The MRB1 complex functions in kinetoplastid RNA processing

Abstract

Mitochondrial (mt) gene expression in Trypanosoma brucei entails multiple types of RNA processing, including polycistronic transcript cleavage, mRNA editing, gRNA oligouridylation, and mRNA polyadenylation, which are catalyzed by various multiprotein complexes. We examined the novel mitochondrial RNA-binding 1 (MRB1) complex that has 16 associated proteins, four of which have motifs suggesting RNA interaction. RNase treatment or the lack of kDNA in mutants resulted in lower MRB1 complex sedimentation in gradients, indicating that MRB1 complex associates with kDNA transcripts. RNAi knockdowns of expression of the Tb10.406.0050 (TbRGGm, RGG motif), Tb927.6.1680 (C2H2 zinc finger), and Tb11.02.5390 (no known motif) MRB1 proteins each inhibited in vitro growth of procyclic form parasites and resulted in cells with abnormal numbers of nuclei. Knockdown of TbRGGm, but not the other two proteins, disrupted the MRB1 complex, indicating that it, but perhaps not the other two, is required for complex assembly and/or stability. The knockdowns resulted in similar but nonidentical patterns of altered in vivo abundances of edited, pre-edited, and preprocessed mt mRNAs, but did not appreciably affect the abundances of mRNAs that do not get edited. These results indicate that MRB1 complex is critical to the processing of mt RNAs, and although its specific function is unknown, it appears essential to parasite viability.

FIGURE 1.

The MRB1 complex binds to kDNA transcripts. Western analysis of mt vesicles isolated from wt PF cells, lysed in the presence (+) or absence (−) of 0.1 mg/mL RNase A, and fractionated on 10%–30% glycerol gradients. Every other 30 μL fractions were probed with (A) mAb53, which is specific for the Tb912.7.2750 protein, or (B) a mix of mAbs against the KREPA1 and KREPA2 20S core editosome. Western analysis of glycerol gradients fractions from whole-cell lysates of (C) wt BF cells and T. evansi AnTat 3/3 Dk cells, or (D) BF RKO-KREN2 cells grown in the presence (KREN2 expressed) or absence (KREN2 repressed) of tet for 3 d, all examined with mAb53.

FIGURE 2.

TbRGGm, Tb927.6.1680, and Tb11.02.5390 are important for the in vitro growth of procyclic parasites. Growth curves of a representative RNAi cell lines of (A) TbRGGm, (B) Tb927.6.1680 (C2H2 Zn finger), and (C) Tb11.02.5390 (unknown function) grown in the presence (filled squares) or absence of tet (open squares). The cumulative cell numbers were calculated by multiplying the cell densities by the dilution factor. The insets show Northern analyses of the corresponding mRNAs with the days sampled indicated, and stained gels of rRNAs in the lower panel serving as loading controls.

FIGURE 3.

TbRGGm is required for MRB1 complex assembly and/or stability. Western analysis of mt lysates from whole PF RNAi cells in which (A) TbRGGm, (B) Tb927.6.1680, or (C) Tb11.02.5390 were either expressed or repressed for 3 or 5 d as indicated. Glycerol gradients fractions were probed with either mAb53 or a mix of mAbs specific for KREPA1 and KREPA2 core proteins of 20S editosomes.

FIGURE 4.

Effects of repression of expression of TbRGGm, Tb927.6.1680, or Tb11.02.5390 on the in vivo abundance of mt RNAs. qRT- PCR analysis of total RNA isolated from PF cells, in which expression of (A) TbRGGm, (B) Tb927.6.1680, or (C) Tb11.02.5390 was repressed for 4 d by RNAi. For each target amplicon, the relative change in RNA abundance was determined by using either β-tubulin mRNA (black bars) or 18S rRNA (with bars) as an internal control. 1 indicates no change in relative amount of RNA, while bars above 1 indicate an increase and bars below 1 indicate a decrease in relative RNA amount on this log-scale graph. The assays were of pre-edited (P) and edited (E) NADH dehydrogenase subunit 7 (ND7), cytochrome oxidase subunits 2 (COII) and 3 (COIII), cytochrome reductase subunit b (Cyb), ribosomal protein S12 (RPS12), maxicircle unknown reading frame 2 (Murf2) and ATPase subunit 6 (A6) mRNAs, and the never-edited COI and ND4 mRNAs. The preprocessed regions linking 9S rRNA (9S) with ND8, Cyb with A6, and RPS12 with ND5 were also assayed.

FIGURE 5.

Microscopy of TbRGGm-depleted procyclic T. brucei. Phase and DAPI Immunofluorescence micrographs of TbRGGm-depleted cells 5 d after induction of RNAi showing (A) the three major cell cycle stages and (B) representative images of abnormal TbRGGm depleted cells. DAPI staining of nuclear and kinetoplast DNA are blue. K Indicates kDNA; N, nucleus.

FIGURE 6.

FACS analysis of T. brucei cells following RNAi knockdown of (A) TbRGGm, (B) Tb927.6.1680, or (C) Tb11.02.5390 expression. The times following RNAi induction are indicated and the DNA amounts, where C is haploid amount DNA content, are indicated by the arrows over each peak of the flow cytometry profiles.