LIN28A binds to meiotic gene transcripts and modulates their translation in male germ cells

The RNA-binding protein LIN28A is required for maintaining tissue homeostasis, including in the reproductive system, but the underlying mechanisms on how LIN28A regulates germline progenitors remain unclear. Here, we dissected LIN28A-binding targets using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse testes. LIN28A preferentially binds to mRNA coding sequence (CDS) or 3'UTR regions at sites enriched wiGAG(A) sequences. Further investigation of Lin28a null mouse testes indicated that meiosis-associated mRNAs bound by LIN28A were differentially expressed. Next, ribosome profiling revealed that the mRNA levels of these targets were significantly reduced in polysome fractions, and their protein expression levels decreased in the Lin28a null mouse testes, even when meiotic arrest in the null mouse testes was not apparent. Collectively, these findings provide a set of LIN28A-regulated target mRNAs, and show that LIN28A binding might be mechanism through which LIN28A acts to regulate undifferentiated spermatogonia fates and male fertility in mammals. Overall design: Triplicate identification of transcripts associated with LIN28A in mice testes, triplicate mRNA profiles profile of Lin28a knockout and control in 10dpp mouse testes were generated by deep sequencing.

RNA结合蛋白LIN28A在维持包括生殖系统在内的组织稳态中发挥必需作用，但其调控生殖系祖细胞的具体分子机制仍不明确。本研究通过对小鼠睾丸中经交联免疫沉淀分离的RNA进行高通量测序（HITS-CLIP，high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation），解析了LIN28A的结合靶标。LIN28A优先结合富含GAG(A)序列位点的mRNA编码区（CDS）或3'非翻译区（3'UTR）。对Lin28a基因敲除小鼠睾丸的进一步研究显示，被LIN28A结合的减数分裂相关mRNA存在差异表达。后续核糖体谱分析结果表明，这些靶标的mRNA在多聚核糖体组分中的水平显著降低，且Lin28a基因敲除小鼠睾丸中这些靶标的蛋白表达水平也随之下降，即便该敲除小鼠睾丸未出现明显的减数分裂阻滞。综上，本研究鉴定出一组受LIN28A调控的靶mRNA，并揭示LIN28A的结合作用可能是其调控哺乳动物未分化精原细胞命运与雄性生育能力的分子机制。整体实验设计：对小鼠睾丸中与LIN28A结合的转录本进行三次重复鉴定；通过深度测序获取产后10日龄（10 days post partum，10dpp）小鼠睾丸中Lin28a基因敲除组与对照组的mRNA表达谱，每组均设置三次生物学重复。