tRNA来源的新型小RNA（tsRNA-01）对成熟配子翻译抑制的功能调节及机理探讨

Global translational repression is a hallmark of mature gametes, which is quickly removed after fertilization, while the underling mechanisms remain elusive. We've recently discovered a novel class of tRNA derived small RNAs, which is evolutionarily conserved and extremely enriched in mature mouse sperm; we therefore called them mature-sperm-enriched tRNA-derived small RNA (mse-tsRNA). We further studied one mse-tsRNA member, tsRNA-01, which have the highest expression level in multiple vertebrate species. We found that tsRNA-01 is also highly expressed in mature oocytes, but its level decreased dramatically after fertilization. Given the specific expression pattern of tsRNA-01 in gametes and zygotes, we hypothesized that it might be actively involved in gametes' translational repression. In present investigation, we aim to test this hypothesis by examining the function of tsRNA-01 in translational repression using early embryos and cell lines. Moreover, we'll examine the localization of tsRNA-01 in oocytes/embryos by using Fluorescence in situ hybridization (FISH) and FITC-labeled RNA in vivo tracking. We'll also explore tsRNA-01' binding protein using RNA-protein pulldown, protein mass spectrum et.al. This study, if successful, will provide novel understandings for the longstanding question regarding the mechanisms of global translational repression in gametes. Moreover，we'll compare the expression of tsRNA-01 in sperm from fertile and infertile men, as to test its potential as an clinical marker for sperm quality.

成熟配子具有广泛翻译抑制的特点，且在受精后迅速解除，然而其具体调节机制不详。申请人前期首次发现一类在成熟精子中高度富集、进化上序列保守、来源于tRNA5'端的新型小RNA，并命名为mse-tsRNA。随后我们对mse-tsRNA中表达量最高的成员tsRNA-01研究发现：其在卵子中也呈高表达，但受精后含量迅速降低。根据tsRNA-01的表达模式及非编码小RNA活跃参与转录后调控的特性，我们推测成熟配子中高度富集的tsRNA-01可能参与调节配子中的翻译抑制。本课题拟采用荧光原位杂交、荧光RNA活体示踪检测其在卵及受精卵中的表达及动态定位；结合RNA显微注射等手段研究其功能；并通过RNA-蛋白共沉淀、蛋白质谱等手段探索其相互作用蛋白，以期对配子实现广泛翻译抑制的机制提出新的见解。我们还将检测tsRNA-01在正常与不育男性患者精子中的表达差异，旨在为男性不育症的临床诊断提供一种潜在的标记分子

本研究在我们前期研究发现tsRNAs在成熟配子中高度富集,以及能够进入受精卵的基础上，进一步对tsRNAs在受精卵及早期胚胎中的功能进行了探索。我们发现父代肥胖小鼠模型中精子tsRNAs的表达谱以及RNA修饰谱均发生了显著的变化。通过分离肥胖小鼠精子中的tsRNAs片段并注射到正常受精卵内，发现tsRNAs能像总RNA一样诱导子代代谢紊乱, 而注射精子其它片段的RNA则不能引发代谢紊乱。这些证据提示精子tsRNAs能够作为一种表观遗传信息的载体而介导父代获得性性状向子代传递。我们进一步发现, 注射肥胖小鼠精子tsRNAs的早期胚胎以及后代小鼠胰岛的转录组发生了明显变化，变化基因集中在代谢通路上；但这些变化与基因CpG岛的DNA甲基化程度并不相关，提示精子tsRNAs的作用并非通过调节DNA甲基化来实现。通过序列匹配检测发现肥胖小鼠精子中差异表达的tsRNAs主要与基因的promotor区结合，提示可能通过调节基因转录进而调节代谢基因表达。