Transcription and RNA editing in a soluble in vitro system from Physarum mitochondria

Abstract

The dissection of RNA editing mechanisms in PHYSARUM: mitochondria has been hindered by the absence of a soluble in vitro system. Based on our studies in isolated mitochondria, insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs is closely linked to transcription. Here we have fractionated mitochondrial lysates, enriching for run-on RNA synthesis, and find that editing activity co-fractionates with pre-formed transcription elongation complexes. The establishment of this soluble transcription-editing system allows access to the components of the editing machinery and permits manipulation of transcription and editing substrates. Thus, the availability of this system provides, for the first time, a means of investigating roles for cis-acting elements, trans-acting factors and nucleotide requirements for the insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs. This methodology should also be broadly applicable to the study of RNA processing and editing mechanisms in a wide range of mitochondrial systems.

Figure 1

RNA synthesis in mitochondrial fractions. Run-on transcription in isolated mitochondria (lane 1), lysed mitochondria (lane 2), cleared mitochondrial lysate (lane 3), pooled TECs (lane 4) and the cleared supernatant from unlysed mitochondria (lane 5) was assayed as described in Materials and Methods. Note that the mito S130 sample (lane 5) represents the amount of RNA synthesized in a transcription reaction that had been scaled up ten-fold relative to the samples in lanes 1–4, supporting the conclusion that the clearing step is sufficient to pellet all unlysed mitochondria.

Figure 2

Fractionation of cleared mitochondrial lysates using Sepharose 4B. (A) Circles, transcription activity in the absence of added template assayed as described in Materials and Methods. Crosses, protein concentration of each fraction. (B) SDS–polyacrylamide gel electrophoresis of even numbered Sepharose 4B fractions, with bands visualized by staining with Coomassie blue.

Figure 3

Characterization of TEC. (A) Dot blot hybridization analysis of RNAs synthesized by TEC. DNAs from the nuclear genes actin and tubulin, the mitochondrial genes coI, coII and α-ATPase, and the cloning vector were immobilized and hybridized to labeled transcripts synthesized by TEC as described in Materials and Methods. (B) RNase H digestion of S1 nuclease protected α-ATPase mRNA synthesized in the absence of a cold nucleotide chase. Lane 1, no oligonucleotide, no RNase H; lane 2, oligonucleotide A in the presence of RNase H; lane 3, oligonucleotide B in the presence of RNase H. Sizes of the expected cleavage products are shown in the diagram to the right. (C) Nucleotide requirements for transcription by TEC. Run-on RNA synthesis in the presence of all four ribonucleotides (lane 1); 20 µM [α-³²P]UTP only (lane 2); 20 µM [α-³²P]UTP, 500 µM CTP and GTP (lane 3); 20 µM [α-³²P]UTP and 500 µM ATP and GTP (lane 4); or 20 µM [α-³²P]UTP and 500 µM CTP and ATP (lane 5).

Figure 4

RNA editing in TEC preparations. (A) [^α-32P]GTP-labeled RNAs were gel purified after S1 nuclease protection with an α-ATPase-specific probe, digested with ribonuclease T1 and the resulting oligonucleotides separated on a denaturing 20% polyacrylamide gel. Oligonucleotide fragments containing editing sites are indicated to the left. For oligonucleotides overlapping sites of nucleotide insertion, each apostrophe designates the presence of an added nucleotide. (B) Nearest neighbor analysis of RNase T1 fragments. RNase T1 oligonucleotides c (11 nt) and c′ (12 nt) were eluted from the gel shown in (A) and digested to mononucleotides, and the resulting 3′ NMPs were separated via two-dimensional thin-layer chromatography as described in Materials and Methods. Bottom, sequence of the RNase T1 oligonucleotides present in the S1 nuclease protected region. The size of each [^α-32P]GTP-labeled fragment visible on the gel is indicated, with the fragments containing editing sites designated with letters corresponding to the unedited (a, b, c, d) and edited (a″, b′, c′, d′) sequence. Nucleotide insertion sites are shown in lower case letters within the sequence. Note that the unedited control RNA contains two 11 nt RNase T1 fragments, while the edited control RNA has an 11mer and a 12mer.