed organisms,indicating its functional conservation in all surveyed organisms.We further constructed a phylogenetic tree for the Anillin genes from silkworm and other organisms by using their complete coding sequences.It is obvious that the topologies of the tree were closely consistent with classical taxonomic divergence.For example,all Anillin genes from the order Lepidoptera,including silkworm BmAnillin gene,were firstly grouped together before grouping with other insect Anillin genes.The phylogenetic tree showed that the BmAnillin was firstly grouped with other Lepidoptera insect Anillin,and then grouped with another insect,last grouped with vertebrates Anillin,suggesting that the function of Anillin is conserved in different species.We profiled spatio-temporal expression patterns of the BmAnillin gene in multiple tissues during silkworm larval development.Quantitative RT-PCR analysis found that on the third day of the last larval instar,the BmAnillin gene exhibited relatively high expression in the tissues,such as gonads and hemocyte,in which cells undergo mitotic cycle.However,the BmAnillin gene presented a very low expression in endocycling silk gland in which cells only execute endoreplication but have no mitosis.We further analyzed the time-course changes of BmAnillin expression in gonad and silk gland during silkworm larval development.Intriguingly,we observed that BmAnillin was persistently expressed in different developmental stages of the gonads and expression could be highly detected during larval molting in silk gland without mitosis,and maintained an undetectable level during the feeding period.2.The function of silkworm BmAnillin in cytokinesisWe performed RNAi-mediated knockdown by transforming the dsRNA against BmAnillin gene into in BmN4-SID1 cells,which is derived from silkworm ovary,to investigate the roles of the BmAnillin gene in silkworm.Immunostaining analysis demonstrated that compared with the control with a treatment of the dsRNA against the RFP gene,BmAnillin RNAi resulted in abnormal cell division and the abnormal cells were up to 21.1%.In detail,chromosome separation exhibited a disorder during mitosis prophase following BmAnillin knockdown,such as multi-polarization.Moreover,BmAnillin knockdown also caused the loss of the F-actin filament at cleavage furrow in anaphase.Taken together,these results revealed that BmAnillin is essential in chromosomal segregation and the final cytokinesis in silkworm BmN4-SID1 cells.3.Aurora B-mediated phosphorylation of Anillin and its function during mitosisThrough Western Blot analysis of migration of Anillin on SDS-PAGE gel after treatment with different conditions,we found that significant phosphorylation occurred during the mitosis of Anillin.Further experiments showed that Aurora B kinase is involved in the phosphorylation of Anillin during mitosis.Inhibiting the activity of Aurora B kinase causes abnormal localization of Anillin on the cell membrane,leading to abnormal cell division.Through co-location analysis of endogenous Anillin and Aurora B and immunoprecipitation analysis,we confirmed that there was an interaction between Anillin and Aurora B in vivo.Combined with the phosphorylation site prediction website and relevant literature,we identified Ser485,Thr1071 and Thr1079 as potential phosphorylation sites of Aurora B kinase at Anillin.Further experiments showed that phosphorylation of Ser485 and Thr1071 was necessary to ensure tight arrangement of chromosomes and correct spindle assembly in metaphase of mitosis.Phosphorylation of Thr1079 is involved in the regulation of nuclear localization of Anillin during interphase and the timely recruitment of Anillin to the cell membrane during cytokinesis.These results suggest that Aurora B may be directly involved in the regulation of cytokinesis by phosphorylation of Anillin.