Epidermal growth factor (EGF) repeats in the extracellular domain of Notch are modified by several O-linked carbohydrates, some of which are important for Notch signaling in various species. In the fruit fly, the O-glucosyltransferase Rumi mediates the attachment of glucose moieties to Notch and is required for Notch signaling, but the functional role of O-glucosylation in mammalian Notch signaling has not been evaluated. Fernandez-Valdivia et al. report that Rumi also catalyzes O-glucosylation of Notch and that this modification is necessary for Notch signaling. Rumi was widely expressed in mouse embryos, and Rumi knockouts died in utero by embryonic day 9.5, exhibiting an expanded neural plate, posterior truncation, and heart defects that were similar to but more severe than mutants lacking canonical Notch signaling components, suggesting that Rumi likely also has Notch-independent functions. In mouse neuroblastoma Neuro2A cells, stable expression of a short hairpin RNA (shRNA) directed against Rumi decreased the expression of the Notch signaling target Hes1 and stimulated neurite outgrowth, phenotypes that required the enzymatic activity of Rumi. Mass spectrometry analysis indicated that knocking down Rumi in mouse myoblast C2C12 cells resulted in decreased O-glycosylation of the EGF repeats in transgenically expressed Notch and reduced the abundance of the γ-secretase–cleaved (activated) form of Notch but did not affect the ability of Notch to bind to Jag1 and Dll1 ligands. Genetic interaction experiments confirmed that Rumi affected Notch signaling in vivo: Animals doubly heterozygous for null alleles of Jag1 and Rumi (Jag1+/-; Rumi+/-) exhibited bile duct phenotypes similar to those of Jag1+/-; Notch2+/- animals. Along with O-GlcNAcylation and O-fucosylation, O-glucosylation of Notch EGF repeats is thus also functionally important for modulating signaling.
R. Fernandez-Valdivia, H. Takeuchi, A. Samarghandi, M. Lopez, J. Leonardi, R. S. Haltiwanger, H. Jafar-Nejad, Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi. Development 138, 1925–1934 (2011). [Abstract] [Full Text]