Effect of RBP42 depletion on cell viability. (A) RNAi-dependent ablation of RBP42 mRNA in T. brucei strain AM1. This strain contains a single intact RBP42 allele, as well as the RNAi construct. (B) Analysis of genomic DNA to assess the AM1 genotype. PCR reactions utilized primers-A, -B, -C, and -D located as shown in panel A. The 0.6-kb DNA in lane 4 and the 0.9-kb DNA in lane 6 confirm that AM1 contains a puromycin cassette in place of one of the two endogenous RBP42 alleles. The 1.3-kb band in lane 2 shows that the remaining RBP42 allele is intact. (Lanes 1,3,5) Controls showing the presence of intact RBP42 alleles in the parental clone 3 cell line. (C) RBP42 is decreased after ablation of mRNA by tetracycline-dependent RNAi induction, as demonstrated by immunoblot analysis. α-Tubulin levels are shown as a loading control. (D) T. brucei requires RBP42 for cell viability. Tetracycline, which activates the RNAi-dependent ablation of RBP42, was added at day 0. Parasites were directly observed and counted daily using a hemocytometer. Three clones were studied and AM1 chosen as a representative example of the observed phenotype. (E) Cellular morphology and cytokinesis are altered as a result of RBP42 depletion. Examples of the predominant phenotypes are shown as fluorescent micrographs containing DAPI-stained nuclei and kinetoplasts (both in blue) of parasites that represent each phenotype observed on day 4 after RBP42 depletion. Quantification of DAPI-stained nuclei per cell is shown for n = 445 cells in the histogram to the right of micrographs. Open bars, normal phenotypic distribution.
