MRBPome Capture Identifies RNA Binding Proteins As The Post-transcriptional Determining Factors In Regulating Spermatogonial Stem Cells

Post-transcriptional regulation plays a central role in the regulation of cell fates,which usually requires RNA binding protein(RBPs)to regulate certain cellular function.Studies have shown that RNA binding proteins play the important roles during the development of germ cells and gametes in mammals(from rodent mice to primates)and the imbalance of RNA-protein complex interactions can cause a variety of diseases or tissue disorders.Several studies have also shown that RNA-binding proteins are involved in the self-renewal,proliferation or differentiation of mouse spermatogonial stem cells(SSCs).However,the scope of RBPs in SSCs has still remained largely unknown.Therefore,it is essential to gain insights into RNA biology from an atlas of mRNA-binding proteins in SSCs for the analysis of post-transcriptional gene regulation.Recently a method of RNA-interactome capture has been developed in the Hela cells and we have further applied this technique in mouse SSCs.By capturing RBPs interacted with mRNA,analyzing protein mass spectrometry,robustly dissecting the bioinformatics and statistics,we have identified 473 RBP candidates(cRBPs)as a repertoire in SSCs.Of them,most genes were related to RNA biology according to the protein database,the isoelectric point,the ratio of disordered residues,gene ontology,and functional domains.We have also compared the RBP list with our RNA sequencing,and found that many RBPs were regulated by GDNF and RA,indicating their association with the functions of self-renewal or differentiation in SSCs.Particularly,we have identified 11 RBPs with specific preferential expression in SSC according to the germline gene expression profiles.Although some RBPs,like LIN28A and DND1,have been shown important functions in SSC self-renewal or differentiation,others,like LIN41,ESRP1 and MEX3A,have not been reported in the reproductive system yet.Our results of immunofluorescence staining in mouse seminiferous tubules had indicated that LIN41,ESRP1,MEX3A were well co-located with ZBTB16,a maker for the undifferentiated spermatogonia.The expression of LIN41 shown in As,Apr,Aal,was identical to the expression pattern of ZBTB16.Western blots showed that LIN41 had the highest testis expression level in 10 days old mice,and the expression of LIN41 protein then went down,followed by the reduced ratio of undifferentiated spermatogonia in the mouse testes.This developmental expression pattern is also identical to the expression of ZBTB16.In addition,other RBPs,like MEX3A,showed the high expression in pup mouse testes,while the expression level was significantly decreased after 14 days;ESRP1 had the same developmental expression pattern in mouse testes as MEX3A,but was regulated by growth factor GDNF,indicating its functional involvement in SSC self-renewal.Next,we found that LIN41,also named E3 ubiquitin ligase TRIM71,colocalized with another RNA binding protein LIN28A in the mouse testes,but the intracellular distribution of two proteins in cells were likely different.LIN41 were mainly found in the cytoplasm of germ cells and LIN2 8A were also found in some granules in the cells-like structures.Functionally,we did not observe the significant changes of initial cells growth after lentivirus mediated knockdown of Lin28a genes in SSC in vitro.On the contrary,knockdown of Lin41 gene resulted in a significant loss of SSCs in the culture,due to significant increase of cell apoptosis in the culture,indicating the functional difference of LIN41 and LIN28A in SSCs.Collectively,we have applied mRNA interactome capture to systematically dissect the RNA binding protein of mouse SSCs.In particular,RBP LIN41 has preferentially expressed in SSCs and was likely associated with SSC functions.These data has provided a set of protein candidates to further dissect the gene regulation at the post-transcriptional levels in the unique male germline stem cells,SSCs.