The Functional Study Of Rbm14 In Early Mouse Embryonic Development

Alternative splicing occurs widely in eukaryotes during the maturation of pre-mRNA.In human,it is estimated that more than 95%of genes are alternatively spliced.By alternative splicing,multiple proteins are coded by a single gene,thus significantly augments the biodiversity of proteins that can be coded by the genome.Previous reports have demonstrated the implication of abnormal alternative splicing events in developmental defects.Alternative splicing is regulated by a system of trans-acting RNA binding proteins that bind to cis-acting elements on pre-mRNA transcripts.As the core trans-acting unit that mediates pre-mRNA splicing,spliceosome is composed of five small nuclear RNAs(snRNAs)and a variety of RNA binding protein factors.In addition to the spliceosome,any other accessory RNA binding proteins,especially RRM family proteins,are involved in the regulation of temporospatial-specific alternative splicing events.RBM14 is an RNA binding protein that is reported to be involved in various cellular functions and processes,such as transcription coactivation,spindle assembly,virus infection,and stem cell differentiation.Recently,we had reported that RBM14 plays an important role in pluripotency maintenance and mesoderm development of mouse embryonic stem cells(ESCs).However,the biological functions of RBM14 in mammalian embryonic development in vivo remain unclear.Using the clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 and embryo microinjection techniques,we generated the Rbml4 knockout allele.We mated the heterozygous male and female mice and discovered that homozygous knockout of Rbm14 in mice was embryonic lethal.Further histological analysis revealed that the depletion of RBM14 resulted in cell cycle arrest and apoptosis of epiblast cells and that the mouse embryonic development was arrested at the gastrulation stage.We also demonstrated that RBM14 was involved in the alternative splicing of DNA damage response(DDR)-related genes.Depletion of RBM14 resulted in alternative splicing of these genes and subsequently the accumulation of DNA damage in mouse ESCs.Hence,our study suggests that RBM14 plays a key role in maintaining the genomic DNA integrity by regulating the alternative splicing of DDR-related genes during early mouse embryogenesis.The key novel findings of our study include:(1)Homozygous knockout of Rbm14 in mice leads to embryonic lethality.(2)Depletion of RBM14 causes cell cycle arrest and apoptosis in post-implantation epiblast cells and leads to disruption of gastrulation during early embryonic development.(3)Depletion of RBM14 results in DNA damage accumulation in the mouse ESCs.(4)RBM14 interacts with splicing factors and regulates DDR-related genes through alternative splicing.In conclusion,we have provided compelling evidence to demonstrate that RBM14-mediated alternative splicing of DDR-related genes is essential for maintaining the integrity of genomic DNA during early mouse embryogenesis.