Large-Scale Screen for Glycosylation-Related Genes Figure 6. Swm regulation of expression and nuclear export of fucTA mRNA. (A) Quantification of FucTA-Myc in control (gfp KD) and swm (swm KD) knockdown eyes. In this figure, all results shown in graphs are the mean 6 SD, n = 2. (B) Expression of FucTA-Myc in the Golgi complex was reduced in swmF14 mutants. FucTA-Myc (green) was well colocalized to the Golgi detected by anti-GM130 (red) in control eyes (arrows, +;fucTA-MycKI). In contrast, FucTA-Myc was hardly colocalized to the Golgi (arrowheads) in swm mutants (swmF14; fucTA-MycKI). Scale bar: 10 mm. (C) In vivo binding of Swm and endogenous fucTA mRNA. Endogenous fucTA mRNA was immunoprecipitated with an anti-Flag antibody (M2) from BG2-c6 cells expressing Swm-Flag and detected by RT-PCR (bottom). Control experiments were performed without Swm-Flag expression (2) or by immunoprecipitation with a mouse IgG antibody (mIgG). (D) Decrease of fucTA mRNA in swm knockdown BG2-c6 cells. Levels of fucTA RNA in swm knockdown cells was normalized to that in control gfp knockdown cells, n = 3. ***p,0.005. (E) Decay of fucTA mRNA in swm knockdown BG2-c6 cells. Levels of fucTA mRNA at each time point after actinomycin D addition were normalized to those at time 0, n = 3. (F) Ratios of fucTA and actin (act5c) mRNA levels in nuclear vs. cytoplasmic fractions in swm knockdown BG2-c6 cells were normalized to those in control gfp knockdown cells. n = 4. (G) PolyA length of fucTA mRNA in swm knockdown BG2-c6 cells. PolyA of fucTA was amplified by PCR and detected by Southern blotting with a probe corresponding to fucTA. doi:10.1371/journal.pgen.1001254.g006 Binding of Swm to fucTA mRNA was thus examined by expressing Flag-tagged Swm in the Drosophila neural cell line BG2-c6 [28] and by performing immunoprecipitation with an anti-Flag antibody. The quantity of endogenous fucTA mRNA that coprecipitated with Swm-Flag was higher in Swm-Flag–expressing cells than in controls in which Swm-Flag was not expressed or immunoprecip- itation was performed using a control IgG (Figure 6C). These results strongly suggest that Swm binds to fucTA mRNA in vivo. The amount of fucTA mRNA under the control of Swm was next examined. dsRNA targeting swm was introduced into BG2-c6 cells, and the amount of endogenous fucTA mRNA was measured. In addition to a decrease in swm mRNA, to 27.3 6 1.9% of the control level (n = 3), the amount of fucTA mRNA also decreased to 60.6 6 10.5% of the control level (Figure 6D). Furthermore, the stability of fucTA mRNA was examined in swm knockdown cells. The amount of fucTA mRNA was measured at 30, 60, and 90 min PLoS Genetics | www.plosgenetics.org 6 December 2010 | Volume 6 | Issue 12 | e1001254