tsRNA的末端修饰及其在遗传信息传递中作用和机制研究

Our previous study showed that a noncoding small RNA, termed tsRNA (transfer RNA-derived small RNAs), mediates intergenerational inheritance of acquired metabolic disorders from obese father, which was published in Science, highlighted by Science and other journals, and awarded “Top Ten Advances in Science and Technology of China in 2016”. Our previous study showed that tsRNA contains many kinds of modifications, and modification profile was altered in sperm tsRNAs of mice fed high-fat diet. This project aims to investigate the 5’ and 3’ terminal modification of tsRNA and the related biological functions. First, we will analyze tsRNA terminal modifications by LC/MS/MS and other methods, and identify the corresponding exogenous demodification enzymes. According to the identified terminal modifications, we will design a strategy to construct libraries for high-throughput sequencing to quantify the terminal modificatins of tsRNA and reveal the real major tsRNA profiles. Furthermore, we will investigate the effect of terminal modifications on the stability of tsRNA, and the roles and underlying mechanisms of sperm tsRNA terminal modifications in intergenerational inheritance of acquired metabolic disorders from obese father. This project will reveal the major terminal modification of tsRNA and tsRNA profiles, discover the important biological functions of tsRNA terminal modificaions, and potentially elucidate the key mechanisms in intergenerational inheritance of acquired metabolic disorders.

申请人前期发现非编码小核酸tsRNA介导了肥胖父代导致子代代谢紊乱，以共同通讯作者发表在Science，并获Science等多个期刊专评，研究成果入选“中国科学十大进展”等。前期发现tsRNA存在多种修饰并且高脂饮食可导致tsRNA修饰谱发生变化。本项目拟深入研究tsRNA 5’和3’末端修饰及其生物学功能。首先通过质谱等技术分析鉴定主要的末端修饰，寻找和验证相应的外源性去修饰酶。根据tsRNA的末端修饰，设计相应的建库策略并进行高通量测序，以揭示tsRNA末端修饰的比例和更完整的tsRNA表达谱。同时探索tsRNA的末端修饰是否影响其稳定性，并深入研究其末端修饰在肥胖小鼠精子tsRNA导致子代代谢紊乱中的作用和机制。本项目的顺利实施，将揭示tsRNA的末端修饰和tsRNA比较完整的表达谱，并发现tsRNA末端修饰的重要生物学功能，为阐明代谢性疾病获得性性状跨代遗传的机理奠定重要基础。

非编码小RNA tsRNA介导了肥胖父代导致子代代谢紊乱，但精子等组织的tsRNA的末端是否存在修饰，该修饰是否通过影响RNA稳定性并有重要的生物学作用目前尚不清楚。本项目以正常饮食小鼠和高脂饮食小鼠精子等组织的tsRNA为研究对象，采用β-内消去反应来初步判断tsRNA的3’末端存在修饰，并发现一些tsRNA等小RNA存在特殊的3’末端修饰并且不能被常规的高通量测序建库方法检测到。通过质谱分析鉴定了小RNA末端存在的特殊修饰，并通过相应的去修饰酶去除tsRNA末端修饰，建立3’末端有该特殊修饰的小RNA的高通量测序方法。揭示了多种组织和细胞中3’末端有该特殊修饰的小RNA和3’末端为羟基的小RNA的表达谱。建立了3’末端有该特殊修饰的小RNA的qPCR定量检测方法，并进行了Northern验证。发现了该3’末端有该特殊修饰的小RNA产生的关键分子机制。合作研究发现Dnmt2通过影响精子非编码小RNA的甲基化修饰介导了父代获得性代谢性状的代际传递。本研究项目对于后续深入了解小RNA的修饰及其生物学功能有重要意义。