knockdown cells (Figure S5). This finding is consistent with the selective decrease of M3F2Gn2 induced by swm knockdown. However, it remains undetermined whether the preferential decrease of glycoforms is caused by an increase in Mgat1, the preferential productivity of FucTA, or both. Regulation of fucTA mRNA by Swm Expression of mRNA is generally regulated at transcription, splicing, 59- or 39-end processing, and nuclear export, and by the stability of the mRNA. The stability of fucTA mRNA was not affected by swm knockdown (Figure 6E). Given that 59-end processing is essential for mRNA stability, the 59-end processing of fucTA mRNA is not regulated by Swm. In addition, splicing of fucTA mRNA does not appear to depend on Swm activity since the misspliced form of fucTA mRNA in the swm knockdown cells could not be detected when we amplified fucTA mRNA using PCR primers that targeted different exons (data not shown). Further- more, both transcription and nuclear export of mRNAs are regulated by the THO/TREX complex [39] and Sus1 [40]; thus, Swm may be a factor participating in both processes, with its depletion resulting in reduced expression and defective nuclear export of fucTA mRNA. We assume that abnormal extension of polyA in swm knockdown cells would be a secondary effect of nuclear export defects, as observed in mutants of genes required for nuclear export [29]. Moreover, Pabp2, Hel25E/UAP56 [41], and Nup358 [42], which participate in the nuclear export of mRNA, were also identified in our screen (Table S4), suggesting that nuclear export might be essential for glycosylation regulation. Nuclear accumulation of polyA(+) RNA in swm knockdown S2 cells was confirmed in our in situ hybridization experiment, as previously reported [43]. However, in neural BG2-c6 cells, weak polyA(+) RNA signals in nuclei were comparable between control gfp knockdown and swm knockdown cells (data not shown), suggesting that only a small portion of mRNA might be exported by Swm. In addition, the ratio of nuclear to cytoplasmic fucTA mRNA was relatively higher than that of actin mRNA in swm knockdown BG2-c6 cells (Figure 6F). Collectively, Swm may have preferred mRNAs for nuclear export. Moreover, the export efficiency and/or target RNA preference of Swm may differ between S2 and BG2-c6 cells. Mammalian homologs of genes identified in this screen Most of the genes identified in our present study have mammalian homologs (Table S5). Among these, Cog3 has been reported to be involved in glycosylation in organisms ranging from yeast to humans [37]. Interestingly, mutations of some COG components are causative for human diseases including congenital disorders of glycosylation (CDG) [44]. Further investigation of the genes identified here will likely provide additional insights into novel glycosylation regulatory mechanisms conserved in organisms ranging from Drosophila to humans and, possibly, into diseases involving glycosylation pathways. Materials and Methods Fly stocks and clone analysis The following fly strains were used: Canton-S as the wild-type strain, GMR-Gal4 (Bloomington Drosophila Stock Center (BDSC)), fucTA mutant Pbac{WH}FucTAf03774 (BDSC), swm mutants swmF14 and swmF15(BDSC), and Pabp2 mutant P{SUPor-P}Pabp2{KG02359} (BDSC). RNAi lines were mainly supplied by the National Institute of Genetics (Japan), with the remainder purchased from the Vienna Drosophila RNAi Center (VDRC). The transgenic flies harboring UAS-dicer2 were also purchased from VDRC. Large-Scale Screen for Glycosylation-Related Genes The swm and Pabp2 mutant mosaic clones were generated using the FLP-FRT system [27]. Adult heads from a cross of w[*]; P{w[+t*] ry[+t*] = white-un1}30C swm[F14] P{ry[+t7.2] = neoFRT}40A/CyO, P{Wee-P.ph0}2 or w[*]; P{w[+t*] ry[+t*] = white-un1}30C swm[F15] P{ry[+t7.2] = neoFRT}40A/CyO, P{Wee-P.ph0}2 with y[1]w[*]; P{w[+mC] = GMR-hid}G1 P{ry[+t7.2] = neoFRT}40A, l(2)CL-L[1]/CyO; P{w[+m*] = GAL4-ey.H}SS5, P{w[+mC] = UAS-FLP1.D}JD2 (BDSC), or y[1]; P{y[+mDint2] w[BR.E.BR] = SUPor-P}Pabp2[KG02359] P{neoFRT}42D/SM1 with y[1], w[*]; P{ry[+t7.2] = neoFRT}42D P{y[+t7.7] ry[+t7.2] = Car20y}44B, P{w[+mC] = GMR-hid}SS2, l(2)CL-R[1]/CyO; and P{w[+m*] = GAL4-ey.H}SS5, P{w[+mC] = UAS- FLP1.D}JD2 (BDSC) were dissected and used for glycosylation analyses as described below. To investigate the localization of FucTA in RNAi-mutant flies, inducible Myc-tagged FucTA-expressing flies (UAS-fucTA-Myc) were generated. fucTA cDNA was a gift from S. Nishihara. A DNA fragment encoding the 3Myc sequence was ligated to the 39end of the coding region of fucTA and inserted into the pUAST vector. The vector was injected into w1118 embryos for the production of transgenic flies. To generate fucTA-Myc knock-in flies (fucTA-MycKI), the procedure was adopted from the ends-out knock in method [45]. Details are shown in Figure S4. Antibodies and lectins Commercial sources provided biotin-conjugated Lotus; HRP- conjugated ConA, WGA, DSA, LCA, UEA, PHAL4, PNA, SBA, and DBA lectins (Seikagaku Kogyo); and HRP-conjugated ABA (Sigma). Anti-HRP antibody was obtained from Jackson Immu- noresearch. In addition, the antibodies used in this study are as follows: streptavidin-POD (Roche); anti-Myc A14 and anti-Myc 9E10 (Santa Cruz); anti-Flag M2 (Sigma); HRP-conjugated anti- mouse IgG, HRP-conjugated anti-rabbit IgG, and normal mouse IgG (Jackson Immunoresearch); Alexa 488-conjugated anti-mouse IgG, Alexa 488-conjugated anti-rabbit IgG, and TOPRO3 (Invitrogen); and Cy3-conjugated anti-mouse IgG and Cy3- conjugated anti-rabbit IgG (Kirkegaard & Perry Laboratories, Inc.). Anti-Chp 24B10, anti-a-tubulin, 22C10, BP102, anti-FasI, anti-FasII, and anti-FasIII were obtained from the Developmental Studies Hybridoma Bank. Anti-Chp antibody N8A [18] and anti- GM130 [46] were generated as described previously. Primers The primer sets used for the generation of transgenic flies are listed in Table S6. The primer sets used for analysis in cultured cells are listed in Table S7. Quantification of ventral nerve cord shape The central nervous systems were manually isolated from CS and fucTAf03774 third instar larvae and fixed in 4% paraformal- dehyde/PBS at 4uC overnight. After several washes with PBS, brain hemispheres were manually removed and placed flatly onto slide glasses. The images were captured using an AxioCam- equipped Zeiss AxioImager M1, and the length and width of VNCs were measured using AxioVision Rel. 4.7 software. First screen The first screen was carried out using a collection of transgenic fly lines carrying inducible dsRNA constructs. To drive expression of dsRNA for each gene in the eye, males from each RNAi fly line were crossed with GMR-Gal4 virgin females. Mating to inducible RNAi males and egg laying were carried out at 25uC for two days, and the resulting embryos were then incubated at 28uC. After 2 PLoS Genetics | www.plosgenetics.org 8 December 2010 | Volume 6 | Issue 12 | e1001254