The Research Of The SRSF1 Interact With Related Puberty MRNA Based On The RNA Immunoprecipitation

In mammals,the puberty onset is regulated by hypothalamus-pituitary-gonadal axis(HPG axis),the secretion of various hormones direct or indirect regulated the puberty onset.In previous,our lab studied the regulation of the puberty onset based on inbred lines strains,C3H/HEJ(C3)and C57BL6/J(B6),which differed significantly in the pubertal timing.A genome-wide Short Tandem Repeat screen was performed in the two strains.Then we constructe the Interval-specific congenic strains,from which found mi R-505-3p as candidate gene related to puberty onset.The targets gene of mi R505-3p was predicted by the construction of stable expression mi R505-3p cell lines,future combined expression profiles and Targetscan.Then,we studied over expression encoding region of the SRSF1 gene in over expression mi R505-3p of the GT1-7 cell lines,in which we found the expression level of puberty related gene that were inhibited by mi-505-3p was recovery in some extent.So SRSF1 maybe involved in the regulation of the puberty related gene expression.Therefore,we want to know how the SRSF1 regulated the puberty by its downstream gene.First we performed the formaldehyde cross link RNA Immunoprecipitation(RIP)using Hela cell lines,in which we studied the conditions of the cell fixed,lysis buffer and sonication,the results indicated that SRSF1 protein and its interaction RNA were obtained in the condition of 0.1% formaldehyde cross-linking immunoprecipitation,1% NP-40 lysis buffer and low-level sonication.Using Real-time Quantitative polymerase chain reaction(q PCR)detect RNA enriched efficiency for SRSF1 based on 0.1% formaldehyde cross-linking immunoprecipitation.0.1% formaldehyde cross-linking immunoprecipitation are used to obtain the direct binding of RNA to SRSF1 protein in Hela cell;Using q PCR detect RNA Immunoprecipitation enriched efficiency for SRSF1 protein with Yeast Parental Strain(BY4741)as reference gene.Results showed that the direct binding of RNA to SRSF1 protein was successfully obtained based on 0.1% formaldehyde cross-linking immunoprecipitation;The differential expression of positive gene achieved 60 fold change in SRSF1 and Ig G sample.The quantification of the RNA Immunoprecipitation enriched efficiency was detected by q PCR with Yeast Parental Strain(BY4741)gene as reference.Next,we obtained the specific binding RNA to SRSF1 protein based on the method as above mentioned.Then,the RNA was detected using the western blot and Coomassie blue,selected the suitable specific antibody to enrich efficient the RNA in vivo,and senting to sequencing.Then,we conducted the quality control for the raw dates of the sequencing in NGStookit and filter the reads in topahat,before mapping the reads to the mouse genome,and those reads were assembled and gained the expression level in cufflink.We selected those gene that maybe interact with SRSF1 protein based on expression level in SRSF1 and Ig G sample,which standard is over 2 for expression level in two sample.We performed the GO and KEGG analysis for the candidate genes to obtained the function information based on the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes database.Finally,we found and identified 13 related puberty candidate genes.These studies lay the foundation for the research of the mechanism in puberty and providing the molecule targets for precocious,prevent,diagnosis and remedy of dysgenesis,as well as have a comprehensive understanding for maintaining reproductive ability and preventing dysgenesis in human beings.