tsRNAs中m5C的定位、定量及其在遗传信息跨代传递中的功能

It has been reported that the influence of environmental factors on the parent may be transmitted to the offspring. The latest studies showed that tsRNAs (transfer RNA-derived small RNAs) of mature sperm might be a carrier of genetic information, which could transmit high-fat diet-induced metabolic phenotype to the offspring. However, the molecular mechanism of intergenerational inheritance remains unclear. Our previous study found that the expression profiling and modification profiling of sperm tsRNAs in high-fat diet mice were changed. Levels of 5-methylcytosine (m5C) and N2-methylguanosine (m2G) were increased only in tsRNAs, which were not observed in other sperm RNAs. It’s suggested that tsRNAs modifications have a relationship with tsRNAs functions. The project intends to research distribution of m5C in sperm tsRNAs from normal and high-fat diet mice by bisulfite sequencing. Then we will separate, purify and identify 2-3 tsRNAs with significant differences in expression level between normal and high-fat diet mice. Subsequently, we will identify and quantify modified nucleosides such as m2G in purified tsRNAs by LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry). According to the experimental results, we will synthesize a series of RNAs with modifications. On the basis of this study, the effects of modifications on the stability of tsRNAs and functions of tsRNAs modifications on the intergenerational inheritance will be also discussed. This project will reveal the functions and roles of modified nucleosides in the process of reproduction and embryo development, and provide scientific evidence for the prevention of obesity and diabetes in future generations from the perspective of epigenetic modification of RNA.

已有文献报道，父代能够将环境因子导致的影响传递给后代。最新研究表明，成熟精子tsRNAs可作为一种表观遗传信息的载体，将高脂诱导的父代代谢紊乱表型传递给子代，然而这种遗传信息跨代传递的分子机制还不是很清楚。前期研究发现，高脂饮食小鼠精子tsRNAs表达谱和修饰谱变化显著，修饰核苷m5C和m2G的含量仅在tsRNAs中升高，精子其他RNA中无变化，这提示修饰可能与tsRNAs的功能有关。本项目拟采用亚硫酸氢钠测序技术研究正常和高脂小鼠精子tsRNAs中m5C的分布；进而分离、纯化、鉴定2-3种差异表达的tsRNAs；利用LC-MS/MS对纯化的tsRNAs中m2G等其他修饰进行定性和定量。在此基础上，合成带有修饰的tsRNAs以探讨修饰对tsRNAs稳定性的影响以及在tsRNAs跨代遗传中的作用。本项目将揭示修饰核苷在生殖与胚胎发育过程中的作用，为后代肥胖、糖尿病等疾病的预防提供科学依据。

成熟精子tsRNAs可作为一种表观遗传信息的载体，将高脂诱导的父代代谢紊乱表型传递给子代，然而这种遗传信息跨代传递的分子机制还不是很清楚。本项目首先建立起高脂饮食诱导的小鼠模型，并以正常饮食小鼠和高脂饮食小鼠精子10-30nt和30-40nt大小的小RNAs为研究对象，利用前期建立的LC/MS检测RNA中修饰核苷的技术对其中的修饰进行定性和定量分析，发现高脂饮食小鼠tsRNAs中的m5C、 m2G显著升高。通过亚硫酸氢钠高通量测序，完成了小鼠精子部分tRNAs中m5C甲基化修饰图谱的绘制，研究发现在 Dnmt2-/-小鼠中，由于缺少Dnmt2的甲基化酶活性，精子和睾丸的tRNAs甲基化水平较低，提示m5C修饰及RNA甲基化酶Dnmt2可能与代谢有密切关系。此外，我们还建立起小鼠组织内特定tRNA及tsRNA的纯化和鉴定方法，并利用LC-MS/MS技术完成对特定tRNA及tsRNAs中修饰核苷的定性和定量分析。通过体外与RNase孵育，发现精子tsRNAs 分子中的核苷修饰对其稳定性有重要作用。本项目的研究结果为修饰核苷在生殖与胚胎发育过程中的作用和机制提供重要的依据，也必将对肥胖、糖尿病等疾病的防治和人类的健康产生积极的影响。本项目自立项以来，共发表研究论文4篇。总体上较好地完成了本项目。