An RNAi library for identifying mitochondrial DNA replication proteins in Trypanosoma brucei.

摘要

Trypanosoma brucei causes African sleeping sickness and is one of the earliest eukaryotes with a mitochondrion. Its mitochondrial genome, kinetoplast DNA (kDNA), is a network of thousands of interlocked DNA circles known as minicircles and maxicircles. During kDNA replication, minicircles are released from the network, they replicate as free minicircles, and the progeny reattach to the network. Approximately 30 kDNA replication proteins are known, but we estimate that there are over 100.; To identify new kDNA replication proteins, we used an RNAi library (trypanosomes containing random genomic fragments in an integrated inducible RNAi vector). Inhibition of replication causes kDNA loss, which is lethal. Therefore, we developed a new procedure for screening for essential genes. We cloned cells from the library, induced RNAi in each clone, screened for kDNA loss, and identified the relevant gene in an uninduced clone by PCR of the RNAi vector insert. In one clone with inducible kDNA loss, the RNAi vector had aberrantly integrated into the genome, leading to over-expression of 10 downstream genes. One of these genes, whose over-expression conferred kDNA loss, is CBRL (cytochrome b5 reductase-like). We found that CBRL is a mitochondrial protein, and that its RNAi or knockout had no effect on kDNA or growth. How does CBRL over-expression cause kDNA loss? We found that over-expression causes oxidation/oligomerization of UMSBP, the minicircle replication origin binding protein. Oligomerized UMSBP cannot bind replication origins. Consequently, minicircles are released from the network but not replicated or reattached. This leads to accumulation of replication intermediates and eventual shrinkage and loss of the network. We also found that maxicircles, which encode some respiratory chain proteins, are rapidly lost, possibly because they also require UMSBP for replication. We proposed that CBRL over-expression depletes mitochondria of tryparedoxin, a redox protein that may reduce UMSBP, thus leading to UMSBP oligomerization. To support this model we partially rescued the growth effect of CBRL over-expression by co-overexpressing tryparedoxin. Additionally, we found that over-expression of mitochondrial tryparedoxin peroxidase, which utilizes tryparedoxin, also caused kDNA loss and UMSBP oligomerization.