Identification And Functional Study On LncRNA Of Bmdsx Gene Locus In Bombyx Mori

Sexually dimorphic is a primarily characteristic of sexual production organisms,which show great differences in physiology,phenotype as well as behavior between female and male individuals.Sex determination and sex differentiation,a basic and key scientific problem,significantly involves in individual development and racial reproduction.The male and female individuals of silkworm have obvious differences in economic efficiency.Compared with the female,the male silkworm has preferable silk yield and quality.Hence one can see that the male has great economic benefit,so study of sex determination regulation mechanism is of great advantage to strengthen the economic value.on the other hand,Bombyx mori is a kind of Lepidopteran insect and most pests in agriculture and forestry are Lepidoptera insects.All in all,the study on sex determination of Bombyx mori is of great significance to provide a theoretical basis for better understanding of pest control.In Bombyx mori,Bmdsx gene is at the bottom of sex determination cascade.The premRNA of Bmdsx is certainly alternatively spliced and transcribed into female-specific（BmDSXF）and male-specific（BmDSXM）protein.The open reading frame of Bmdsx contains five exons,and exon 3 and exon 4 are specifically existed in females by sexspecific splicing but not in males.In 2008,researchers identified a regulatory element,which located at exon 4 of Bmdsx and function as exonic splicing silencers regulate malespecific splicing.CE1 specifically binds to upstream factor BmPSI and caused sexspecific splicing of Bmdsx pre-mRNA.And another regulatory factor BmIMP can bind to BmPSI and facilitate male-specific splicing through enhancing the binding activity of BmPSI and CE1.Increasing studies have demonstrated that long non-codin RNA（lncRNA）play a vital role in gene regulation.In 2016,it was reported that there were lncRNAs in Bmdsx gene loci.And are these lncRNA responsible for the regulation of sex-specific splicing of Bmdsx?In this study,we performed RT-PCR and identified a novel lncRNA at Bmdsx gene loci,termed Bmdsx-AS1,and whose expression is developmentally regulated sex-specific splicing of Bmdsx.The main results are as follows: 1、 Cloning and identification of lncRNA in Bmdsx gene lociTotal testis RNA were extracted with silkworm on the third day of fifth-instar larvae,then synthesis of cDNA from total testis RNA.The primers were designed for the lncRNA sequence of TCONS₀0200625 that given from article,then performed PCR amplification.We were identified three lncRNAs within Bmdsx gene loci via sequencing and sequence analysis,and termed Bmdsx-AS1、Bmdsx-AS2 、Bmdsx-AS3 in turn.BmdsxAS3 is sequence of TCONS₀0200625 that mentioned in the article.Surprisingly,BmdsxAS1 is overlapped with partial sequences of exon 4 and intron 3 of Bmdsx.RT-PCR and fluorescence quantitative PCR were carried out to detect expression pattern of Bmdsx-AS1 in different tissues.The result revealed that Bmdsx-AS1 is preferential to expressed in testis and low expression level in other tissues,suggesting that possible involvement of Bmdsx-AS1 in testis functions.To further study characterization of Bmdsx-AS1,we were performed Fluorescence in situ hybridization（FISH）in cell and tissue level aims at analysis localization of BmdsxAS1.Fluorescence labeled probe was prepared by specific sequence of Bmdsx-AS1.Fluorescence signals were detected in cytoplasm and nucleus of BmE cells,indicating that Bmdsx-AS1 involved in differ functions in cytoplasm and nucleus.The results of FISH showed that Bmdsx-AS1 had strong fluorescence signal in testis.Cytoplasm and nucleus of spermatozoa were all detect fluorescence signals,suggesting that Bmdsx-AS1 plays different functions in spermatozoa.2、 Study the function of Bmdsx-AS1Bmdsx-AS1 is exactly overlapped with partial sequences of exon 4 and intron 3 of Bmdsx,while exon 4 of Bmdsx contains an important regulatory element CE1.For study function of Bmdsx-AS1,RNAi assay was carried out to down-regulate expression level of Bmdsx-AS1.The double-stranded RNA（dsRNA）was transcribed and synthesized in vitro,and the dsRNA were injected into male and female individuals on the first day of fifth-instar larvae.Total RNA was extracted and reverse transcribed into cDNA to detect splicing pattern of Bmdsx after 48 h.The result