Several technical advances have brought improvements to the ability to screen RNAi libraries in BSF T. brucei
since the chromosome 1 RNAi project was reported (22
). That study required a small consortium of labs to score each RNAi clone for many complex phenotypes associated with knocking down all of the T. brucei
genes on chromosome 1 (22
). One improvement to screening RNAi libraries in BSF parasites included the development of a more efficient transformation protocol (23
). Improved transformation efficiency has now made it feasible to construct RNAi plasmid libraries that cover the entire T. brucei
genome, a necessity for genome-wide screens. Recently, a genome-wide RNAi library was created and transfected into BSF parasites employing the more efficient protocol. The library was transfected in plasmid pools sufficient to give 9-fold genome coverage (24
). In that study, the strategy was to induce the RNAi clones with tetracycline while selecting them in the presence of melarsoprol or eflornithine. That functional genomic strategy was suitably designed as a gain-of-function screen aimed at identifying genes implicated in drug uptake or resistance.
The HTS assay described in the current study was designed to identify genes essential for the parasite to proliferate normally. Identifying proliferation-deficient clones in response to gene silencing represented an efficient way to screen for potential targets in BSF parasites. Kinases are attractive targets because of their druggable properties. They are also important regulators of physiological pathways in T. brucei
. To identify potential kinase targets, we constructed a small RNAi library consisting of 31 previously uncharacterized kinases and used them to generate 30 stable RNAi clones. One clone was removed from the library because no stable clone could be made from it despite several attempts. The promoters of the pZJM vector can “leak” producing low background levels of dsRNA (11
). The fact that this clone could not be made may indicate that the mRNA message being targeted by this RNAi construct has some important physiological function. It should be possible to productively examine this RNAi construct with an RNAi vector system with tighter promoter regulation (27
Pair-wise analysis of the library revealed several kinases that appeared to be important for normal proliferation. The kinases identified from that analysis included three cdc2-related kinases, CRK1, CRK 8 and CRK 12, one extracellular signal-related kinase, ERK 8, and one uncharacterized kinase unique to kinetoplastids, Tb927.7.4090. Upon more detailed analysis of each clone identified from the pair-wise comparison, we discovered that only two of the kinases were essential for normal proliferation.
Screening the same RNAi library in an HTS format however only identified two kinases, ERK8 and CRK12, that were essential for normal proliferation in trypanosomes. Further analysis of these two clones after RNAi by northern blot analysis revealed that the mRNA messages of these two kinases were indeed silenced after RNAi induction, validating them as essential for normal proliferation in T. brucei
. This represents the first study to identify TbERK8
as an essential gene in BSF T. brucei
is homologous to the human ERK8
gene. There are three subfamilies of mitogen-activated protein kinases (MAPKs); the ERKs, c-jun N-terminal kinases (JNK) and the p38 Map kinases. The human ERK 8 represents the latest member of the ERK subfamily of MAPK to be discovered (28
), thus little is known about the functions of this kinase in comparison to the other ERK family members. In mammalian cells, Erk8 activity becomes rapidly up-regulated in response to DNA single-strand breaks caused by hydrogen peroxide. Its activity is also up-regulated by single-strand breaks caused by other agents such as MMS, however at a reduced rate (29
). It was recently demonstrated that in human cells ERK8 regulates proliferation and DNA repair through a proliferating cell nuclear antigen (PCNA) mechanism (31
). It interacts with PCNA through a PCNA-interacting protein box (PIP) sequence aa 297-QA
-308. Interaction between the human Erk8 and PCNA is required in order to maintain the stability of PCNA in the cell, preventing PCNA from interacting with the E3 ligase HDM2 (31
). TbErk8 also contains a putative PIP box at aa 293-TAEQ
-306. However, further studies are needed to determine if TbErk8 interacts with TbPCNA through its putative PIP box.
Several cdc2-related kinases (CRKs) in Trypanosoma brucei
function analogously to their homologues in other eukaryotes as master regulators of cell cycle progression (32
). For example, cyclins E1 and CRK1 regulate G1/S transition and the complexes of cyclin E1 and CRK3 as well as that of cyclin B2 and CRK3 control passage of the G2/M boundary during cell cycle progression (34
). Like these other CRKs, it is possible that CRK12 may also be involved in cell-cycle regulation. This may explain why silencing its gene product by RNAi resulted in reduced proliferation in trypanosomes. Further studies of ERK8 and CRK12 will be necessary to confirm these hypotheses and further elucidate their function in T. brucei
Now that these two kinases have been identified as potential targets, we will take advantage of small-molecule libraries to further characterize them.
In addition to identifying two potential targets, we have tested and validated a luciferase-based assay for its suitability in screening T. brucei RNAi libraries. Here, we demonstrated that a single-step luciferase-based assay was sufficient to screen a small RNAi library. Three clones K19, K23, and K24 showed a significantly lower growth rate than the other clones present in the screen under un-induced conditions using a pair-wise comparison. The lower RLU reading for K19 occurred because this clone grew slower than the other clones present on plate 5. A plausible explanation for the slow growth rate of K19 was that the RNAi targeting vector was misincorporated in the genome. In this scenario, the vector integrates into a genomic locus and disrupts a gene that is important for normal proliferation. The reduced growth rates of clones K23 and K24 on plate 6 appeared to be caused by loading errors in the primary plate on day 3. The RLU values of these two un-induced clones were comparable to values of the other clones on plate 6 by days 6 and 9, indicating that these two clones grew normally under control conditions. These same three clones were also screened in an HTS format that included the entire set RNAi clones. The performance of the un-induced clones in the HTS format was comparable to their performance in the pair-wise format (data not shown).When RNAi was induced in this screening format, only the RLU values of K5 and K24 shifted away from the mean significantly. The RLU value of K19 shifted away from the mean, but its Zscore was less than 1.96 once again indicating that its was not significant enough to be counted as a hit. These results suggest that the luciferase-based assay was unaffected by small changes in experimental methods or parameters during both formats. Overall, we conclude that the HTS format was more robust for screening RNAi libraries than the pair-wise comparison.
The larger implication of this project is the potential for the assay to be scaled up to screen a genome-wide RNAi library consisting of the roughly 9,000 genes that make up the T. brucei genome. Identifying every essential T. brucei gene will be critical for prioritizing the targeting environment of this parasite. Having a suitable HTS assay in hand eliminates a barrier that has been a critical bottleneck in T. brucei drug discovery, and can provide the HAT community with the ability to select the best druggable targets from the complete repertoire of genes.