ELAVL1与microRNA相互作用调控精子发生过程中靶mRNA延迟翻译分子机制的研究

One unique event of spermiogenesis is the chromatin condensation which leads to cessation of transcription in elongating spermatids. Thus, translation of proteins required for late stage spermatids depends upon mRNAs transcribed long before elongation starts. This uncoupling characteristic of transcription and translation is essential for the successful development of mature spermatozoa. However, the mechanism of this ‘delayed translation’ expression pattern is still unknown. In our published paper previously, we reported that there was a cluster of mRNAs with delayed translational expression pattern during spermatogenesis and their expression pattern was regulated by the binding status of their targeting miRNAs on their 3’UTRs. Our recent research showed that the str8-cre, Elavl1 conditional knockout mouse line has the phenotype of disruption of spermiogenesis and RNA-seq data showed changes of mRNAs expression pattern comparing with wildtype mice data. Also, Elavl1 is one of the RNA-binding proteins who has binding sites on the 3’UTRs close to miRNAs. Thus, we hypothesize that interactions between ELAVL1 and miRNAs at the 3’UTRs determine the fate of ELAVL1 target mRNAs during spermiogenesis. To test our hypothesis, we propose the following experiments: Identify the ELAVL1-targeting mRNAs and their binding sites by iCLIP-seq; Clarify the relationship between ELAVL1and miRNAs in regulating their targeting mRNAs expression pattern by bioinformatic analysis; Verify the interactions of ELAVL1 and miRNAs in vivo by luciferase assay. By studying the mechanism of the ELAVL1-miRNAs regulation pathway, we may be able to develop a general model of RBP-miRNA interactions in spermatogenesis. Furthermore, serve the clinic demands.

精子形成晚期转录停止,但精子蛋白合成依旧进行,这种“延迟翻译”对精子的发育成熟至关重要。然而目前对于调控“延迟翻译”的分子机制仍然不明。我们前期文章报道:精子发生过程中存在一簇mRNA,其3’UTR上miRNA结合位置的改变可调控其延迟翻译。预实验发现:ELAVL1条件敲除小鼠精子形成停滞,RNA-seq显示部分mRNA延迟翻译失败; ELAVL1结合位点同样位于靶mRNA 3’UTR。因此我们提出假说:精子发生过程中,ELAVL1与miRNA在靶mRNA 3’UTR上的竞争/协同性结合作用调控靶mRNA的延迟翻译。本课题将通过CLIP-seq明确ELAVL1靶mRNA及其作用位点;生物信息学分析明确ELAVL1-miRNA相互作用关系;荧光素酶体外实验验证两者在调控靶mRNA翻译模式中的作用,深层解析RBPs-miRNAs调控基因延迟翻译中的分子机制,为男性不育诊疗提供新思路。

精子形成晚期转录停止,但精子蛋白合成依旧进行,这种“延迟翻译”对精子的发育成.熟至关重要。延迟翻译调控通过靶mRNA 3’UTR及其上的RNA结合蛋白与miRNA结合位点的改变来实现。RNA结合蛋白ELAVL1条件敲除小鼠精子形成停滞,RNA-seq显示部分mRNA延迟翻译失败。本项目使用ELAVL1条件敲除小鼠作为模型，通过RNA immunoprecipitation sequencing （RIP-seq）联合生物信息学分析，鉴定出精子发生过程中与ELAVL1相互作用的靶mRNA 33个，以及miRNA 80个；通过三代全长转录组测序（PAPA-seq），明确miRNA通过调控靶mRNA poly(A)尾的脱腺苷化调控其长度的动态变化，进一步调控靶基因的稳定性和延迟翻译；在Drosha 条件敲除小鼠中，通过三代全长转录组测序，验证在缺失miRNA的状态下，靶mRNA的poly(A)尾长度变化失败，最终导致延迟翻译失败，生精异常；通过RIP-seq鉴定出精子发生过程中与ELAVL1相互作用的关键miRNA，并在临床非梗阻性无精子症患者精浆中验证其表达模式，筛选出4个可作为显微取精术前预判睾丸生精状态的miRNA(mir-34b,mir-34c,mir-3065,mir-4446)作为标志物，通过机器学习建立模型，预判睾丸内生精状态，指导临床显微取精手术。.