Validation Of Mouse Rbm15 Core Promoters And Preliminary Exploration Of Transcriptional Regulators

N6-adenosine methylation,referred to as m6A methylation,is the most conservative and extensive modification of RNA.It widely exists in m RNA,ribosomal RNA,and long noncoding RNA.It can regulate the processes of RNA export,variable splicing,translation,degradation,and so on.It is an important link in the life process and is also the current research hotspot.M6 A has three processes of writing,erasing and reading.RNA binding motif protein15(RBM15)is a component protein of the writer complex.It participates in important regulatory processes such as selective splicing of m RNA and inactivation of X chromosome mediated by Xist RNA,and plays an important role in maintaining the homeostasis of hematopoietic cells,nerve and brain development.Although there have been many studies on the function of RBM15,the transcriptional regulation mechanism of the Rbm15 promoter is still unclear.Exploring the transcriptional regulation mechanism of the Rbm15 promoter will help to further understand the regulatory mechanism of m6 A.The research team found in the early stage that the m RNA expression level of RBM15 gene in different tissues of the regenerative organ of red deer(Cervus canadensis)is in the order of antler skin>cartilage>mesenchymal>prochondral.However,due to the immaturity of cell platform technology related to antler,further genetic and cellular molecular biology research cannot be conducted in antler cells.For the more,mouse is a commonly used model species in biological research,with a high degree of similarity to the human genome and rich research data.Therefore,this study used prediction software and web pages to predict the core promoter,transcription factors,and transcription factor binding sites of mouse Rbm15.In 3T3 and 293T cells line,real-time quantitative polymerase chain reaction(QPCR),Western Blot(WB),Dual Luciferase Reporter Gene Assay and other methods were used for research.The main research results are as follows:(1)A luciferase vector with segment-by-segment truncation of the mouse Rbm15 promoter was constructed,and the core promoter of the mouse Rbm15 gene was found,in the region of-347 to-171 upstream of the starting codon.(2)The transcription factors that regulate mouse Rbm15 were predicted,the possible binding sites of TRIM28 on the Rbm15 promoter were predicted,and the inhibitory effect of TRIM28 on the transcription of mouse Rbm15 was verified in 3T3 cells.(3)TRIM28 was found and verified to promote transcription levels and protein expression levels of human Rbm15 within the 293 T cell line.(4)POU5F1 was found and verified as a transcriptional activator of human RBM15 within the 293 T cell line,promoting the expression of Rbm15 at the m RNA and protein levels.The study of the promoter of Rbm15 gene in this study lays a foundation for further research on the regulation of Rbm15 gene,aiming to provide a new way to study the regulation of m6 A methylation and a new idea for the study of diseases involved in m6A methylation.