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  • The correct deposition of m C into RNAs is essential

    2018-10-24

    The correct deposition of m5C into RNAs is essential for normal development. Loss-of-function mutations in the NSUN2 gene in both mouse and human cause growth retardation and neurodevelopmental deficits including microcephaly, as well as defects in cognition and motor function (Blanco and Frye, 2014). In the developing mouse brain, expression of NSUN2 is highest in the cerebral cortex, hippocampus, and striatum, and all of these areas show decreased global protein synthesis, increased cellular stress, and reduction in size in the absence of Nsun2 (Blanco et al., 2014). Importantly, cleaved 5′ tRNA fragments are required and sufficient to induce the cellular stress responses, and both cellular stress and microcephaly can be rescued through inhibition of angiogenin (Blanco et al., 2014). Here, we set out to dissect the underlying cellular process leading to the selective reduction in size of the cerebral cortex in the absence of NSUN2. In the developing mouse brain, deletion of Nsun2 does not affect radial glia but delays differentiation into upper-layer neurons. In humans, NSUN2 is expressed in early neuroepithelial progenitors during development and cultured neuroepithelial stem/progenitor cells. Repression of NSUN2 is sufficient to inhibit neural migration and, in the presence of angiogenin, impairs neural lineage commitment. Thus, cytosine-5 RNA methylation pathways are required akt inhibitor for the efficient cellular response toward neural lineage-inductive stimuli.
    Results
    Discussion Loss of function of NSUN2 impairs normal akt inhibitor development and leads to microcephaly in mouse and human (Blanco et al., 2014; Martinez et al., 2012). Here, we reveal that the microcephaly phenotype can be explained by a reduced number of upper-layer neurons in the cerebral cortex. The precise timing of self-renewal and differentiation of neural progenitor cells as well as their migration and correct expansion are essential for normal mammalian brain development (Kriegstein and Alvarez-Buylla, 2009). Disturbance of any of these processes can lead to microcephaly (Manzini and Walsh, 2011; Thornton and Woods, 2009). Similar to other tissues in the Nsun2−/− mice, the brain developmental deficits were not due to impaired cellular proliferation (Blanco et al., 2011; Hussain et al., 2013b). On the contrary, we found an increase in TBR2-positive intermediate progenitors in the Nsun2 cortex. The increase in progenitors but decrease of SATB2-positive upper-layer neurons strongly suggested a delay in differentiation. To understand how NSUN2 and its methylated RNA targets interfere with neural differentiation pathways, we identified all NSUN2-dependent 5-methylcytosines in total RNA of the developing forebrain. In line with our previous studies in normal skin and skin tumors, we only find a robust loss of m5C in tRNAs (Blanco et al., 2014, 2016). The overall lower methylation rate at distinct sites within cRNAs and ncRNAs might have several reasons: (1) the sites could be bisulfite conversion artifacts; (2) the sites are more difficult to detect due to the lower RNA stability compared with tRNAs; (3) the low percentage of methylation at distinct sites might indicate that only a small proportion of the expressed cellular RNA carried the modification. Thus, we cannot exclude that the few cRNAs and ncRNAs carrying NSUN2-dependent m5C sites contribute to the brain developmental deficits. However, we now demonstrate that the impaired cellular differentiation is at least in part dependent on tRNA methylation. Differentiation of cultured human NES cells was less efficient in the absence of NSUN2, and the decreased capacity to differentiate was potentiated by angiogenin. Angiogenin was the first human tumor-derived protein with in vivo angiogenic activity (Fett et al., 1985). Since then angiogenin has been implicated in a wide range of cellular responses including cell migration, invasion, proliferation, and formation of tubular structures (Gao and Xu, 2008). Small ncRNAs derived from angiogenin-mediated tRNA cleavage inhibit endothelial cell migration and tube formation (Li et al., 2016). Deletion of NSUN2 in induced tRNA cleavage, and reduced migration of human epidermal cells (Blanco et al., 2016).