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Abstract
In eukaryotes, cyclin-dependent kinases (CDKs) control the cell cycle and critical steps in gene expression. The lethal parasite Trypanosoma brucei, member of the phylogenetic order Kinetoplastida, possesses eleven CDKs which, due to high sequence divergence, were generically termed CDC2-related kinases (CRKs). While several CRKs have been implied in the cell cycle, CRK9 was the first trypanosome CDK shown to control the unusual mode of gene expression found in kinetoplastids. In these organisms, protein-coding genes are arranged in tandem arrays which are transcribed polycistronically. Individual mRNAs are processed from precursor RNA by spliced leader (SL) trans splicing and polyadenylation. CRK9 ablation was lethal in cultured trypanosomes, causing a block of trans splicing before the first transesterification step. Additionally, CRK9 silencing led to dephosphorylation of RNA polymerase II and to hypomethylation of the SL cap structure. Here, we tandem affinity-purified CRK9 and, among potential CRK9 substrates and modifying enzymes, discovered an unusual tripartite complex comprising CRK9, a new L-type cyclin (CYC12) and a protein, termed CRK9-associated protein (CRK9AP), that is only conserved among kinetoplastids. Silencing of either CYC12 or CRK9AP reproduced the effects of depleting CRK9, identifying these proteins as functional partners of CRK9 in vivo. While mammalian cyclin L binds to CDK11, the CRK9 complex deviates substantially from that of CDK11, requiring CRK9AP for efficient CRK9 complex formation and autophosphorylation in vitro. Interference with this unusual CDK rescued mice from lethal trypanosome infections, validating CRK9 as a potential chemotherapeutic target.
Highlights
Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are unicellular, vector borne, human parasites belonging to the early-diverged phylogenetic order Kinetoplastida whose hallmark, the kinetoplast, is a network of catenated mitochondrial DNA
We recently discovered that the cyclin-dependent kinase (CDK) cdc2-related kinase 9 (CRK9) of the kinetoplastid Trypanosoma brucei is essential for trans splicing and parasite viability
The tripartite nature of the CRK9 complex and sequence insertions that disrupt both kinase and cyclin domains suggest that CRK9 deviates structurally from human CDKs
Summary
Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are unicellular, vector borne, human parasites belonging to the early-diverged phylogenetic order Kinetoplastida whose hallmark, the kinetoplast, is a network of catenated mitochondrial DNA. Kinetoplastid parasites collectively affect millions of people worldwide primarily in developing countries, causing both debilitating and fatal human diseases. Trypanosomatid parasites share a unique mode of protein coding gene expression that is distinct from their hosts. Their genomes are organized in large gene clusters of tandemly linked protein coding genes which are transcribed polycistronically. In T. brucei, the cdc2-related kinase 9 (CRK9) appears to be of crucial importance for this unusual mode of gene expression since depletion of this cyclin-dependent kinase (CDK) led to a lethal block of trans splicing in both the insectstage procyclic (PF) and the mammalian-infective bloodstream form (BF) of the parasite [10]
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