Dissecting the role of sperm transcriptome dynamics in intergenerational inheritance through native RNA nanopore sequencing

ongoing

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. 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.