LncRNA Platr10 Regulates Stem Cell Pluripotency At The Translation Level Through The EIF4G2-MAP3K3 Pathway

Background and objectives:In 1981,researchers isolated embryonic stem(ES)cells from early mouse embryos successfully.They quickly recognized the research and clinical application potential of ES cells,due to the unique features of self-renewal and differentiation into any kind of cells in the body.However,the establishment of the first human ES cell line 17 years later sparked a huge ethical firestorm,which hindered the research and clinical usage of ES cells.Until 2006,based on somatic cell nuclear transfer experiments,the Yamanaka team in Japan introduced Oct3/4,Sox2,c-Myc and Klf4(OSKM)factors into mouse fibroblasts,and induced the cells into induced pluripotent stem cells(iPSCs).The usage of iPSCs avoids the ethical issues and immune rejection of allograft,and brings stem cell research into a new era,thus winning the 2012 Nobel Prize in Physiology or Medicine.The discovery of iPSCs was the biggest innovation in regenerative medicine.Since then,more than 100 clinical trials of iPSCs have been registered worldwide until now,and cell therapy programs for iPSCs-derived cells are also carried out.But limitations of reprogrammed cells still exist,such as low induction efficiency,long cycle of induction and poor proliferation ability,which impede the clinical application of iPSCs and are urgent problems to be solved.Therefore,since the discovery of ESCs,researchers have dedicated to explore the mechanisms of stem cell pluripotency and reprogramming,and the number of studies has increased sharply.The pluripotency and self-renewal of stem cells are controlled by an extremely complex network composed of intracellular and extracellular signaling pathways.To date,most studies have focused on how these characteristics of stem cell are regulated at the transcriptional level,including DNA methylation,histone modification,transcription factors,RNA splicing,and metabolism-epigenetic interactions,etc.However,in recent 30 years,an extensive body of literature has shown that the translation process may also play a crucial role in the regulation of stem cell fate.Long noncoding RNAs(lncRNAs)are important regulatory factors that can participate in epigenetic and transcriptional regulation of stem cells by shaping chromatin conformation,binding transcription factors and chromatin modification factors.However,their regulatory effects and mechanisms on stem cells at the posttranscriptional and translational levels remain unclear.Researches have shown that most lncRNAs will be exported to the cytoplasm after generation,and they can bind ribosomes.These ribosome-associated lncRNAs can regulate translation process through their non-coding functions.Therefore,lncRNAs have great potential in translation regulation and may control the fate of stem cells at the translational level.In order to map ribosome-associated pluripotent lncRNAs,we used RNA-Seq of polysomes and whole cells to screen lncRNAs that are enriched in mouse E14 cell polysomes and iPSCs by using fibroblasts as a control.Using these strategies,we identified lncRNA Platr10.Then we explored the expression patterns,functions and mechanisms of Platr10 to clarify its role in stem cell pluripotency and reprogramming.In this study,we revealed a new picture of how lncRNAs regulate stem cell pluripotency at a translational level.Methods:1.Ribosomes were extracted using sucrose-gradient centrifugation,and curves of ribosomal fractionation were drawn.RNA of polysomes was extracted and RNA-Seq was performed(Ribo-Seq).At the same time,RNA of the whole cell was extracted and RNA-Seq was also performed.By integrating these two data,we identified lncRNA Platr10 that was highly enriched in the polysomes of E14 embryonic stem cells.2.In order to explore the correlation between Platr10 expression and cell pluripotency,PCR and/or qPCR were used to confirm the expression of lncRNA Platr10 in E14 cells,fibroblasts,and other differentiated tissues,as well as during the embryoid body(EB)formation and in the LIF/2i culture.3.Polysome profiling and qPCR were used to find the distribution of Platr10 in different ribosomal fractions.4.To investigate the effect of Platr10 on stem cell pluripotency,Platr10-knockdown plasmid was constructed,and was transfected into E14 cells.The expression of pluripotent genes was detected by qPCR and Western blot.5.Platr10-overexpression(Platr10-OE)plasmid was constructed,and Platr10-OE and control vector plasmids were transfected into DOX-OSKM-MEFs,respectively.qPCR was used to test the level of Platr10.NANOG immunofluorescent staining was used to identify iPSC clones.Alkaline phosphatase staining and assay were used to explore whether Platr10 could promote reprogramming.6.To explore the mechanism of Platr10,surface sensing of translation(SUn SET)assay was used to