Analysis of edited MURF2 RNA non-encoded tails.

<p>A. Percentage of the total population possessing each tail type in cells collected 3 days post-induction of RNAi and in uninduced cells. Total number of tails in each of the three populations ranged from 17 to 26. B. For each non-encoded tail obtained from each cell type, the percentage U in the tail was calculated and plotted. “Control population” is comprised of sequences derived from RNA of <i>uninduced</i> cultures of the TbRND, KPAP1, and KPAP1/TbRND RNAi cell lines.</p

Gene expression to mitochondrial metabolism: Variability among cultured <i>Trypanosoma cruzi</i> strains

<div><p>The insect-transmitted protozoan parasite <i>Trypanosoma cruzi</i> experiences changes in nutrient availability and rate of flux through different metabolic pathways across its life cycle. The species encompasses much genetic diversity of both the nuclear and mitochondrial genomes among isolated strains. The genetic or expression variation of both genomes are likely to impact metabolic responses to environmental stimuli, and even steady state metabolic function, among strains. To begin formal characterization these differences, we compared aspects of metabolism between genetically similar strains CL Brener and Tulahuen with less similar Esmeraldo and Sylvio X10 strains in a culture environment. Epimastigotes of all strains took up glucose at similar rates. However, the degree of medium acidification that could be observed when glucose was absent from the medium varied by strain, indicating potential differences in excreted metabolic byproducts. Our main focus was differences related to electron transport chain function. We observed differences in ATP-coupled respiration and maximal respiratory capacity, mitochondrial membrane potential, and mitochondrial morphology between strains, despite the fact that abundances of two nuclear-encoded proteins of the electron transport chain are similar between strains. RNA sequencing reveals strain-specific differences in abundances of mRNAs encoding proteins of the respiratory chain but also other metabolic processes. From these differences in metabolism and mitochondrial phenotypes we have generated tentative models for the differential metabolic fluxes or differences in gene expression that may underlie these results.</p></div

Mitochondrial transcript abundances in TbRND, KPAP1, and dual-TbRND/KPAP1 RNAi cell lines.

<p>Relative transcript levels of indicated transcripts compared to levels prior to RNAi induction, as determined by quantitative RT-PCR normalized to 18S RNA. A. Edited transcripts. p, amplification with primers specific to the pre-edited form of the transcript; e, amplification with primers specific to the edited form. B. Never-edited transcripts. The abundance values for 9S and 12S rRNAs for the TbRND RNAi were published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037639#pone.0037639-Zimmer1" target="_blank">[26]</a>, but included here for the purposed of comparison. C. Pre-processed transcripts. Amplification with primers spanning two adjacent genes.</p

Different strains have unique mitochondrion morphologies.

<p>Mitochondrion morphologies of four <i>Trypanosoma cruzi</i> strains were determined using MitoTracker Deep Far Red FM probe (shown in green for easier visualization). Exponentially growing parasites in normal medium (LIT) were stained with 250 nM MitoTracker in PBS, fixed with paraformaldehyde and counter stained with DAPI following fixation. Cells were visualized and qualitatively described for three biological replicates. Representative images for each strain are presented. Arrows in Esmeraldo point to punctate mitochondrial structures, and arrows in Sylvio X10 indicate larger mitochondrial blobs positioned close to the cell membrane.</p

Analysis of pre- and partially-edited RPS12 RNA non-encoded tails.

<p>A. Percentage of the total population that possesses each tail type for RPS12 pre-edited transcripts in cells collected 3 days post-induction of RNAi and in uninduced cells. Total number of tails in each of the three populations ranged from 29 to 41. B. For each non-encoded tail obtained from each cell type for the pre-edited RPS12 transcripts, the percentage U in the tail was calculated and plotted. “Control” population is comprised of sequences derived from RNA of <i>uninduced</i> cultures of the TbRND, KPAP1, and KPAP1/TbRND RNAi cell lines. C. Average length of U tail in indicated cell lines with standard error shown. D. Same as B, except comparing the percentage U in tails from RPS12 transcripts prior to editing to those that were in the process being edited from both Control and KPAP1 RNAi cells. Pre-edited KPAP1 RNAi data was transposed from B for comparison. Tails in the control population for partially-edited RPS12 were obtained only from KPAP1 RNAi <i>uninduced</i> cells only.</p

Differential gene expression analysis of key metabolic pathways between <i>Trypanosoma cruzi</i> strains.

