Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei

Figures

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  Figure 1.. Quantitative RT-PCR analysis of PCF and BSF mRNAs. Relative mRNA levels for total, pre-edited (Pre) and edited (Edit) mitochondrial mRNAs in four BSF cell lines (427, 90–13, SM-1 and SM-2) relative to those in the 29–13 PCF cell line. Values were normalized to the average of three controls (TERT, PFR2 and actin). Data shown were generated from three biological replicates of each sample with three technical replicates per biological replicate. The gray shaded area represents the range from one log2 increase (2.00) and one log2 decrease (0.50). Error bars indicate standard error of the mean.

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  Figure 2.. Reversibility of BSF-stage edited mRNA levels. (A) Western blot analysis of undifferentiated BSF 427 cells, BSF 427 cells treated with 3 mM citrate/3 mM cis-aconitate (CCA) for 2, 4 or 6 days, and PCF cells. Western blots of SM-2 cells showed similar trends (not shown). (B–F) Quantitative RT-PCR analysis of relative mRNA levels for total, pre-edited (Pre), and edited (Edit) (B) COII, (C) COIII, (D) CYb, (E) A6 and (F) ND8 mRNAs in two undifferentiated BSF cell lines (427 and SM-2) and BSF 427 and SM-2 cells treated with CCA for 2, 4 or 6 days relative to those in the PCF 29–13 cell line. Undifferentiated BSF cells compared to PCF are used as a reference starting point for each cell line. Values were normalized to the average of three controls (TERT, PFR2 and actin). Data shown were generated from two biological replicates of each sample with four technical replicates per biological replicate. The gray shaded area represents the range from one log2 increase (2.00) and one log2 decrease (0.50). Error bars indicate standard error of the mean.

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  Figure 3.. Proportions of pre-edited, partially edited, and fully edited CYb, COIII, and A6 mRNA in procyclic and bloodstream cells. (A) Schematic for the placement of CYb, COIII and A6 high throughput sequencing (HTS) primer pairs. The primers are represented by red arrows. The never-edited regions of the mRNAs are indicated in black; the pre-edited regions are indicated in gray; the edited regions of the mRNAs are indicated in yellow. (B) Pie charts illustrating the breakdown for the percentage of CYb, COIII and A6 mRNAs by editing status in procyclic (PCF) 29–13 and bloodstream cell lines 427, 90–13, SM-1 and SM-2 as determined by HTS/TREAT analysis. Red arrows denote samples containing fully edited mRNA reads. Note that ‘fully’ edited reads for A6 and COIII refer to those reads that are canonically fully edited up to the end of the forward primer.

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  Figure 4.. Editing profiles of COIII and A6 mRNA intermediates organized by editing stop site. Editing peak profiles for PCF 29–13 (upper left), BSF 427 (upper right), BSF SM-1 (lower left), and BSF SM-2 (lower right) for (A) COIII mRNA intermediates and (B) A6 mRNA intermediates. The Y axes represent the number of normalized read counts for mRNAs at each editing stop site (note some differences in scales). Red diamonds indicate the final major peak in COIII mRNA or the most prominent peak in A6 mRNA. The yellow diamonds in (B) indicate peaks at ES26 generated by alternative A6 mRNA editing in BSF 427 and SM-2 at ES27, ES29 and ES33.

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  Figure 5.. Statistical and bioinformatic analyses of COIII mRNA editing intermediates at the final prominent EPS in PCF and BSF 427 cells. (A) Schematic illustrating the positions of exacerbated pause sites (EPSs) in BSF COIII mRNA in relation to the positions of gRNA families. gRNA anchor regions are illustrated as a box while the coding information region is illustrated with a line. The hatchmarked boxes at gRNA 5′ and 3′ ends represent variation in gRNA lengths within a gRNA family. The BSF 427 cells have an alternative editing consensus sequence that differs from the canonical sequence (shown in pink). The alternative guide RNA (1-Alt) that directs the editing of this alternative sequence is shown as a purple gRNA. Every 10 ESs are indicated above the sequence. Underscores at ES10 and ES40 are inserted to clarify the position of the ES. The yellow shaded regions indicate where the reverse COIII HTS primer anneals. The red diamond represents the final prominent EPS at ES32 in this cell line. (B) Sequence alignments of the 10 most abundant junction sequences at ES32 in COIII mRNA of PCF and BSF 427 cells. The canonical edited sequence is shown red and white letters with gray or black…

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  Figure 6.. Statistical and bioinformatic analyses of COIII mRNA editing intermediates at the final prominent EPS in PCF and BSF SM-1 cells. (A) Schematic illustrating the positions of exacerbated pause sites (EPSs) in BSF COIII mRNA in relation to the position of gRNAs. Symbols as in Figure 5. The red diamond represents the final prominent EPS at ES37 in this cell line. (B) Sequence alignments of the 10 most abundant junction sequences at ES37 in COIII mRNA of PCF and BSF SM-1 cells. Symbols and shading as in Figure 5. (C) Schematic of terminator guide RNAs found in PCF (gRNAs A–C) and BSF (gRNAs D–E) annealed to COIII mRNA with the non-productive consensus junction sequence at ES37. The ratio for the counts for the canonical gRNAs that direct the editing past ES37 and the terminator gRNAs are shown as determined by previously published gRNA reads (61,62).

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  Figure 7.. Statistical and bioinformatics analysis of COIII mRNA intermediates at the final prominent EPS in PCF and BSF SM-2 cells. (A) Schematic illustrating the positions of exacerbated pause sites (EPSs) in BSF COIII mRNA in relation to the position of guide RNAs (gRNAs). Symbols as in Figure 5. The red diamond represents the final prominent EPS at ES53. (B) Sequence alignments of the 10 most abundant junction sequences at ES53 in COIII mRNA of PCF and BSF SM-2 cells. Symbols and shading as in Figure 5.

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  Figure 8.. Statistical and bioinformatics analysis of A6 mRNA editing intermediates at ES58 in PCF and BSF SM-1 cells. (A) Schematic illustrating the positions of exacerbated pause sites (EPSs) in BSF A6 mRNA in relation to the position of guide RNAs (gRNAs). Symbols as in Figure 5. The red diamond represents the most prominent EPS in SM-1 cells at ES58. (B) Sequence alignments of the 10 most abundant junction sequences at ES58 in A6 mRNA of PCF and BSF SM-1 cells. Symbols and shading as in Figure 5.

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  Figure 9.. Quantitative RT-PCR analysis of induced RBP6OE cells compared to uninduced PCF. (A) Western blot analysis of RBP6 levels at 0–8 days post-induction. Mitochondrial (mt) Hsp70 is a loading control. (B) Immunofluorescence of RBP6OE cells on day 2 post-induction (using anti-procyclin antibody), day 4 post-induction (using anti-BARP antibody) and day 6 post-induction (using internalized fluorescently-labeled dextran). DNA was visualized by DAPI staining. (C, D) Relative mRNA levels for total, pre-edited, and edited mitochondrial mRNAs between cells that were induced to ectopically express RBP6 for (C) 2 days to generate an EMF-dominant culture or (D) 8 days to generate a mixed culture of MCF and PCF cells compared to the uninduced PCF cells. Values were normalized to the average of three controls (TERT, PFR2, and actin). Data shown were generated from three biological replicates of each sample with three technical replicates per biological replicate. The gray shaded areas represent the range from one log2 increase (2.00) and one log2 decrease (0.50). Error bars indicate standard error of the mean.