Fig. 3: SLAP1 regulates SL-RNA production with knockdown resulting in an ES transcription block.
From: An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes
a SR-SIM imaging of a BF T. brucei cell line with nuclear signals corresponding to mNG::SLAP1 (SLAP1 mNG, green) or SNAP3::6xHA (SNAP3-HA, red), with the SL-RNA intron (magenta) visualised with RNA-FISH. SLAP1 RNAi was induced for the time in hours (h). b) RNA quantitation using qPCR from cells in (a) after induction of SLAP1 RNAi for the time in hours (h). Nascent RNA is from the promoter (Prom) or telomere (Telo) of BES1, or from the actin or rRNA loci. Average values are from three biological replicates with error bars indicating ± SD. **P ≤ 0.01; *P ≤ 0.05. For SLAP1 mRNA, P = 0.006, BES1 telomere pre-mRNA P = 0.02 and actin pre-mRNA P = 0.0051. c Fluorescence microscopy imaging of a BF T. brucei cell line, with mNG::SLAP1 (SLAP1 mNG, green) and SNAP3::6xHA (SNAP3-HA, red) signal shown, with VSGΨ transcript (magenta) detected with RNA-FISH. SLAP1 RNAi was induced for the time in hours (h). d Quantitation of the percentage (%) of cells in (c) with VSGΨ RNA-FISH signal after induction of SLAP1 RNAi for the time in hours (h). Values are the average of three biological replicates, with error bars indicating ± SD. At least 297 1K1N cells were counted for each condition. *P = 0.029. e Fluorescence microscopy imaging of a BF T. brucei cell line with a signal corresponding to mNG::SLAP1 (SLAP1-mNG, green) or TdT::RPA2 (RPA2-TdT, red), and a white arrowhead indicating the ESB. SLAP1 RNAi was induced for the time in hours (h). f Quantitation of the percentage (%) of cells in (e) with a discrete ESB after induction of SLAP1 RNAi for the time indicated in hours (h). Averages of three biological replicates are shown, with error bars indicating ± SD. At least 247 1K1N cells were counted for each condition. **P = 0.0031. In all microscopy images nuclei (dashed lines) are stained with DAPI (blue). On bar graphs, the values from individual replicates are shown with circles. All P values were determined using a two-tailed paired Student’s t test. All scale bars = 1 µm. Source data are provided as a Source Data file.