<p>The functional categories analyzed are listed in the first column; with the identities of genes within these categories provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197983#pone.0197983.s003" target="_blank">S3 Table</a>. The total number of mRNAs that are at least three-fold differentially expressed between three pair-wise comparisons are shown in the next three columns. For the Esmeraldo vs. Sylvio X10 comparison, the percentage of analyzed <i>T</i>. <i>cruzi</i> genes that were found to be differentially expressed for each category is also shown. If the categories contained two or more differentially expressed Esmeraldo vs. Silvio X10 genes, the directionality of the regulation is presented (up or down). For regulation to be considered directional, zero or one gene only was permitted to be regulated in the opposite direction as the rest of the differentially expressed genes within that category. Strains analyzed are CL Brener (CLB); Sylvio X10 (Syl); Esmeraldo (Esm).</p

Three models explaining indirect stabilizing effects of TbRND depletion on mRNAs in a KPAP1-depleted background.

<p>A. TbRND recruits exoribonucleases to nonadenylated transcripts. B. Upon TbRND/KPAP1 co-depletion, RNAs that are normally targets of TbRND build up and generally dilute the effects of mitochondrial ribonucleases on other transcripts, thus stabilizing them. C. TbRND degrades uridylated antisense transcripts that stabilize an mRNA.</p

Analysis of ND4 RNA non-encoded tails.

<p>A. Percentage of the total population that possesses each tail type in cells collected 3 days post-induction of RNAi and in uninduced cells. Total number of tails in each of the three populations ranged from 34 to 49. B. For each non-encoded tail obtained from each cell type, the percentage U in the tail was calculated and plotted. “Control population” is comprised of sequences derived from RNA of <i>uninduced</i> cultures of the TbRND, KPAP1, and KPAP1/TbRND RNAi cell lines.</p

Mitochondrial electron transport chain (ETC) utilization and membrane potential in <i>Trypanosoma cruzi</i> strains.

<p>A mitochondrial stress test was used to determine ETC utilization among different strains in exponential growth in regular growth medium and after 4 days in restricted medium. Oxygen consumption rate measurements are normalized to SYBR Green 1 fluorescence. <b>A.</b> Basal respiration measured prior to ETC inhibitor addition. <b>B.</b> ATP-coupled respiration. <b>C</b>. Proton leak. <b>D.</b> Spare respiratory capacity. Data show the means of 3 biological replicates (A-D). <b>E.</b> Mitochondrion membrane potential (ΔΨm) was measured by detecting the incorporation of the TMRM in both regular growth medium and in restricted medium cultures. ΔΨm of <i>T</i>. <i>cruzi</i> strains were plotted relative to strain CL Brener. Data show the means of 4 biological replicates. <b>F.</b> Percent change in the mitochondrial membrane potential (ΔΔΨm) was calculated by subtracting the potential values in the restricted medium from the potential values in the exponentially growing cultures. Means of absolute values from 4 biological replicates were plotted. <b>G.</b> <i>T</i>. <i>cruzi</i> lysates of different strains from exponentially growing cells in regular growth medium were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with <i>Leishmania major</i> COXIV (complex IV) antibody and <i>Trypanosoma brucei</i> β-subunit (ATP synthase) antibody. α-tubulin was used as a loading control. Representative blots are presented along with the normalized densitometry analysis of 4 biological replicates. Error bars represent the standard error of the mean. p-values shown below each plot were generated using one-way analysis of variance (ANOVA). <i>Post-hoc</i> Tukey’s test was performed to compare each data set to each other. * represents p ≤ 0.05 and ** represents p ≤ 0.01.</p

Differential gene expression between cultured <i>Trypanosoma cruzi</i> strains.

<p>Bar plots of the number of genes at least three-fold differentially expressed in each two-strain comparison listed on the y axis. ESM vs. SYL, expression in strain Esmeraldo relative to Sylvio X10; CLB vs. SYL, expression in strain CL Brener relative to Sylvio X10; CLB vs. ESM, expression in CL Brener relative to Esmeraldo.</p