A sperm-enriched 5’fragment of tRNA-Valine regulates preimplantation embryonic transcriptome and development

Sperm small RNAs have been implicated in intergenerational epigenetic inheritance of paternal environmental effects; however, their biogenesis and functions remain poorly understood. We previously identified a 5’ fragment of tRNA-Valine-CAC-2 (tRFValCAC) as one of the most abundant tRNA fragments in mature mouse sperm. We previously reported that tRFValCAC is specifically enriched in sperm during post-testicular maturation in the epididymis, and extracellular vesicles secreted by the epididymis epithelial cells can deliver tRFValCAC to sperm. Here, we investigated the mechanistic basis of tRFValCAC delivery from epididymis to sperm and its functions during early embryonic development. We show that tRFValCAC interacts with an RNA binding protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB), in the epididymis and that hnRNPAB regulates the levels of tRFValCAC in the vesicles secreted by epididymis epithelial cells. Inhibition of tRFValCAC in preimplantation embryos altered the transcript abundance of genes involved in RNA splicing and mRNA processing. Importantly, tRFValCAC-inhibited embryos showed altered mRNA splicing, including alternative splicing of various splicing factors and genes important for proper preimplantation embryonic development. Moreover, our data suggest that tRFValCAC regulates its target genes via interaction with RNA-binding proteins. Finally, we find that inhibition of tRFValCAC in zygotes delayed preimplantation embryonic development. Together, our results reveal a potential function of a sperm-enriched tRF in regulating alternating splicing and preimplantation embryonic development and shed light on the mechanism of sperm small RNA-mediated epigenetic inheritance. We examined the regulatory roles of tRFValCAC in early preimplantation embryos by inhibiting tRFValCAC using an antisense locked-nucleic acid (LNA) containing oligo targeting tRFValCAC (Val_LNA). Embryos were microinjected with either H3.3-GFP mRNA with LNA-containing scramble oligo (Ctrl_LNA) (control group) or H3.3-GFP mRNA plus tRFValCAC antisense LNA oligo (Val_LNA) (experimental group). GFP-positive embryos were cultured until the late 2-cell stage for mRNA-seq (28 hours post-IVF).