The Study Of RNA Binding Protein LIN28A In Regulating Mouse Spermatogonial Stem Cell Proliferation

Spermatogonial Stem Cells(SSCs)support life-long sperm production in male gonad.Self-renewal of SSCs,together with the fragmentation of undifferentiated spermatogonia contributes to the potential germline stem cell pool in the testes.RNA-binding protein LIN28A,first identified in the nematode C.elegans,marks the spermatogonial progenitor population and regulates their cyclic expansion in the mouse testes.Conditional deletion of Lin28a in the mouse germline resulted in a reduction in testis weight,sperm number and spermatogonia number.Although the functions of LIN28 A in mouse testes have been reported,the underlying mechanisms about how LIN28A regulates undifferentiated spermatogonia were unclear.We previously found LIN28A was exclusively expressed in mouse SSCs and undifferentiated spermatogonia.In the present experiments,knockdown Lin28a in SSC culture through lentivirus-mediated small hairpin RNA resulted in the decreased proliferation in Lin25a-KD-S SCs in vitro with 20-day culture,but functional transplantation showed that reduction of LIN28A did not affect their capacity of self-renewal of these cells.We next generated Lin28a-null-SSCs from Lin28a germline knockout mice.The proliferation of Lin28a-null-SSCs was identical to Lin28a-KD-SSCs without significant difference in cell-cycle assay.Although we found that Lin28a in SSCs was negatively regulated by GDNF in the 18h GDNF withdrawn and 8h refreshment experiment.It has been reported that LIN28A post-transcriptionally regulated mRNAs therefore affecting gene transcription and/or translation.In order to profile mRNA targets of LIN28A in SSCs,we next conducted a high-throughput sequencing of RNA transcripts from 10 day-old mouse testes with Lin28a germline knockout and wild type mice.We found 70 genes upregUlated and 70 genes downregulated.Gene Ontology analysis of the upregulated genes indicated that these regulated genes were likely related to germ cell meiosis.We also showed that the mRNA levels of Sycp1,Sycp2,Meiobt Ccnb3 and Dmc1 were significantly increased,suggesting that LIN28 A may bind these meiosis genes and affect their translation.Since several binding sequences of LIN28A in cells have been reported,we then performed high-throughput sequencing by crosslinking immunoprecipitation(HITS-CLIP)experiments.Of total 19929 genes,?65%were overlapped among the three repeats,and we found the binding motif was significantly enriched in the exons and 3' untranslated region(3'UTR)of these mRNA targets.We further analyzed binding sequences and the motif,GGAGA,which has been detected by HITS-CLIP in human embryonic stem cells(hESC)and 293T cells.By analysis of combined data of RNA-seq with HITS-CLIP,we found that 133 LIN28A-regulated genes were also potentially binded by LIN28 A.Gene ontology analysis indicated,of these 133 genes,genes associated with meiosis cell cycle were enriched.Collectively,we have explored the potential function and mechanism of LIN28A in determining spermatogonial stem cell pool by cell proliferation assay,polysomeassay,RNA-seq and HITS-CLIP.We found the loss of LIN28A did block mouse SSC proliferation,which is likely by affecting the translation of meiosis gene in SSCs.