Identification And Functional Study Of RNA Binding Proteins Of Nanog And Oct4

Embryonic stem cells have a unique capacity to self-renew and differentiate into all cell types.Several transcription factors are required for pluripotency,including Oct4,Nanog and Sox2,which collaborate and form a core regulatory circuitry to control the ES cell transcriptional network.This transcriptional regulatory circuitry,collaborated with other transcription factors,play a fundamental role in maintaining stemness and pluripotency of ES cells ^1,2.Recent work indicates that the posttranscriptional control of eukaryotic gene expression is much more influential than previously thought,on every step of messenger RN A（m RNA）metabolism in an m RNA-specific manner,including selectively splicing,nuclei transporting,cleavage,location and translation.Intereastingly,there is often little or no transcription in the early embryo,a time in development when many crucial decisions such as cell division and cell-fate determination are made.For this reason,the control of gene expression in embryogenesis and later developmental processes such as stem-cell proliferation often relies on post-transcriptional control.Our purpose is to explore the possible mechanism of stem cell pluripotency maintenance and differentiation through studing on post-transcriptional regulation.Through their metabolism,m RNAs are escorted by a set of associated RNA binding proteins（RBPs）,some of which remain stable bound with target RNAs.So we decided to identify the RNA binding proteins ascciating with the key transcription factor-Nanog and study their functional roles played in maintaining the pluripotency of embryonic stem cells,through which we explore the m RNA metabolism of Nanog and the network regulating Nanog and other key factors which are important in controlling the pluripotency and differentiation of ES cells.By the way,we also identified the RNA binding proteins interacting with Oct4 m RNA,which sets a base for exploring the regulation network of Oct4.We applied an in vitro approach namely“Strepto Tag”affinity purification stretagy to isolate RNA binding proteins interacing with functional m RNAs of Nanog and Oct4,after Mass Spectra,we got 118 RBPs associated with Nanog m RNA and 114 RBPs associated with Oct4 m RN A.Then we found 8 most reliable RBPs from Nanog RBPs list as our candidates for further validation and functional study.Western Blot demonstrated these 8 candidates actually exist in the elution from affinity purification,which represents the complexes associated with Nanog RNA.RN A immunoprecipitation results showed that interaction relationship between the 8 candidates and Nanog RNA also exists in vivo.We also applied an in vivo RBPs isolation approach named MS2-Bio TRAP,followed by Western Blot,results demonstrated the RBPs candidates were also found in the elution from in vivo RBPs isolation approach,further indicating these RBPs interact with Nanog m RNA in vivo.After that we selected two RBPs which had outstanding performance in RNA immunoprecipitation,YBX1 and ILF3,as our further functional study candidates in maintaining the pluripotency and regulating the differentiation of embryonic stem cells.RNAi experiment was performed to knock down the expression of YBX1 and ILF3,we found that the expression of pluripotency-related genes were reduced heavily in YBX1-KD and ILF3-KD cells,and YBX1 and ILF3 gene expression knockdown leads the ES cells differentiating into mesoderm cells.Genome-wide microarray profiling of ILF3-KD cells showed that a large number of genes whose expressions heavily changed in ILF3-KD cells analyzed by Microarray profiling hit the factors whose encoding genes are downstream targets of the core pluripotency factors Nanog,Oct4,and Sox2.This result shows that ILF3 play an important role in regulating the downstream target genes of Nanog,Oct4,and Sox2,which further demonstrates that RNA binding proteins may indirectly regulate the downstream target genes of key transcription factors by directly interacting with RNA targets of these TFs.As ILF3 is also the downstream target gene of Oct4 and Sox2,our data further indicates the transcription of Nanog may be regulated by Oct4 and Sox2through controlling the expression of ILF3,which further regulates the translation of Nanog by interacting with elements within 3’UTR,that’s may be an evidence to explain a fact that the transcription of Nanog is mainly dependent on Oct4 and Sox2.