Identification of Genes Required for Neural-Specific Glycosylation Using Functional Genomics Miki Yamamoto-Hino1,2, Yoshimi Kanie1¤a, Wakae Awano3, Kiyoko F. Aoki-Kinoshita4, Hiroyuki Yano1¤b, Shoko Nishihara4, Hideyuki Okano 2 , Ryu Ueda5, Osamu Kanie1¤c, Satoshi Goto1,2* 1 Research Group of Glycobiology and Glycotechnology, Mitsubishi-kagaku Institute of Life Sciences, Tokyo, Japan, 2 Department of Physiology, Keio University, Tokyo, Japan, 3 Mutant Flies Laboratory, Mitsubishi-kagaku Institute of Life Sciences, Tokyo, Japan, 4 Department of Bioinformatics, Faculty of Engineering, Soka University, Tokyo, Japan, 5 Genetic Strains Research Center, National Institute of Genetics, Shizuoka, Japan Abstract Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, a1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific a1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating a1,3-fucosylation. Further analyses revealed that an RNA–binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells. Citation: Yamamoto-Hino M, Kanie Y, Awano W, Aoki-Kinoshita KF, Yano H, et al. (2010) Identification of Genes Required for Neural-Specific Glycosylation Using Functional Genomics. PLoS Genet 6(12): e1001254. doi:10.1371/journal.pgen.1001254 Editor: Norbert Perrimon, Harvard Medical School, Howard Hughes Medical Institute, United States of America Received May 3, 2010; Accepted November 19, 2010; Published December 23, 2010 Copyright: 2010 Yamamoto-Hino et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to SG (No.60280575) and by the Core Research for Evolutionary Science and Technology (CREST) of Japan Science and Technology Agency (JST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sgoto@a7.keio.jp ¤a Current address: Chemical Resources Laboratory, Tokyo Institute of Technology, Kanagawa, Japan ¤b Current address: Structural Biology Center, National Institute of Genetics, Shizuoka, Japan ¤c Current address: RIKEN Advanced Science Institute (ASI), Japan Science and Technology Agency (JST), ERATO Glycotorilogy Project, Saitama, Japan Introduction Neural cells require correct glycosylation patterns for their development, function, and viability. An example of this is the attachment of an a1,3-fucose moiety to an N-glycan core via a1,3- linkage [1], a process which is prevalent in neural cells in Drosophila [2]. This a1,3-fucose moiety can be detected with an anti-horse radish peroxidase (HRP) antibody and therefore has also previously been referred to as an HRP epitope. The a1,3-fucose is thought to be essential for neural development, function, and behavior because a nac (neurally altered carbohydrate) Drosophila mutant that lacks this a1,3-fucose moiety exhibits deformation of the eyes [3], the misrouting of wing sensory neurons [4], and abnormal grooming behavior [5]. However, as it remains unclear that the nac mutation impairs only a1,3-fucosylation, the necessity of a1,3- fucosylation for neural development and/or function in Drosophila has not been conclusively demonstrated. The enzyme a1,3-fucosyltransferase (FucTA) [6], which is mainly expressed in neural cells, directly catalyzes a1,3-fucosyla- tion. In addition to FucTA, other glycosylation-related proteins such as UDP-GlcNAc: a-3-D-mannoside-b -1,2-N-acetylglucosa- minyltransferase I (Mgat1) [7], GDP-mannose 4,6-dehydratase (Gmd) [8], and a GDP-fucose transporter (Gfr) [9,10] are required for a1,3-fucosylation. Whereas Mgat1 provides a preferred substrate for FucTA by adding N-acetylglucosamine to the nonreducing end of an N-glycan, Gmd and Gfr are responsible for the synthesis and transport, respectively, of GDP-fucose, another substrate for FucTA. These genes, in contrast to the gene encoding FucTA, are widely expressed in various tissues and also utilized for other glycosylation processes such as O-fucosylation of Notch, a1,6-fucosylation of N-glycans, and formation of complex type N-glycans. Hence, the neural-specific expression of FucTA appears to account for the neural-specific regulation of a1,3- fucosylation. However, the mechanisms regulating FucTA expres- sion have remained largely unknown. Forward genetic approaches have proven to be powerful methods of elucidating novel mechanisms. For example, the study of Drosophila genetics has yielded important contributions to our understanding of the developmental significance of proteoglycans [11,12] and Fringe-dependent Notch glycosylation [13]. Genetic screens for mutations affecting morphogenesis and growth factor signaling have now identified a number of genes involved in Notch glycosylation and/or proteoglycan formation. Most of these genes are conserved in mammals, suggesting that Drosophila is a useful model system for the study of glycosylation in metazoans. However, although previously performed screens of this nature have identified glycosyl enzymes and nucleotide sugar transporters, to date they have not been used to uncover regulators of these molecules. PLoS Genetics | www.plosgenetics.org 1 December 2010 | Volume 6 | Issue 12 | e1001254