Author Summary Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. Accordingly, some glycans are generated in a stage- and tissue-specific manner. To address how such distinct glycosylation is regulated in different tissues, we performed a large-scale screen for genes involved in glycosylation of a neural-specific protein. We identified 109 genes, 95 of which are assigned for the first time as directly or indirectly involved in glycosylation. We further found that neural-specific glycosylation is regulated at the RNA level, which is a novel regulatory mechanism of tissue- specific glycosylation. To elucidate novel regulatory mechanisms underlying neural- specific glycosylation, we performed a genetic screen in Drosophila and identified 109 genes required for glycosylation of a retinal neural cell-specific protein. These included 95 genes that are newly implicated in this process and 9 functional groups. Furthermore, 17 genes were identified to be specifically required for a1,3-fucosylation. Among these genes, we further analyzed the function of second mitotic wave missing (Swm), which contains an RNA-binding motif. Here, we show that Swm directly binds to fucTA mRNA, upregulates fucTA mRNA and protein levels, and facilitates the nuclear export of fucTA mRNA in neural cells. These results indicate that Swm is involved in neural-specific glycosyl- ation in addition to the cell cycle, in which its involvement has been previously reported [14]. This report, the first large-scale screen for glycosylation in a multicellular organism, has thus identified a number of new genes directly or indirectly involved in glycosylation and unveiled a novel regulatory mechanism of neural-specific glycosylation. Results Biological significance of a1,3-fucosylation As a1,3-fucosylation has not proven to be essential for neural development and/or function in Drosophila, we first aimed to determine the neural function of a1,3-fucosylation. A phenotype of a piggyBac insertional mutant for the fucTA gene, fucTA f03774, Large-Scale Screen for Glycosylation-Related Genes was examined. To validate this mutant, its central nervous system (CNS) was stained using anti-HRP antibody. The mutant CNS was negative for anti-HRP staining, suggesting that a1,3- fucosylation was compromised (data not shown). CNS morphology was then compared between wild-type and fucTA mutant third instar larvae. The longitudinal length-to-width ratio of the ventral nerve cord (VNC) was significantly lower in fucTA mutants than in wild-type larvae (Figure 1A and 1B), indicating that a1,3- fucosylation plays an important role in neural development. However, the staining patterns of antibodies such as 22C10, BP102, anti-FasI, anti-FasII, and anti-FasIII were not noticeably different in the fucTA mutant (data not shown). Screening strategy To reveal novel regulatory mechanisms underlying neural- specific glycosylation, a genetic screen was performed in adult Drosophila using the RNAi-mediated tissue-specific gene knock- down method. The procedure is summarized in Figure 2A. We chose eye-specific knockdown because eyes bear the neural- specific glycan a1,3-fucose. Additionally, the ablation of genes essential for development and/or cell viability in eyes might not cause knockdown fly lethality, whereas their ablation in the central and/or peripheral nervous systems would be lethal. DsRNAs targeting different genes were expressed in Drosophila eyes by crossing the eye-specific Gal4 driver, GMR-Gal4, with the corresponding strains. These strains harbored inverted repeats of a portion of the corresponding cDNA downstream of the yeast upstream activating sequence (UAS), which is activated by the Gal4 protein (http://www.shigen.nig.ac.jp/fly/nigfly/about/ aboutRnai.jsp). In our screen, the glycosylation of a model eye-specific glycoprotein was examined, as this allowed us to accurately measure the number of glycans added. We chose Chaoptin (Chp) [15] as our model glycoprotein based on the following advantages: (i) Chp is modified by neural-specific a1,3-fucosylation; (ii) detailed information is available on both the structures of the N-glycans and their attachment sites on Chp (described below in detail) [16]; and (iii) simple protein affinity purification is possible using an anti-Chp antibody (24B10). As shown in Figure 2B, Chp protein Figure 1. FucTA is required for proper shape of the ventral nerve cord. (A) The wild-type (CS) and fucTA mutant (fucTAf03774) third instar larval CNS. (B) The ratio of longitudinal length to width in fucTA mutants was significantly lower that that in wild-type larvae. Results are the mean 6 SD (n = 19 CS, n = 10 fucTAf03774 larvae). **p,0.01. doi:10.1371/journal.pgen.1001254.g001 PLoS Genetics | www.plosgenetics.org 2 December 2010 | Volume 6 | Issue 12 | e1001254