of PCR showed that dsRNA caused a consistent other splicing pattern and reduce male specific splicing of Bmdsx in males,suggesting Bmdsx-AS1 refers to regulation of sex-specific splicing of Bmdsx.To further study whether Bmdsx-AS1 involves in regulating male-specific splicing of Bmdsx,we tried transgene technology to increase Bmdsx-AS1 expression in silkworm.Bmdsx-AS1 was examined at newly mo lted fifth larva by qPCR.Bmdsx-AS1 was abundantly expressed in female and male transgenic individuals.Up-regulation of BmdsxAS1 by transgenic approach significantly increased male-specific splicing of Bmdsx in females.The nuclear extracts from HeLa cells revealed that female-specific Bmdsx mRNA is the default splicing product,while male-specific form of Bmdsx is due to regulation of splicing silencer.The treatment by the above approaches affect sex-specific splicing of Bmdsx,suggesting a relative specificity of Bmdsx-AS1 may function as exonic splicing silencer in responsible for male-specific splicing of Bmdsx.Vitellogenin gene（Vg）and Storage protein 1（SP1）is a direct target gene of BmDSX protein.Increasing expression of Bmdsx-AS1 caused the expression of Vg and SP1 was decreased significantly in male and female,indicating that Bmdsx-AS1 may directly or indirectly regulate the expression of Vg and SP1.When the phenotype of transgenic silkworm was observed,it was found that the testis of male silkworm was larger than that of normal silkworm,which indicated that Bmdsx-AS1 might affect the development of testis.3、 The screening of Bmdsx-AS1 binding proteinAlternative splicing is a complex biological process that need multiple proteins and regulatory elements.Therefore,it is imperious to identify other factors Bmdsx-AS1 responsible for male-specific splicing of Bmdsx.Previous chapter showed that BmdsxAS1 refers to male-specific splicing of Bmdsx,and it may coordinate the alternative splicing of Bmdsx by binding to other splicing factors.RNA-protein pull down was performed to screen Bmdsx-AS1 in testis,and further analysis of the screened proteins was carried out to identify relevant splicing factor.The screened proteins were subjected to LC-MS / MS analysis,in which heterogeneous nuclear ribonucleoprotein H（hnRNPH）protein was identified.The hnRNPH protein belongs to the hnRNPs protein family and its mainly function is participated in alternative splicing.Therefore,we regard hnRNPH protein as a candidate factor.4、 Verification of HnRNPH protein binding to Bmdsx-AS1The accession number of heterogeneous nuclear ribonucleoprotein H protein of silkworm in NCBI is XP₀21203948.1.This gene encodes 350 amino acids,molecular weight is 38 kDa,it’s isoelectric point of 6.5 and it is an acidic protein.Primers were designed according to the CDS sequence of hnRNPH.The hnRNPH gene was successfully cloned from the cDNA of silkworm on the third day of the fifth-instar,and then cloned into pET-28a（+）vector to construct the recombinant prokaryotic expression vector.At last the recombinant vector was transformed into BL（DE3）expression strain for prokaryotic expression.HnRNPH protein can express active protein in the supernatant at 16℃,and then obtained relatively pure target protein by protein mass expression and affinity chromatography.Previous study revealed that hnRNPH is functionally conserved and participates in the regulation of alternative cleavage by binding to the poly（rG）sequence.After further sequence analysis,there is a poly（rG）sequence on Bmdsx-AS1,and then this poly（rG）sequence was prepared for a biotinylated RNA probe.We performed an in virto electrophoretic mobility shift assay to testify whether hnRNPH would bind to the poly（rG）sequence positioned in Bmdsx-AS1.The experimental results showed that hnRNPH protein could specifically bind to the poly（rG）,suggesting that hnRNPH may function by forming a complex with the poly（rG）positioned in BmdsxAS1.In summary,using a variety of experimental techniques,we identified a novel lncRNA within the region of the Bmdsx in silkworm,and termed Bmdsx-AS1.To our knowledge,it is the first lncRNA whose transcript composition is testis-specific in silkworm.RNAi and transgenic technology analysis revealed that Bmdsx-AS1 function as a splicing inhibitor responsible for male-specific splicing of Bmdsx.This study will provide new ideas for the study of sex determination and other lncRNA functions in silkworm.