Project Summary Emerging evidence has shown that small non-coding RNAs (sRNAs) harbor a diversity of RNA modifications. RNA modifications have the potential to store a secondary layer of labile biological information that is responsive to various environmental exposures and can modulate RNA properties such as stability and interaction potential, thus contributing to complex physiological/pathological processes. In our previous mouse model of paternal high- fat diet (HFD)-induced intergenerational inheritance, we found that tRNA-derived small RNAs (tsRNAs) and RNA methylatranserase (Dnmt2)-mediated site-specific RNA modification established a “sperm RNA code” that is required for intergenerational transmission of paternally acquired metabolic disorders (Science 2016; Nat Cell Biol 2018). These data, along with others, support an emerging concept that RNA modifications in sperm small RNAs serve as an additional layer of paternal hereditary information that can be modulated by environmental input, and is essential for regulating offspring phenotype via embryo development. These advances have set the stage to further examine whether a wider range of paternal environmental exposures, such as tributyltin (TBT) and arsenite (both are known to associate with obesity and metabolic disorders) will similarly alter sperm RNAs to confer offspring phenotype. This concerns the nature of the core sperm RNA code (i.e. a group of modified tsRNAs) shared by different exposure that is responsible for the intergenerational phenotype transmission; and also the molecular mechanism by which the modified sperm tsRNAs regulate embryo development to dictate offspring’s metabolic performance. In present project, we aim to first decipher the essential sperm tsRNAs & associated RNA modifications that responsible for programming offspring metabolic health, by comparatively studying different paternal environmental stressors (HFD, TBT & arenite exposure) with improved small RNA- seq protocol, which reduces sequencing bias by enzymatically removing RNA modifications that block reverse transcriptase and terminal adaptor ligation; we also explore the upstream regulators of the altered sperm tsRNAs, with a focus on RNA modifications enzymes (Aim 1). We will further isolate individual tsRNAs followed by RNA modification quantification using Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS), and test their function in conferring offspring phenotype by zygotic RNA injection and offspring phenotype tracking (Aim 2). Mechanistically, we will test the hypothesis whether modified sperm tsRNAs can program the metabolic state by regulating ribosome heterogeneity that control distinct translational pool of mRNAs (Aim3). In other words, we propose that environmental stressor-induced “sperm RNA code” is transformed into an “embryonic ribosome code”, which generates translational specificity to define the metabolic phenotype of offspring. Data from the proposed study may not only reveal...