Loss Of Rbm15 Induces Hyper MTORC1 Activity And Impairs Hepatic Maturation

The liver plays an important role in modern biology and medical research because of their unique structure,function and important role in life activities.The liver is the largest digestive organ and plays the center role in metabolism.It is responsible for the secretion of bile,synthesis of plasma proteins,detoxification and glycogen synthesis and storage.The liver is a highly complex and ordered organ,mainly formed by hepatocytes,bile duct cells,stellate cells.Hepatocytes are the mainly cell type of liver and undertakers of liver function.Abnormal hepatic function or cell death can result in a variety of liver disease,it has become a major problem in human health.Therefore,it is important to find out the mechanism that regulates liver development and regeneration.mTOR,a mammalian target of rapamycin,gets its name because it can be specifically inhibited by rapamycin.mTOR protein is a typical serine/threonine kinase,which can form complex mTORC1/2 with RAPTOR(complex 1),RICTOR(complex2),LST8,and m Sin1,etc.This complex can phosphorylate downstream target genes,such as S6k1,4EBP1,Akt,and PKC.mTOR is the major regulatory factor for cell metabolism.It can respond to nutrients,growth signals and environmental pressures in the extracellular environment to regulate cell metabolism,proliferation,protein synthesis,immunity,aging and other life processes.Abnormal mTOR signaling has been linked to various human diseases,such as diabetes and cancer.Therefore,mTOR signal is a promising target for drugs development.Zebrafish is a model animal with a great prospect of scientific application.It is widely used in the field of organ development and regeneration due to its advantages of easy feeding,short sexual maturity,easy to observe and high similarity with human genome.In order to study liver development and regeneration,we construct a liver-specific transgenic line Tg(lfabp:Dendra2-NTR).This transgenic line can specifically label hepatocytes and can induce liver damage by adding Methazolamide to egg water.Based on this transgenic line,we can get the mutants which affect liver development and regeneration by using the positive genetic mutagenesis screening methods.In our experiment,we obtain a very promising mutant cq97,the confocal images show extremely weak and heterogeneous fluorescence in hepatocyte.Meanwhile,in situ hybridization results indicate that the expression of liver-specific genes are significantly reduced,but the development of pancreas and intestine are normal.These experimental results mean that the mutant is a liver-specific mutant.In order to check the early development and the early specialization of the liver in this mutation,we perform the Insitu experiment to check the expressions of hhex,foxa3,gata6 and prox1 at 60 hpf.The results demonstrate that the development of endoderm and the specification of liver are normal.Further antibody staining results show that the development of bile duct system of the mutant is normal,but the protein levels of two important liver transcriptional factors Hnf4 a and Prox1 are significantly decreased,indicating that the mutant gene specificity affects hepatic maturation.During organ development,cell proliferation and apoptosis are strictly regulated,and abnormal proliferation and apoptosis often bring negative effects to organ development.Antibody staining results of PCNA and TUNEL staining indicate that the mutation doesn't affect the proliferation and apoptosis of liver cells.Through mapping cloning,we find out the mutant gene rbm15.Rbm15 is an RNA binding protein that plays an important role in RNA methylation and m RNA nuclear export.The results of Insitu hybridization and fluorescence in situ hybridization show that the gene is highly expressed in the liver at5 dpf,which explains why the deletion of the gene affects liver development.In order to verify this result,we knock out the gene by CRISPR/Cas9,and successfully repeat the cq97 mutant phenotype.At the same time,over-expressing line Tg(Hsp70l:rbm15-flag)is constructed to overexpress Rbm15 protein in mutants by 38-degree heat shock for 30 min.48 hours post heat shock,confocal images show that the fluorescence signal of the mutants is restored,and the expression of hepatic genes are significantly increased.These results confirm that loss of Rbm15 induce liver maturation defects in the mutants.Since mTOR can affect protein synthesis,we check the protein level of mTORC1 downstream protein 4Ebp1.The antibody staining results show that the level of p-4Ebp1 in the mutant was significantly higher than wild type,suggesting that the abnormal activation of mTORC1 might be the cause of abnormal liver cell maturation.We then use rapamycin to suppress mTORC1 signaling and successfully rescue liver maturation defects in rbm15 mutant.Meanwhile,Hnf4 a protein levels and expression of liver-specific genes are also recovered.Combined with the above experimental results,our study firstly find that loss of RBM15 specifically affects liver maturation,but not affects the formation of liver bud and development of pancreas and intestine.Deletion of this gene leads to hyper mTORC1 activation and thus inhibits the maturation of hepatocytes.This result indicates that the activity of mTORC1 should be strictly regulated.This research is important for understanding the development mechanism of liver,especially for liver maturation.