Delineating the roles of NSun proteins at the onset of mouse embryogenesis

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Fertilisation transforms two cells that are destined to die - the gametes, sperm and egg - into one that engenders an entire individual. The transformation integrates complex intracellular events in ways that we hardly understand. Our ignorance of this is remarkable given the fundamental nature of the gamete-to-embryo transition and its implications for health and disease - including stem cells and cancer - and compels us to develop a comprehensive model explaining how the transition occurs.In ongoing MRC-funded work, we made the unexpected discovery that mammalian (porcine and mouse) sperm introduce NSun1 into oocytes during fertilisation. This preliminary finding is remarkable. NSun1 is the prototype of a small protein family that includes RNA methyl-transferases and although the general importance of NSun family members is only recently becoming appreciated, it is clear that they are involved in multiple developmental pathways, regulating cell division and proliferation. These are key events in the gamete-to-embryo transition and strongly imply a link to NSuns that would strengthen the over-arching hypothesis in the applicant laboratory, that carcinogenesis and embryogenesis share mechanisms of initiation.New unpublished evidence shows that most members of the NSun family are up-regulated immediately after fertilisation in the mouse. The present proposal seeks to investigate this in an integrated embryological and biochemical approach to produce a model of NSun function in reprogramming to totipotency during the mammalian gamete-to-embryo transition.Our output will include careful characterisation of NSun transcripts and proteins during and immediately after mouse fertilisation and will show how interfering with NSun expression during this period affects embryo development. NSuns have a highly-conserved RNA methyl-transferase catalytic domain, enabling us to employ a technique recently developed by our collaborator to capture and characterise NSun RNA substrates. This will reveal the RNA substrates of different NSuns at single-base resolution and allow us to begin an analysis of early embryonic roles played by NSun targets. Our work identified NSun1 by its DNA binding ability, which we have confirmed in living oocytes, but DNA binding by NSuns has not so far been reported. We shall therefore characterise the binding of NSuns in oocytes and embryos to DNA and RNA using a novel in vivo microbead assay; the assay will also identify the domains in NSuns responsible for binding. Together, this work is expected to indicate how maternal mRNA are regulated immediately after fertilisation and whether regulatory mechanisms are shared by cellular potency changes in other contexts.These experiments will reveal the phenotypic consequences of NSun disruption and target RNA networks at the onset of mammalian development. A better understanding of NSuns in the emergence of totipotency has the potential to impact the controlled induction of pluripotency with two clear additional translational applications that we will start to investigate. First, because of their roles promoting proliferation, NSuns may be oncogenic. We will work with clinicians to investigate whether there is a link that can be used in the diagnosis and treatment of cancer, particularly breast cancer. Secondly, altered NSun activity may impair fertility. Our collaboration with a major IVF clinic will determine whether NSuns represent (a) novel markers, and (b) treatment targets of impaired fertility.