Alternative Splicing Based On RNA-Seq Data In Mouse Neural Development

Alternative splicing is a major mechanism which regulates gene expression and generates much of the enormous protein diversity. It occurs to transcription process of pre-m RNA to mature m RNA. Alternative splicing is not only widespread in eukaryotes, but, splicing patterns of the gene in different stages and different tissues or organs are changing to regulate cell proliferation, differentiation, migration and apoptosis. Development of nervous system is extremely complex and rigorous process which relates to protein variety. Due to alternative splicing to play an important role in various stages of nervous system development, changes in splicing modes for specific periods and cells could result in function abnormality of nervous system. Smek protein having high conservation, which could regulate Wnt/Ryk signal pathway that affects molecular mechanisms of mice embryonic cerebral cortex early development, and other functions. Proliferation and differentiation of neural stem cells in the early mouse embryos are crucial for normal neural development, therefore, this paper studies alternative splicing events caused by knocking out Smek gene in the process of neural stem cell differentiation, and effect on neural development.Firstly, build a library contains poly(A) tail RNA which resource from neural stem cells and was single sequenced at 50 bp length by next-generation technology. These samples are divided into two groups: wild type and knockout Smek genes, separately exist three biological replicates. In addition, these sequenced data takes sampleof three time points Day0\Day2\Day6s for better study alternative splicing in embryonic neural stem cells differentiation processes(partners sequence RNA). Then, we pre-process this data, through align them to the whole genome and assemble the corresponding transcriptome and select genes\primary transcripts\isoforms have significantly different expression, which makes preparations for analysis of different expression and alternative splicing.Secondly, analyze significantly different expression of pre-processed sequenced data using cuffdiff. Filter genes which have lower variant expression, and their functions are analysized by gene ontology software GO to get impact on biological function caused by genes related to Smek. In addition to this, we also research various expression genes from two samples of different time points which result in functions changes. In the results, it could be found these different expression genes affect neurons formation, such as Egr2\Dlx2 participate in the formation of neurons, and Miat\Gsx1 take part in neuronal differentiation which expression values significant change.Thirdly, based on pre-processed sequenced data selects genes that occur alternative splicing by Jensen-Shannon different method, similarly, it needs to analyse these genes’ function of GO. At the same time, compared to other means which could identify signal alternative splicing modes, it can gain multiple modes by way of Jensen-Shannon different method. According to analysis results, an organism that missing Smek gene induces related genes occurring alternative splicing and produces various function protein, affects neural stem cells differentiation, such as Ank3\Clasp2 participate in cell morphogenesis involved in neuron differentiation, and Ppfibp2\Ncam1take part in neuron formation which translate into function protein other than normal organism.Finally, summarize relationships between biological function of Smek gene and neural stem cells differentiation. Through analysis alternative splicing caused by knockout Smek gene, on the one hand, it could verify action of Smek. On the other hand, study Smek playing an important role in neural stem cells differentiation. At the same time, research work in this paper can also lay a solid foundation for diagnosis and treat diseases related to nervous system diseases.