Mammalian sperm RNA is increasingly recognized as an additional source of paternal hereditary information beyond DNA. Environmental inputs, such as diet and stress, can reshape the sperm RNA signature and induce offspring phenotypes that relate to paternal environmental stressors. However, how, when and to what extent sperm RNA populations change, and what is the role that RNA modifications and other post-transcriptional regulatory layers play in shaping sperm RNA dynamics, remains poorly understood.
Here, we propose to characterize the dynamics of RNA populations during sperm formation and maturation using native RNA nanopore sequencing. This technology is suited to provide an integrative and comprehensive view of the transcriptome, epitranscriptome, degradation patterns and tailing dynamics simultaneously, and with single molecule resolution. We will establish novel library preparation methods that can capture the full sperm (epi)transcriptome, and will capitalize on our recently developed algorithms to map and quantify RNA modifications in individual RNA molecules. We will then apply these methods to reveal how paternal dietary exposures affect sperm RNA populations and the metabolic phenotypes of their offspring, and test whether the novel identified RNA candidates can transmit diet-induced paternal phenotypes to the subsequent generation. The integrative nature of our approach, which combines transcriptomics, epitranscriptomics, fragmentomics and tailomics, has the potential to provide answers to how environmental traits are inherited across generations, as well as reveal previously unidentified carriers of intergenerational inheritance, which we will experimentally test and orthogonally validate as part of this proposal. Finally, we propose to expand our previous work on direct RNA multiplexing to establish single cell direct RNA nanopore sequencing, to characterize the diversity and heterogeneity of the sperm RNA (epi)transcriptome at an unprecedented single cell and single molecule resolution. This technology will allow us not only to examine the sperm epitranscriptome and its heterogeneity at an unprecedented resolution, but will also have a major impact in many other scientific disciplines, such as cancer therapies. Moreover, the possibility of studying the epitranscriptome at single cell and single molecule resolution will greatly facilitate the functional dissection of the many RNA modifying enzymes whose functions are yet to be elucidated, but are known to be the cause of diverse human diseases when dysregulated.
With EPISPERM, we propose to obtain a comprehensive and integrative multi-omics view (transcriptomics, epitranscriptomics, fragmentomics and tailomics) of the dynamics of sperm RNA across maturation stages (Aim 1), upon different diets (Aim 2) and across individual cells (Aim 3). These aims will include experiments to functionally test whether identified RNA modifications and RNA biotypes play a role in sperm maturation and in intergenerational inheritance.
