| Background and objectives: When the concept of stem cells was first proposed,it did not attract widespread attention.Until 1997,when the sheep Dolly was born,the stem cell field successfully attracted worldwide attention.However,there are ethical issues about the source of embryonic stem cells.If human embryonic stem cells are used for research,some people think that this is a disregard for life and will lead to a series of social problems.Therefore,stem cell research has also fallen into a bottleneck period.It was not until 2006 that Japanese scholar Yamanaka and his colleagues overexpressed transcription factors(Oct3/4,Sox2,Klf4,and c-Myc,OSKM)into mouse embryonic fibroblasts for the first time and transformed them into induced pluripotent stem cells(iPSCs).The emergence of iPSCs solves the ethical problem of the source of embryonic stem cells,but the low efficiency of reprogramming to generate iPSCs also limits its application in the field of regenerative medicine.In the decade since the iPSCs were discovered,efforts have been made to explore the mechanisms of reprogramming and how to improve reprogramming efficiency.Studies have found that the addition of small molecular compounds such as vitamin C can promote reprogramming.The addition of novel transcriptional regulators,such as various proteins and RNAs,can affect important signaling pathways and regulate epigenetic modification to improve the efficiency of reprogramming.Long non-coding RNA(lncRNA)can regulate different cell biological processes.Previous studies have shown that lncRNA can regulate epigenetic changes such as gene imprinting and chromatin remodeling,and plays an important role in pluripotent maintenance and reprogramming.However,previous researches prove that lncRNA can regulate the fate of stem cells mainly at the transcriptional level instead of translation.Some people have proved that stem cells proliferate rapidly and require high translation efficiency to maintain their active pluripotent state.Inhibiting the translation of stem cells will lead to a decrease in the expression of some important unstable proteins such as Nanog and other stemness factors,resulting in cellular differentiation and therefore regulation of the translational level of stem cells is also crucial.Studies have reported that lncRNA binds to ribosomes,suggesting that it may regulate the stem cell translation.In order to search for pluripotent lncRNAs that may regulate the translation process,we extracted RNA from the polysomes of different cells and screened a new lncRNA with high expression in embryonic stem cells by RNA-Seq,and named it ribosomeassociated lncRNA 1(Rslnr1).We propose that Rslnr1 may regulate stem cell pluripotency at the translation level,promoting pluripotency maintenance and reprogramming.A series of studies are needed to explore the expression,localization,function and mechanism of Rslnr1.Through this study,we intend to propose a new idea that lncRNA can regulate stem cell pluripotency through translation,and gain a deeper understanding of the reprogramming mechanism.Methods: 1.We used sucrose gradient centrifugation to separate different components of ribosome,and then draw the polysome profiling curves.Polysomes RNA was extracted and RNA-Seq was sent to screen out the highly expressed lncRNA Rslnr1 in mouse embryonic stem cells.2.Real time quantitative polymerase chain reaction(RT-qPCR,qPCR)was used to detect the expression of Rslnr1 in stem cells,fibroblasts,and other differentiated tissues and organs;the Rslnr1 expression lever was also detected during embryoid body(EB)formation.3.The sublocation of Rslnr1 in cells was determined by RNA fluorescence in situ hybridization(FISH)and cytoplasmic and nuclear fractions analysis;and qPCR was used to explore the Rslnr1 distribution of different ribosome components.4.The Rslnr1 knockdown vector was constructed and transferred into stem cells,and the pluripotent factors were detected by western blot(WB)and qPCR.Meanwhile,the proliferation ability of stem cells was detected by clone formation assay and CCK8 proliferation assay.5.The Rslnr1 overexpression vector was constructed.And the overexpression group(Rslnr1-OE),random control group(CT-OE)and blank control group(Vector)were set during the reprogramming process.qPCR was used to detect the difference of pluripotent gene expression between different groups.Nanog immunofluorescence staining and alkaline phosphatase staining were used to determine whether Rslnr1 could promote reprogramming.6.Rslnr1 was overexpressed in fibroblasts to explore its influence on stem cell genes.7.Surface sensing of translation(Sunset)was used to explore the protein synthesis efficiency after Rslnr1 knockdown.8.Polysome shift was used to explore the protein synthesis efficiency of specific genes.9.RNA pull-down assay was performed and mass spectrometry was performed to search for proteins interacting with Rslnr1.10.The binding of eIF4G1 and Rslnr1 was further verified by RNA immunoprecipitation(RIP).Results: 1.We discovered Rslnr1 was a novel lncRNA that located on the X chromosome with a length of 479 bp.2.Rslnr1 was highly expressed in stem cells but not in fibroblasts and other differentiated tissues and organs.The expression of Rslnr1 was down-regulated with embryoid differentiation,and the trend was consistent with pluripotent factors.3.The result showed that Rslnr1 was mainly located in the cell cytoplasm;and it was expressed not only in polysome fraction,but also in monosome fraction.4.Knockdown of Rslnr1 in stem cells resulted in down-regulation of the m RNA and protein levels of pluripotency factor Oct4,Sox2 and Nanog;and the ability of cell clone formation and proliferation were weakened.5.After the overexpression of Rslnr1 during reprogramming,the stemness gene expression,Nanog immunofluorescence staining positive iPSCs,and alkaline phosphatase staining were all stronger than those in the control group,showed that Rslnr1 promoted reprogramming.6.Overexpression of Rslnr1 in fibroblasts had no significant effect on stemness gene expression.7.We used Sunset method to prove that the overall protein synthesis efficiency decreased after Rslnr1 knockdown.8.Using "polysome shift" method,we found that Sox2 m RNA moved from the "heavy polysome" with higher translation efficiency to the "light polysome" with reduced translation efficiency after Rslnr1 knockdown,proved that the translation efficiency of Sox2 decreased.9.Rslnr1 RNA pull-down and mass spectrometry analysis showed that the proteins interact with Rslnr1 were enriched in pathways related to ribosome synthesis and translation,and Rslnr1 may bind to various translation initiation factors such as eIF4G1.10.RIP-qPCR proved that Rslnr1 can indeed bind to eIF4G1.Conclusions: 1.LncRNA Rslnr1 is extracted from the polysome fraction of ribosome,mainly located in the cytoplasm,highly expressed in mouse embryonic stem cells,not expressed or lowly expressed in mouse fibroblasts and other tissues and organs,and decreases with cell differentiation.2.LncRNA Rslnr1 is essential for stem cell pluripotency maintenance,and knockdown of Rslnr1 leads to cell differentiation.And it can promote cell reprogramming to improve reprogramming efficiency.3.LncRNA Rslnr1 plays an important role in the translation of stem cells,and knockdown Rslnr1 can reduce the overall protein especially the Sox2 synthesis efficiency.LncRNA Rslnr1 functions by interacting with eIF4G1. |
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