MRB8620 plays a role in RNA editing. (A) RNA was isolated from PS cultured in SDM80 to Day 4 post-induction. RNA abundance was quantified by qRT-PCR using primer pairs specific for selected never-edited, minimally edited, pan-edited, and ribosomal RNAs, as labeled on the x-axis. The bar graph depicts the relative abundance of RNA levels in tetracycline-induced T. brucei compared to uninduced-control cells. RNA levels were standardized to β-tubulin (black bar) or 18S (striped bar) cDNA. Whiskers denote range of obtained relative abundances within technical triplicates. (B) Levels of gRNAs in MRB8620 RNAi T. brucei grown in SDM80. Nylon membranes were probed with 5′ end labeled oligonucleotides complementary to gA6[14], gMURF2[II], or gCO3 gRNAs, with 5.8S rRNA set as a loading control for each membrane. The signals were quantified and normalized to the levels of 5.8S RNA from the same sample. The bar graph on the right depicts fold change in RNAi-induced cells compared with uninduced-control T. brucei and whiskers the error between duplicates. (C) As in A, where the cells were cultured in SDM79. Top of the graph indicates whether species of RNA assayed is never-edited (NE), minimally, pan-edited, or mitochondrial rRNA. The following abbreviations were used for mRNAs assayed by qPCR: ATPase subunit 6 (A6), cytochrome c oxidase subunits 1 (CO1), 2 (CO2), and 3 (CO3), cytochrome reductase subunit b (CYB), maxicircle unknown reading frame 2 (MURF2), NADH dehydrogenase subunits 4 (ND4) and 7 (ND7), and ribosomal protein S12 (RPS12). (P) Pre-edited RNA; (E) edited RNA.
