Trypanosoma brucei Tb927.2.6100 is an essential protein associated with kinetoplast DNA

Abstract

The mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.

Fig 1

Tb927.2.6100 associates with the kDNA. The tagged protein was visualized by fluorescence microscopy using polyclonal anti-c myc antiserum coupled with fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Panels show phase-contrast light microscopy of PF T. brucei cells, anti-c-myc antibody coupled with FITC-conjugated secondary antibody showing localization of the target protein, DAPI staining of nucleus and kDNA, and merge.

Fig 2

Effects of Tb927.2.6100 RNAi. (A) Cell growth curves of a representative RNAi cell line of Tb927.2.6100 grown in the presence (open circles) or absence (filled squares) of Tet. The cumulative cell numbers were calculated by multiplying cell densities by the dilution factor. (B) qPCR analysis of total RNA isolated from cells, in which expression of Tb927.2.6100 was repressed for 4 days by RNAi. The relative change in Tb927.2.6100 mRNA abundance was determined by using either β-tubulin mRNA (right bar) or 18S rRNA (left bar) as an internal control and is presented in log scale. (C) Effect of RNAi on kinetoplast size. DAPI staining shows kinetoplasts from cells without RNAi or after 4 days of RNAi. n, nucleus; k, kinetoplast. (D) Quantitation of the different cell cycle stages during RNAi of Tb927.2.6100 showing that the proportion of 200 randomly chosen cells having one kinetoplast/one nucleus (1K1N) or two kinetoplasts/one nucleus (2K1N) is reduced during RNAi of Tb927.2.6100, whereas the proportion of these cells having no kinetoplast/one nucleus (0K/1N) is increased dramatically, as is the proportion having a reduced-size kinetoplast/one nucleus (1ksmall1N).

Fig 3

Quantitative PCR of kDNA. (A) Threshold cycles using different amounts of DNA from DK164 cells (no mitochondrial DNA) and kDNA from PF 29.13 cells. Primers that target the maxicircle (max1, A6 pre) and minicircle (min2) DNA showed no significant amplification without mitochondrial DNA (DK164), whereas good amplification was obtained using kDNA as the template. Error bars represent standard deviations of measured C_T replicates. (B) RNAi of Tb927.2.6100 results in decreases of both maxicircle and minicircle DNAs, with the maxicircle decreasing to a larger extent (∼20%) than the minicircle (∼30%). (C) Maxicircle and minicircle abundance is not altered by KREX2 repression by RNAi, a control that indicates that effects of Tb927.2.6100 RNAi are specific. β-Tubulin was used as the internal reference for relative comparisons.

Fig 4

Quantitative PCR of mtRNA. RNAi of Tb927.2.6100 results in the strong decrease of the examined mt transcripts over the course of 6 days. RNAs examined included both pre-edited and edited ATPase subunit 6 (A6 pre and A6 ed, respectively), never-edited COI, and the polycistronic junction regions of primary transcripts corresponding to RPS12 and ND5 or 9S and ND8.