Opposing Effects of Polyadenylation on the Stability of Edited and Unedited Mitochondrial RNAs in Trypanosoma brucei

Fully edited RPS12 transcripts degrade more rapidly than their unedited counterparts. RPS12UE and RPS12FE RNAs were in vitro transcribed and internally labeled with [α-³²P]GTP. These RNAs were incubated with partially purified mitochondrial fractions at 27°C for the indicated times. (Upper panel) Products were resolved on a 7 M urea-6% acrylamide gel and visualized by autoradiography. (Lower panel) Percent full-length RNA remaining was determined by densitometry of a nonsaturated autoradiograph and was plotted for each time point.

An edited cis-acting sequence encompassing only six editing sites is sufficient to facilitate decay of RPS12 transcripts. (A) Schematic representation of unedited, fully edited, and two partially edited RPS12 RNAs (shown 5′ to 3′, not to scale). RPS12PE6 is edited at 6 of 77 editing sites (∼10% edited), and RPS12PE45 is edited at 45 of 77 editing sites (∼60% edited). Black regions indicate fully edited sequence. Gray indicates junction regions containing partially edited sequence. (B) Internally labeled RPS12UE, RPS12PE6, and RPS12PE45 transcripts were incubated with partially purified mitochondrial fractions at 27°C for the indicated times. Products were resolved on a 7 M urea-6% acrylamide gel and visualized by autoradiography (left panel). Percent full-length RNA remaining was determined by densitometry of a nonsaturated autoradiograph and was plotted for each time point (right panel).

A poly(A)₂₀ tail destabilizes unedited RPS12 RNAs but stabilizes fully edited RPS12 RNAs. RPS12UE (A) and RPS12FE (B) RNAs with or without a 3′ 20-A tail were synthesized in vitro. RNA degradation reactions were performed as described in the legend for Fig. 2. The left panels show the autoradiographs. The percent full-length RNA remaining for each time point as determined by densitometry is plotted in the right panels.

Presence of edited cis-acting sequences encompassing six editing sites is sufficient to switch a poly(A)₂₀ tail from a destabilizing to a stabilizing element for RPS12 transcripts. RPS12UE (A), RPS12PE45 (B), and RPS12PE6 (C) either with or without a 20-A tail were transcribed and internally labeled in vitro. Degradation of these RNAs was measured in vitro over a time course as described in the legend for Fig. 2. Products were resolved on a 7 M urea-6% acrylamide gel and visualized by autoradiography (left panels). Percent full-length RNA remaining was determined by densitometry and was plotted for each time point in the right panels.

Effects of two in vivo tails on stability of fully edited RPS12 RNAs. RPS12FE RNAs with no tail, a poly(A)₂₀ tail, a poly(A)₂₀₀ tail (A), or a poly(AU)₂₀ tail (B) were synthesized in vitro. These RNAs were incubated with partially purified mitochondrial fractions at 27°C for the indicated times. Products were resolved on a 7 M urea-6% acrylamide gel and visualized by autoradiography (left panels). Percent full-length RNA remaining was determined by densitometry and was plotted for each time point (right panels).

A poly(A)₅ tail and a poly(U)₂₀ tail fail to stabilize fully edited RPS12 RNA. RPS12FE RNAs with either no tail or a 20-A, 5-A, or 20-U tail were transcribed and internally labeled in vitro. These RNAs were incubated with partially purified mitochondrial fractions at 27°C for the indicated times. Products were resolved on a 7 M urea-6% acrylamide gel and visualized by autoradiography (left panel). Percent full-length RNA remaining was determined by densitometry and was plotted for each time point (right panel).

Poly(A) homopolymers fail to stimulate decay of RPS12FE-20A. RPS12FE-20A RNAs were incubated with partially purified mitochondrial fractions in the presence of 0, 10, 100, 250, or 500 ng of poly(A) homopolymers at 27°C for 60 min. Products were resolved on a 7 M urea-6% acrylamide gel and visualized by autoradiography, and percent full-length RNA remaining was determined by densitometry for each poly(A) concentration. Relative percent full-length RNA remaining with poly(A) addition was calculated by using the 0-ng poly(A) reaction mixture as 100%.

Degradation of fully edited RPS12 transcripts involves both endonuclease and 3′-to-5′ exoribonuclease activities. (A) RPS12FE RNAs with or without a poly(A)₂₀ tail were labeled at their 5′ or 3′ ends and incubated with partially purified mitochondrial fractions for 15, 30, 45, or 60 min. Products were resolved on a 7 M urea-6% acrylamide gel and visualized by phosphorimager analysis. Shown on either side of the gel is the schematic representation of RNA molecules, both full-length substrates and proposed degradation intermediates. Corresponding endonucleolytic cleavage products are represented as X and Y (5′ products) and X′ and Y′ (3′ products). Arrowheads indicate minor degradation intermediates. (B) Products from the in vitro degradation assays using 3′-labeled RNAs as substrates were spotted onto a polyethyleneimine-F-cellulose plate, developed using 0.75 M Tris and 0.45 M HCl, and visualized by phosphorimager analysis. The migration positions of unlabeled 5′-AMP, ADP, and ATP standards were visualized by UV shadowing and are indicated on the left.