measure the global protein synthesis in Platr10-knockdown E14 cells.7.The changes of pluripotent signal pathways after Platr10 knockdown were detected by Western blot.Western blot and qPCR were used to detect the expression of MAP3K3 in Platr10-knockdown E14 cells,clarifing the regulatory role of Platr10 on key proteins affecting cell pluripotency at the translation level.8.To explore the underlying mechanisms,RNA pull-down and mass spectrometry were performed to search for Platr10-interacting proteins.9.The specific binding of eIF4G2 and Platr10 was verified by RNA binding protein immunoprecipitation(RIP).Western blot was used to figure out the distribution of eIF4G2 in different ribosomal fractions.And crosslinking-immunoprecipitation(CLIP)was used to determine the specific binding sites of eIF4G2 on Platr10.10.The binding of eIF4G2 and Map3k3 mRNA was confirmed by RIP.Western blot was used to determine the expression of eIF4G2 in Platr10-knockdown E14 cells.11.Bicistronic reporter system was used to determine whether the 5’-UTR of Map3k3 has the IRES activity,and the effect of Platr10 on the Map3k3 5’-UTRmediated translation.The result may indicate whether Platr10 can specifically regulate Map3k3 translation by influencing Map3k3 5’-UTR-mediated cap-independent translation initiation.Results:1.LncRNA Platr10 was identified by Ribo-Seq and RNA-Seq as a ribosomeassociated pluripotent lncRNA.Ribo-Seq showed that Platr10 was highly enriched in the polysomes of E14 cells.RNA-Seq showed that Platr10 was highly expressed in iPSCs.2.Using qPCR,we showed that Platr10 was abundantly expressed in E14 cells,but not expressed in fibroblasts and other differentiated tissues.The expression of Platr10 gradually decreased during the embryoid body(EB)formation,and it increased in LIF/2i culture.The change trend of Platr10 was consistent with those of pluripotent genes.These results suggest that the expression patterns of Platr10 were related to cell pluripotency.3.Polysome profiling showed that Platr10 was mainly distributed in 40 S small ribosomal subunit and light polysomes.4.After knockdown of Platr10 in E14 cells,the mRNA and protein levels of pluripotent genes were down-regulated,suggesting that Platr10 is important for the maintenance of E14 cell pluripotency.5.The overexpression of Platr10 in DOX-OSKM-MEFs increased the number of iPSC clones and the activity of alkaline phosphatase during the reprogramming process,and enhanced the reprogramming efficiency.6.SUn SET assay showed that Platr10-knockdown decreased the global protein synthesis in E14 cells.7.Platr10 knockdown in E14 cells significantly reduced the phosphorylation level of AKT and increased the phosphorylation level of MEK.8.Platr10 knockdown resulted in an increase of protein level of Map3k3,but no significant changes in its mRNA level in E14 cells.The expression of Map3k3 increased gradually during EB formation.These results suggest that Platr10 inhibits the expression of pro-differentiation protein MAP3K3 at the translation level.9.RNA pull-down and mass spectrometry analysis showed that the Platr10-interacting proteins were enriched in pathways including ribosome biogenesis in eukaryotes,PI3K-AKT signaling pathway,and translational initiation.The eukaryotic translation initiation factors(e IFs)that may bind to Platr10 were screened by mass spectrometry analysis combined with the databases.10.RIP-qPCR showed that Platr10 specifically bound to eIF4G2,and CLIP-qPCR as well as database predictions identified the 776-827 nt of Platr10 as the potential binding site.11.RIP-qPCR showed that eIF4G2 bound to Map3k3 mRNA,and it mediated the translation of Map3k3.Knockdown of Platr10 had no effect on the protein level of eIF4G2.12.The results of the bicistronic reporter system assays showed that the 5’-UTR of Map3k3 had the IRES activity,which could mediate the translation of Firefly luciferase(Fluc).The overexpression of Platr10 significantly reduced the activity of Fluc,suggesting that Platr10 could specifically regulate the translation of Map3k3 by inhibiting Map3k3 5’-UTR-mediated cap-independent translation initiation.Conclusions:1.The expression of lncRNA Platr10 is closely related to the pluripotency of stem cells.2.LncRNA Platr10 in the cytoplasm binds to ribosomes.3.LncRNA Platr10 is required for the maintenance of stem cell pluripotency.Knockdown of Platr10 leads to the down-regulation of pluripotent gene expression.Overexpression of Platr10 enhances the process of reprogramming.4.LncRNA Platr10 regulates the global protein synthesis in E14 cells,and specifically inhibits the translation of the pro-differentiation protein MAP3K3.5.Mechanistically,Platr10 regulates stem cell pluripotency through the eIF4G2-MAP3K3 pathway.Platr10 specifically interacts with eIF4G2 and inhibits the capindependent translation of Map3k3 mediated by eIF4G2 via the 5’-UTR of Map3k3 mRNA.