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. 2020 Nov 9;9:e52416. doi: 10.7554/eLife.52416

Figure 4. SUMO depletion leads to derepression of both interrupted and intact rDNA transgenes.

Figure 4.

(A) SUMO knockdown depresses rDNA transgenes. Expression of rDNA transgenes in ovaries as measured by RT-qPCR upon germline knockdown of SUMO (shSmt3) and in control (shW). Data shows three biological replicates normalized to rp49 mRNA. Error bars indicate standard deviation of xxxx biological replicates. Statistical significance is estimated by two-tailed Student’s t-test; ***p<0.001. (B) Transgenic pre-rRNA expression is repressed by SUMO as detected by HCR-FISH Expression of rDNA transgenes in nurse cells is detected upon germline-specific knockdown of SUMO (shSmt3) or control (shWhite) gene using a probe against the UID sequence (Red). Vasa mRNA (green) is detected in parallel. Scale bar: 10 µm. Bottom row shows magnification of individual boxed nuclei. (C) rDNA transgenes are expressed in more nuclei upon SUMO KD as detected by HCR-FISH Shown is the number of nurse cell nuclei with positive HCR-FISH signal per egg chamber (out of 15 nuclei per stage 7/8 chamber). Each circle represents data from one egg chamber. Both intact and interrupted transgenes are expressed in all nuclei upon SUMO knockdown. Error bars indicate the standard deviation of 5 egg chambers. Statistical significance is estimated by two-tailed Student’s t-test; ***p<0.001. (D) Transgenic rDNA HCR-FISH signal is increased upon SUMO KD The total intensity of HCR-FISH signal was measured in individual nuclei that have positive signal. Error bars indicate the standard deviation of 5 nuclei. Statistical significance is estimated by two-tailed Student’s t-test; ***p<0.001. (E) SUMO knockdown increases pre-rRNA expression. Expression of pre-rRNAs were measured by RT-qPCR using primers that target ETS region normalized to rp49 mRNA. Germline-specific knockdown of SUMO (shSmt3) or control (shW) gene was induced by small hairpin driven by maternal-tubulin-Gal4 driver. In S2 cells, knockdown of SUMO (dsSmt3) or control (dsGFP) was induced by double stranded RNA. Error bars indicate the standard deviation of three biological replicates. Statistical significance is estimated by two-tailed Student’s t-test; **p<0.01, ***p<0.001.