High-throughput analysis of an RNAi library identifies novel kinase targets in Trypanosoma brucei

Abstract

New drugs are needed to treat human African trypanosomiasis because the currently approved treatments are toxic or limited in efficacy. One strategy for developing new drugs involves discovering novel genes whose products can be targeted for modulation by small-molecule chemotherapeutic agents. The Trypanosoma brucei genome contains many genes with the potential to become such targets. Kinases represent one group of genes that regulate many important cell functions and can be modulated by small molecules, thus representing a promising group of enzymes to screen for potential therapeutic targets. RNAi screens could help identify the most promising kinase targets, but the lack of suitable assays represents a barrier for optimizing the use of this technology in T. brucei. Here, we describe an RNAi screen of a small RNAi library targeting 30 members of the T. brucei kinome utilizing a luciferase-based assay. This screen both validated the luciferase-based assay as a suitable method for conducting RNAi screens in T. brucei and also identified two kinases (CRK12 and ERK8) that are essential for normal proliferation by the parasite.

Figure 1. Flow of luciferase-based RNAi Screen

31 partial cDNA fragments were PCR amplified from genomic DNA and subcloned into RNAi plasmids. The plasmids were linearized with NotI and transfected into T. brucei 90-13 to make stable RNAi clones A). Each of the T. brucei RNAi clones was counted then diluted with HMI-9 and transferred to 96-well plates so that each well contained 10⁴ clones. Each row contained 12 replicates of RNAi clones that were maintained in either control medium or induction medium containing 100 ng/mL tetracycline, allowing 4 RNAi clones to be tested per plate. At the appropriate time point (days 3, 6 or 9 post-induction), luciferase reagent was added to the plates and RLU values were obtained with read with a luminometer B).

Figure 2. Pair-wise comparison of RNAi clones

Mean RLU (RLU_Mean) values of control vs. tetracycline induced RNAi clones as graphed in histograms. Bars in the histogram represent the RLU_Mean from an entire row of RNAi clones. Each plate contains the data from 4 clones in which the control and tetracycline-induced RLU_Mean was evaluated in a pair-wise manner. Replicas were made from each plate prior to reading and then assayed on every third day for nine days, as represented by each column (Day 3, Day 6, Day 9). The mean and standard deviations were calculated for each row. The Student’s t- test was used to determine which RNAi clones had significant differences in the RLU_Mean in response to tetracycline induction. Clones in which the RLU_Mean values were significantly different are represented by the hatched bars.

Figure 3. Pair-wise counting of RNAi clones

The 5 RNAi clones that showed a significant change in growth rate over all 3 reads were each expanded into individual flasks and counted every day for 9 days. The numbers were plotted as a line graph with the log₁₀ cell number plotted on the y-axis. For each clone, the closed diagrams (● ▲ ◆) represent the respective clones growing under control conditions whereas open diagrams (○ △ ◇) represent the cones being induced with tetracycline at 100 ng/ul. TbK5 and TbK24 represent the clones with strong growth phenotypes after RNAi induction A). TbK6, TbK8 and TbK16 represent the clones with subtle growth phenotypes after RNAi induction B). The values on the graph represent the averages of 3 separate experiments.

Figure 4. HTS assay format for RNAi library

Each of the wells tested were inoculated with 10⁴ RNAi clones in medium containing 100 ng/µL tetracycline. Duplicated screens of the RNAi library were conducted on each 96-well plate. The data points graphed on the scatter plot represent the log-transformed RLU values for each of the replicated RNAi clones tested in the screen. A). The RLU_Mean values for all of the RNAi clones with SD were calculated and then graphed as a separate scatter plot B). The Z_score at 1.96 below the mean RLU depicted by the dashed line represents the cut-off point for the p =0.05.

Figure 5. Northern blot analysis of K5 and K24

The two proliferation-deficient clones identified from screening the 30 RNAi clones in HTS format were further analyzed by northern blot analysis. Total RNA was isolated from control or tetracycline-induced clones after 48 hours post induction. Labeled gene specific probes were hybridized to the blots and visualized by autoradiography. CRK12 A). ERK8 B).