Determining the Postfertilization Regulatory Functions of a Sperm microRNA Cluster

ABSTRACT: As specialized cells with limited cytoplasm, sperm have long been viewed as contributing only the paternal genome to progeny. However, in the past decade experiments in mice have revealed that sperm microRNAs (miRNAs) influence early embryogenesis and are sufficient for the paternal transmission of non-genetically inherited phenotypes. miRNAs, along with their protein cofactor Argonaute (Ago), bind messenger RNA (mRNA) targets and subsequently downregulate their expression through mRNA decay and translational repression. However, the mechanism underlying how sperm miRNAs act as inherited information remains undetermined. We hypothesize that sperm miRNAs modulate early development through the direct regulation of key mRNA targets like chromatin modifiers or signal transduction pathways, thus altering downstream gene expression programs in a manner that persists through development. Due to the difficulty of identifying targets of miRNAs in the embryo, the targets of sperm miRNAs are still currently unknown. The Fx-miRs are a cluster of miRNAs that are highly expressed in the sperm of every mammalian species analyzed. When wildtype eggs are fertilized with Fx-miR deficient sperm, resulting embryos have decreased developmental potential and demonstrate significantly altered gene expression in preimplantation embryogenesis. Despite these phenotypes, the mechanistic functions of the Fx-miRs in the early embryo, especially what mRNAs these miRNAs target and how these binding events impact overall gene expression during early development, remain unknown. I hypothesize that sperm miRNAs directly target and regulate a subset of maternally provided or zygotically expressed mRNAs postfertilization which then leads to downstream gene expression effects that modulate embryonic development. In Aim 1a, I will use ribosome profiling to evaluate the impact of individual Fx-miRs and the whole cluster on global translation to discover additional regulation by these miRNAs not captured by mRNA-seq. Here, I will use mESCs as a model for early embryonic development, as my preliminary data has demonstrated that mESCs are an efficient model to profile the molecular functions of sperm miRNAs. In Aim 1b, I will use Ago enhanced Crosslinking and Immunoprecipitation (Ago eCLIP) to identify the direct targets of the Fx-miRs on the level of individual miRNAs, also using mESCs as a model. Finally, in Aim 2, to dissect direct versus secondary effects, I will knockdown specific Fx-miR targets identified in my preliminary data via siRNA in embryos fertilized with Fx-miR deficient sperm and perform single embryo RNA-seq. Downregulation of specific Fx-miR targets and the analysis of the resulting differential gene expression will allow for the separation of direct versus downstream effects of the Fx-miRs in vivo. This project will be the first to identify the postfertilization gene regulatory functions and direct targets of an important cluster of sperm miRNAs and thus reveal mechanistically how sperm miRNAs can program embryonic development to initiate the non-genetic inheritance of offspring phenotypes.