Complete cycles of bloodstream trypanosome RNA editing in vitro

Abstract

RNA editing in kinetoplastid protists is required for the maturation of mitochondrial pre-mRNAs and occurs by protein-catalyzed cycles of uridylate insertion and deletion. During the complex life cycle of Trypanosoma brucei this process is differentially regulated in the mammalian bloodstream and insect procyclic stages. Complementary guide RNAs (gRNAs) direct editing, but the abundance of these transcripts is not developmentally controlled. The establishment of in vitro systems that recreate efficient RNA editing in bloodstream T. brucei would be valuable for mechanistic studies of regulation. Here we describe a robust in vitro system that reconstitutes full cycles of both U insertion and U deletion in bloodstream trypanosomes, and the first direct comparisons of the in vitro systems for strains of mammalian and insect stages.

FIGURE 1.

Complete cycles of Bf RNA editing. A6 deletion and insertion cycles in Bf crude mitochondrial extracts using 3′ -end-labeled pre-mRNA and enhanced gRNAs. (A) Deletion reaction with and without gRNA. (B) Insertion reaction with gRNA and UTP or without either gRNA or UTP (lanes 1–3). Upon editing reaction completion and deproteinization, the RNA transcript was treated or not with calf intestinal alkaline phosphatase (CIP) and loaded (lanes 4 and 5, respectively). The phosphatase treatment removes all 5′ tri- and 3′ mono-phosphates in 3′ -end-labeled RNA transcripts, thereby significantly slowing down their gel mobility. Accurate deletion and insertion products are indicated as −3 and +3, respectively. Partially edited products are also detected. (A6 in) Input A6 pre-mRNA.

FIGURE 2.

Isopycnic centrifugation of Bf and Pf editing activities. Crude mitochondrial extracts (0.5 mL at ~2 mg/mL) from strains (Bf) ILTat1.3 and (Pf) TREU667 were fractionated in glycerol gradients and each fraction assayed for full cycles of A6 deletion and insertion, and adenylylation activities (top, middle, and bottom panels, respectively). Bf (A) and Pf (B)editing activities. Some sedimentation fractions generate a +1 band (*) not related to editing (see text). The Pf ligases most commonly termed REL1 and REL2, or Bands IV and V, respectively (Rusché et al. 1997; Schnaufer et al. 2001; Simpson et al. 2004; see also McManus et al. 2000), and corresponding Bf adenylylated proteins (with [α-³²P]-ATP as in Sabatini and Hajduk 1995) are indicated as “lig”. Fraction one is at the bottom of the gradient. (C) The adenylylated Pf and Bf ligases, run side by side, exhibit the same gel mobility. Two isoforms (doublet) of the larger ligase (Rusché et al. 1997) are also present in the strain Bf ILTat1.3.

FIGURE 3.

Isopycnic centrifugation of Bf (A) and Pf (B) editing activities. Crude mitochondrial extracts (0.5 mL at ~2 mg/mL) from Bf (90.13) and Pf (29.14) cell lines of the strain 427 were fractionated in glycerol gradients and each fraction assayed for full cycles of A6 insertion.

FIGURE 4.

ATP titrations of Pf and Bf full editing cycles of A6 pre-mRNA in ~20S fractions. (A) Bf ILTat1.3 and Pf TREU667 editing reactions (left and right panels, respectively). (−3) Accurate A6 deletion products (top panels). (+3) Accurate A6 insertion products (bottom panels). Lanes 1–5 contain 3, 0.3, 0.03, 0.003 mM, and no ATP, respectively. (B) Percent of substrate accurately edited at decreasing ATP concentrations. Empty and filled symbols denote Bf and Pf reactions, respectively. Conditions not supporting visible editing are not represented in the plots. ATP concentrations higher than 3 mM (up to 4 mM) had no additional effect (not shown). The percent of accurate editing (−3 and +3 bands for deletion and insertion, respectively) at different ATP concentrations relative to the remaining input (In) was quantitated with a PhosphorImager. Some fractions generate a prominent +1 RNA band, which is not a product of editing (see text and Fig. 2 ▶ legend) and remained constant throughout the ATP titration. Each point in the plots represents the average of duplicate ATP titrations performed simultaneously, with standard deviations (a single experiment). The data is representative of at least two independent experiments.

FIGURE 5.

ATP titrations of Pf and Bf full editing cycles of A6 pre-mRNA in ~20S fractions. (A) Bf and Pf cell lines of the strain 427 (left and right panels, respectively). (B) Percent of substrate accurately edited at decreasing ATP concentrations. The titrations and analyses were performed as in Figure 4 ▶.