Non-templated addition of nucleotides to the 3' end of nascent RNA during RNA editing in Physarum

Abstract

RNAs in Physarum: mitochondria contain extra nucleotides that are not encoded by the mitochondrial genome, at least in the traditional sense. While it is known that insertion of non-encoded nucleotides is linked to RNA synthesis, the exact nature of this relationship remains unclear. Here we demonstrate that the efficiency of editing is sensitive not only to the concentration of the nucleotide that is inserted, but also to the concentration of the nucleotide templated just downstream of an editing site. These data strongly support a co-transcriptional mechanism of Physarum: RNA editing in which non-encoded nucleotides are added to the 3' end of nascent RNAs. These results also suggest that transcription elongation and nucleotide insertion are competing processes and that recognition of editing sites most likely involves transient pausing by the Physarum: mitochondrial RNA polymerase. In addition, the pattern of nucleotide concentration effects, the context of editing sites and the accuracy of the mitochondrial RNA polymerase argue that the mechanism of Physarum: editing is distinct from that of other co-transcriptional editing systems.

Fig. 1. Effects of NTP concentration on C insertion within the coI gene. Top: schematic representation of the editing assay. Middle: separation of RNase T1 fragments from hybrid-protected coI mRNAs synthesized under varying NTP concentrations in isolated mitochondria. Lanes 1 and 7, digestion patterns of unedited (lane 1) and edited (lane 7) S1-protected control transcripts. Lanes 2–6, RNAs synthesized in isolated mitochondria in the presence of the indicated nucleotides. Lane 2, 0.2 µM [α-³²P]CTP and 200 µM ATP, GTP, UTP; lane 3, 0.2 µM [α-³²P]CTP and 20 µM ATP, GTP, UTP; lane 4, 0.2 µM [α-³²P]CTP and 2 µM ATP, GTP, UTP; lane 5, 0.2 µM [α-³²P]CTP and 0.2 µM ATP, GTP, UTP; and lane 6, 0.2 µM [α-³²P]GTP and 200 µM ATP, CTP, UTP. Each reaction was pulse-labeled for 3 min, then chased for 12 min with 200 µM NTPs. Note that due to differences in salt concentrations, the control digests migrate with a slightly increased mobility relative to the mitochondrial samples in this experiment. Bottom: sequence of the region of the coI mRNA analyzed, with individual RNase T1 fragments indicated. Inserted C residues are underlined, lower case letters. Oligonucleotides containing sites of nucleotide insertion, which differ in length in the two control RNAs, are labeled with lower case letters; each prime (′) designates an added nucleotide. Oligonucleotides b′ and c′, which are RNase T1 fragments containing one, rather than two, added nucleotides (Visomirski-Robic and Gott, 1997b), are indicated by arrowheads. Note that band f′ consists of a non-edited 15 nt oligonucleotide as well as the edited version of oligonucleotide f. Because RNA synthesis is extremely limited under these conditions and only RNAs that were extended to the end of the S1 probe were isolated for RNase T1 analysis, fragments near the 3′ end of the hybrid-protected RNA are more heavily labeled than the 5′ fragments, which are less likely to have been fully extended.

Fig. 2. C insertion editing in mtTEC is sensitive to the concentration of CTP. (A) Quantification of the extent of editing at C insertion sites within the α-atpase mRNA under low GTP (hatched bars) or low CTP (solid bars) conditions. RNAs synthesized by mtTEC in the presence of 20 µM [α-³²P]GTP, 500 µM CTP, ATP and UTP or 20 µM [α-³²P]CTP, 500 µM GTP, ATP and UTP were hybrid protected and digested with RNase T1 as described for Figure 1, and the resulting oligonucleotides separated in two dimensions. The RNA fingerprints from which the data are derived are presented as Supplementary data. The extent of editing at sites 24 and 25 is not included in the graph because the edited and unedited versions of the RNase T1 oligonucleotides containing these sites do not resolve well on RNA fingerprints. (B) Sequence of the region of the α-atpase mRNA analyzed, encompassing editing sites 17–26 of the α-atpase mRNA, with individual RNase T1 oligonucleotides indicated. Sites of C insertion (es17–es26) are underlined.

Fig. 2. C insertion editing in mtTEC is sensitive to the concentration of CTP. (A) Quantification of the extent of editing at C insertion sites within the α-atpase mRNA under low GTP (hatched bars) or low CTP (solid bars) conditions. RNAs synthesized by mtTEC in the presence of 20 µM [α-³²P]GTP, 500 µM CTP, ATP and UTP or 20 µM [α-³²P]CTP, 500 µM GTP, ATP and UTP were hybrid protected and digested with RNase T1 as described for Figure 1, and the resulting oligonucleotides separated in two dimensions. The RNA fingerprints from which the data are derived are presented as Supplementary data. The extent of editing at sites 24 and 25 is not included in the graph because the edited and unedited versions of the RNase T1 oligonucleotides containing these sites do not resolve well on RNA fingerprints. (B) Sequence of the region of the α-atpase mRNA analyzed, encompassing editing sites 17–26 of the α-atpase mRNA, with individual RNase T1 oligonucleotides indicated. Sites of C insertion (es17–es26) are underlined.

Fig. 3. The effect of nucleotide concentration on C insertion is context dependent. Lanes 1 and 2, digestion patterns of edited (lane 1) and unedited (lane 2) S1-protected control transcripts. Lanes 3–6, RNase T1 products from S1 nuclease-protected α-atpase transcripts synthesized by mtTEC under varying NTP concentrations. Lane 3, 20 µM ATP, 30 µM [α-³²P]GTP, 500 µM CTP, UTP; lane 4, 20 µM CTP, 30 µM [α-³²P]GTP, 500 µM ATP, UTP; lane 5, 20 µM UTP, 30 µM [α-³²P]GTP, 500 µM CTP, ATP; lane 6, 30 µM [α-³²P]GTP, 500 µM CTP, UTP, ATP. Editing sites (es #) within RNase T1 fragments resolved on this gel are indicated at the left. Bottom: sequence of the region analyzed, encompassing editing sites 48–54 of the α-atpase mRNA, with individual RNase T1 oligonucleotides indicated. Sites of C insertion are underlined.

Fig. 4. The Physarum mtRNAP does not stutter at a homopolymer tract. RNA fingerprints from an unedited T7 control transcript synthesized in the presence of 75 µM [α-³²P]ATP, 500 µM CTP, GTP and UTP after (A) and before (B) S1 nuclease protection. The fingerprinted region is the same as that shown in the Supplementary data for Figure 2, but because all of the spots of interest fall within the lower third of the fingerprint, only this region is shown here to allow for enlargement. Mobility in the first dimension is determined largely by base composition, whereas migration in the second dimension is influenced primarily by size. Extra spots are indicated by asterisks. (C and D) Fingerprints of RNAs synthesized by mtTEC in the presence of 75 µM [α-³²P]ATP, 500 µM CTP, GTP and UTP (C) or 20 µM [α-³²P]ATP, 500 µM CTP, GTP and UTP (D). The sequence of the fingerprinted region is given in the bottom panel of Figure 2. The oligonucleotide indicated by the arrowhead has the sequence UCAAAAAAAAAG. Secondary analyses of the extra oligonucleotides are presented as Supplementary data.