after total transcription was inhibited by addition of actinomycin D. Decay rates of fucTA were indistinguishable between swm knock- down and control cells (swm, t1/2 = 97.72; control, t1/2 = 100.48, Figure 6E). These data suggest that Swm is involved in the expression of fucTA mRNA but not in its stability. Swm was recently reported to incorporate into the complex that regulates nuclear export of mRNAs [29]. The necessity of Swm for the nuclear export of fucTA mRNA was therefore examined in BG2-c6 cells. Following separation of cell lysates into nuclear and cytoplasmic fractions, fucTA mRNA levels were measured in each fraction of swm knockdown and control cells. The amount of fucTA mRNA in the nucleus increased by 9.6-fold in swm knockdown cells compared to control gfp knockdown cells (Figure 6F). The nuclear export of actin (act5c) mRNA was also examined in swm knockdown cells. The amount of actin mRNA in the nucleus increased by 1.7-fold in swm knockdown cells compared to control cells. However, the fold change in nuclear accumulation of actin mRNA was significantly lower than that of fucTA mRNA in swm knockdown cells (Figure 6F). These results suggest that relatively more Swm is required for nuclear export of fucTA mRNA than for nuclear export of actin mRNA. Since nuclear export defects have been reported to result in an abnormally longer polyA length of mRNAs [29], the polyA length of fucTA mRNA was examined in swm knockdown cells. The polyA length of fucTA mRNA was clearly longer in swm knockdown cells than in control cells (Figure 6G). Collectively, these results indicate that Swm positively regulates a1,3-fucosylation by promoting nuclear export of fucTA mRNA in neural cells. Discussion The current literature on glycosylation regulatory mechanisms is remarkably limited, and our current study is in fact the first report of a large-scale screen for glycosylation genes using a multicellular organism. We identified 109 genes in our screen, including 95 genes that have not been previously associated with glycosylation. In silico analyses using the available Drosophila databases suggested that these gene products participate in a variety of cellular processes (Figure 3), among which RNA regulation is, for the first time, shown to play an important role in the context of glycosylation. Moreover, the candidate genes belonging to the RNA regulation group were enriched by rescreening for a1,3-fucosylation-specific genes. Biological significance of a1,3-fucosylation In the current study, the fucTA mutant showed an imbalanced VNC. The VNC longitudinal length-to-width ratio was signifi- cantly shorter in fucTA mutants compared to wild-type larvae. To our knowledge, this is the first report of such shortened VNC in the loss-of-function mutant, although elongated VNC has already been reported in mutants for integrin [30], collagen IV [31], DHR3 [32], worniu [33] and C1GalTA [34]. Interestingly, C1GalTA encodes a core 1 galactosyltransferase that adds galactose to O- GalNAc attached to Ser or Thr protein residues. Taken together with our results, glycosylation patterns might affect the VNC shape. Although how a1,3-fucose affects VNC formation remains to be revealed, we think that two possible defects may result in a shortened VNC in fucTA mutants. (1) The aberrantly elongated VNC in the C1GalTA mutant is proposed to result from deglycosylation of laminin, a component of the extracellular matrix. In addition, overexpression of FucTA has recently been reported to enhance cell migration in the embryonic nervous system [35]. By analogy with the case of C1GalTA, cell migration in the larval VNC may be impaired in fucTA mutants because of Large-Scale Screen for Glycosylation-Related Genes aberrant interactions between migrating cells and extracellular matrix. (2) Another possibility is that neural activity may be impaired in fucTA mutants because neural activity has been reported to be necessary for VNC condensation in embryos [31]. This possible defect is consistent with the finding that one of the major a1,3-fucosylated proteins is a b-subunit of Na+/K+ ATPase essential for the maintenance of neural cell membrane potentials [36]. It will be interesting to examine whether neural activity is controlled by glycosylation in the future. Insight into the genes identified in this screen Three types of genes were identified in this screen: (1) genes that specifically regulate glycosylation such as glycosyltransferases, (2) genes with multiple functions including regulation of expression, activity and/or localization of glycosyltransferases, and (3) genes with primary functions other than glycosylation that secondarily affect glycosylation. For example, knockdown of cytoskeletal regulation and mitochondrial function genes may secondarily affect glycosylation as a result of damage to cell structures and viability. For several reasons, swm might be the second type of gene. Although swm has been previously reported to show involvement in the cell cycle [14], no glycosylation defects were observed with the knockdown of other known cell cycle regulators such as cdc2 (CG5363) or cdk4 (CG5072) (Table S2 and S3). In addition, no cell cycle defects could be detected in the fucTA mutant. These data suggest that cell cycle and glycosylation are regulated indepen- dently. Therefore, swm would play at least two independent roles in cell cycle and glycosylation. Traffic genes identified in this screen may be another example of the second type of genes. The conserved oligomeric Golgi (COG) complex has been reported to participate in the Golgi localization of glycosyltransferases and membrane traffic [37], but no involvement of the COG complex in glycosylation has been reported in Drosophila. Our screen identified Cog3 as a gene whose knockdown impaired a1,3-fucosylation. Thus, localization of FucTA protein was examined in cog3 knockdown photoreceptor cells. Expressed FucTA was hardly localized to the Golgi complex in knockdown photoreceptor cells (data not shown). Moreover, Golgi localization of FucTA was also preliminarily found compromised by knockdown of vps2 and vps20, other traffic genes (data not shown). These data raise the possibility that some traffic genes may be involved in glycosylation by localizing glycosyl- transferases to appropriate Golgi and/or endoplasmic reticulum sites. Preferential requirement of Swm in glycoforms Our detailed analysis of glycan structures using mass spectrom- etry revealed that one (M3F2Gn2) of the two forms bearing an a1,3-fucose (M3F2Gn3 and M3F2Gn2) is more sensitive to the decrease of swm and Pabp2 than the other (M3F2Gn3). Two reasons may account for this difference, which are not mutually exclusive. (1) FucTA productivity differs between these two glycoforms. The structural difference between the glycoforms consists of a GlcNAc moiety attached to the nonreducing end of N- glycans. Given that this GlcNAc moiety has been shown to increase the reactivity of FucTA to the core of N-glycans [6], we assume that M3FGn3 is preferentially modified by FucTA to form M3F2Gn3. Therefore, under the condition of reduction, not complete loss, of FucTA by swm and Pabp2 knockdown, M3F2Gn2 might be preferentially lost. (2) The other possibility is that Swm or Pabp2 regulate Mgat1 or Fused lobes (Fdl) [38], the enzymes that add and remove the GlcNAc moiety, respectively, from the nonreducing end. In fact, the amount of mRNA encoding Mgat1, but not fdl, was slightly but significantly increased in swm PLoS Genetics | www.plosgenetics.org 7 December 2010 | Volume 6 | Issue 12 | e1001254