Figure 4. Swm and Pabp2 are required for neural-specific a1,3- fucosylation. (A) Glycan structures attached to N1012 of Chp as revealed by mass spectrometry analysis. The percentages of each structure in the control (left, gfp KD), swm (middle, swm KD), or Pabp2 (right, Pabp2 KD) knockdown condition are listed. The glycans bearing a1,3-fucose are highlighted (gray). ND, not detected. (B) a1,3- fucosylation in swm and Pabp2 mutants. Anti-HRP antibody reactivity normalized to Chp levels was markedly decreased in swmF14, swmF15, and Pabp2KG02359 mutants. Results are the mean 6 SD of 3 experiments. ***p,0.005. doi:10.1371/journal.pgen.1001254.g004 The observed requirement of Swm for most a1,3-fucosylation was also supported by the immunostaining of eyes with anti-HRP antibody. Adult photoreceptor cells from swm knockdown and control flies were simultaneously stained with anti-HRP and anti- Chp antibodies (Figure 5C). Control photoreceptor cells appeared yellow in color because the intensities of green signal (anti-HRP) and red signal (anti-Chp) were nearly equal in this condition. In contrast, swm knockdown cells appeared relatively red in color, suggesting that the intensity of green signal was reduced compared to the red signal. These data clearly indicate that the signal strength of anti-HRP relative to anti-Chp was reduced in the knockdown photoreceptor cells compared to control cells. Since a1,3-fucosylation is directly catalyzed by FucTA, the Swm-dependence of FucTA protein expression was examined. Because no anti-FucTA antibody was available, a knock-in fly was generated in which the fucTA locus was replaced by a Myc-tagged fucTA gene (Figure S4A and S4B). When swm dsRNA was expressed by GMR-Gal4, FucTA-Myc protein levels were reduced to 47.562.4% of control values as revealed by immunoblot analysis (n = 2) (Figure 6A and Figure S4C). The reduction of FucTA-Myc levels in swm mutant tissue was also examined. We costained the CNS of first instar larvae with anti-Myc and anti- GM130, a Golgi marker. The number of Golgi harboring FucTA- Myc protein was markedly decreased in swmF14 mutants (2.0% of total Golgi) compared to wild-type larvae (43.1% of total Golgi, Figure 6B). The different ratios of FucTA-Myc reduction observed Large-Scale Screen for Glycosylation-Related Genes Figure 5. Immunoblot analysis. (A) Immunoblot analysis of whole- eye extracts from swmF14 and swmF15 mutants (swmF14 and swmF15). The anti-HRP antibody detected several bands, including Chp (Band 1). Anti- HRP antibody reactivity was decreased in swm mutant homozygotes (2/2) compared with heterozygotes (+/2). Loading controls with anti- Chp and a-tubulin antibodies are shown. (B) Quantified immunoblot band intensity. HRP-positive band intensities were normalized to the intensities of corresponding anti-a-tubulin signals. The values normal- ized to those in swmF15 heterozygotes (+/2) are represented. (C) Anti- HRP signal (green) relative to anti-Chp signal (red) in swm knockdown mutants (GMR . swmRNAi) was obviously reduced compared to that in control knockdown mutants (GMR . gfpRNAi). Scale bar: 100 mm. doi:10.1371/journal.pgen.1001254.g005 in the biochemical and immunostaining experiments may be due to different levels of Swm depletion in knockdown and mutant flies. Functional analyses of Swm Although Swm has an RNA recognition motif (RNP-1) that is present in a wide variety of other RNA binding proteins [26], it remained undetermined whether Swm directly bound to mRNA. PLoS Genetics | www.plosgenetics.org 5 December 2010 | Volume 6 | Issue 12 | e1001254