精子tsRNA及其RNA修饰在精子跨代遗传信息传递中的作用

Sperm RNA mediated transmission of phenotypes has drawn increasing attention as an important type of non-genetic (epigenetic) inheritance. However, the exact carrier of such RNA-mediated information remains elusive and remains the open question in the field. By analyzing small RNA deep-sequencing data, we recently discovered that the mammalian mature sperm contain a unique subset of highly enriched small RNA families derived from 5’ halves of mature tRNAs, mostly being 29-34nt in length, which are named tsRNAs (tRNA-derived small RNAs). In a paternal mouse model given a high-fat diet (HFD), we showed that sperm tsRNAs exhibited changes in expression profiles and RNA modifications. Injection of sperm tsRNA fractions from HFD males into normal zygotes generated metabolic disorders in the F1 offspring, demonstrating that sperm tsRNAs represent a paternal epigenetic factor that may mediate intergenerational inheritance of diet-induced metabolic disorders. These discoveries raise the open question that how sperm tsRNAs, along with their RNA modifications, mediate the embryonic developmental programming that affect the offspring phenotype, thus are the emphases of this present project. Here, we propose to (1) test sperm tsRNAs’ target by injecting sperm tsRNAs (from HFD and control male) into zygote, followed by comparative single-cell embryo RNA-seq of 2- to 4-cell embryos and bioinformatics analysis. (2) Because significant alteration of m5C (5-methylcytidine) occurs in sperm tsRNAs from HFD males, we will further test whether the m5C cytosine RNA methyltransferase, DNMT2, is essential for sperm tsRNAs’ ability to transmit acquired metabolic disorder to offspring. Utilizing the HFD model in Dnmt2-/- and Dnmt2+/+ male mice, we will inject sperm tsRNAs into normal zygotes and examine the metabolic phenotype of F1 offspring. The overall aim of present project is to determine the mechanisms by which sperm tsRNAs establish transcriptional changes in early embryos that lead to offspring phenotype and to identify the RNA modification (and the enzyme) essential for transmitting the paternally acquired phenotype.

越来越多的证据表明，父辈在环境暴露、营养状况等改变下，某些获得性性状可通过精子遗传给下一代。然而，目前对该现象背后的具体表观遗传信息载体尚缺乏清晰的认识。申请人实验室近期在哺乳动物成熟精子和血清中发现了一类进化上保守、来源于tRNA5’端序列、且高度富集的新型小RNA：tsRNAs；并进一步在高脂饮食诱导的父代肥胖小鼠模型中，发现tsRNAs及其携带的RNA修饰m5C可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 (Science, 2016)。然而，精子tsRNAs在调节早期胚胎发育中的靶基因是什么？精子tsRNAs上的修饰m5C受何种酶调控并参与跨代信息的传递？本课题将针对上述两个问题，通过RNA测序，受精卵RNA注射，RNA修饰谱差异筛选、以及RNA修饰酶DNMT2敲除小鼠等多种方法和模型，力争对精子tsRNAs调控获得性性状的跨代遗传的分子机理提供更深入的认识。

越来越多的证据表明，上一代在环境压力下产生的某些获得性性状可以“记忆”在配子中并遗传给下一代。我们实验室在2012年于哺乳动物成熟精子中首次发现了一类来源于tRNA的新型小RNA--tsRNAs（tRNA-derived small RNAs）,并且发现tsRNAs可以将父代高脂饮食导致的获得性代谢紊乱性状传递到子代（Science，2016）。研究的过程中，我们发现高脂饮食刺激引起了小鼠精子30-40nt RNA中m5C 和m2G 这两种RNA修饰的显著上调。化学合成的不带修饰的tsRNAs在受精卵裂解液中极易降解，且不能引起子代代谢紊乱表型的发生，提示精子tsRNA及其RNA修饰是介导父代获得性表型向子代传递的关键因素。Dnmt2负责tRNA C38 位的m5C修饰。我们推测Dnmt2调控的tRNA m5C修饰可能参与了精子RNA介导的父代高脂饮食代谢紊乱表型向子代传递的调控，因此构建了Dnmt2敲除的高脂饮食小鼠模型。研究发现高脂饮食条件下Dnmt2敲除小鼠精子总RNA和30-40nt RNA失去了将父代高脂饮食所诱导的获得性代谢紊乱表型传递给子代的能力。进一步研究发现，Dnmt2缺失抑制了精子30-40nt RNA中m5C 和m2G这两种修饰水平在高脂饮食刺激下的上升，并且改变了精子小RNA的表达谱，使之恢复到正常饮食条件下的小鼠精子小RNA表达模式。我们还发现Dnmt2调控tRNA C38 位的m5C修饰对tsRNA的结构和功能都具有重要的调控作用，为tsRNAs的作用机制研究打开了新思路。以上结果表明Dnmt2缺失破坏了高脂饮食刺激下精子RNA中具有表观遗传信息传递能力的“编码指纹”的形成及存储，从而无法将父代的获得性代谢紊乱性状传递给子代。

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