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Chapter 22 : RNA Editing in Mitochondria

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Abstract:

In vitro approaches have facilitated mechanistic studies designed to look at the timing, accuracy and efficiency of insertional editing of newly synthesized RNAs. Because different assays are required for these distinct RNA populations, the two approaches are discussed separately. Importantly, upon incubation under editing conditions, nucleotide insertion into previously synthesized, unedited RNA was not observed, even though newly transcribed RNA made during the ‘’chase’’ reaction was fully edited . The lack of unedited regions in RNAs labeled in isolated mitochondria under standard, non-limiting nucleotide concentrations and the proximity of RNA editing to the site of transcription, coupled with the inability of the editing activity to insert nucleotides into previously synthesized RNAs, argue that there is a limited ‘’window of opportunity’’ for editing in mitochondria. Given the proximity of nucleotide insertion to the active site of transcription, it is likely that the mitochondrial RNA polymerase plays some role, direct or indirect, in the editing process. Nonencoded nucleotides are known to be added very close to the growing end of the RNA chain in mitochondria , and it is possible that they are added concurrently with RNA synthesis. Although transcription normally is templated, many RNA polymerases are capable of adding nucleotides in a nontemplated or pseudotemplated manner. Much of the mitochondrial genome of has yet to be sequenced, and it is possible that additional types of editing events may yet be discovered upon characterization of the remaining genes and their RNA products.

Citation: Gott J, Visomirski-Robic L. 1998. RNA Editing in Mitochondria, p 395-411. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch22

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Figures

Image of Figure 1
Figure 1

Functional outcome of editing in mitochondria. A region of the DNA sequence is shown, with gaps at sites of nucleotide insertion at the RNA level. Nucleotides added at each site are indicated (+N), as are the four sites of apparent С to U changes (C → u). Predicted translation products in all three reading frames (single-letter symbols) are given below the mitochondrial DNA sequence. Underlined amino acids are those that match the consensus sequence for CoI proteins from a wide range of organisms ( ).

Citation: Gott J, Visomirski-Robic L. 1998. RNA Editing in Mitochondria, p 395-411. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch22
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Image of Figure 2
Figure 2

Analysis of labeled RNAs synthesized in vitro. Run-on transcripts are labeled in isolated mitochondria with [ P] NTP prior to isolation of mitochondrial RNA. Total RNA is then hybridized to ssDNA corresponding to the region of interest and digested with S1 nuclease. Hybrid-protected RNAs are gel-purified and digested with RNase Τ. T oligonucleotides are separated in one dimension (on 15–20% acrylamide denaturing gels) or in two dimensions via RNA fingerprinting (low-pH gel and homochromatography). Black spots indicate oligonucleotides which are present in both edited and unedited controls. Oligonucleotides whose mobility is affected by insertional or substitutional editing are indicated by hatched or shaded spots, respectively. Individual bands or spots can be isolated and analyzed further with additional nucleases, if desired. In the example shown, an [-P]ATP-labeled T oligonucleotide, [CpUUp(c)CpUUpGp, where (c) indicates the inserted С residue and underlining indicates labeled nucleotides] was digested with RNase T and the resulting 3′ monophosphates (Np) were separated in two dimensions via thin-layer chromatography. The labeled phosphates are transfered to the nucleotide 5′ of each A residue. Examples of each of these techniques can be found in .

Citation: Gott J, Visomirski-Robic L. 1998. RNA Editing in Mitochondria, p 395-411. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch22
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Image of Figure 3
Figure 3

Schematic illustration of S1 nuclease protection experiments used to determine whether nascent RNAs synthesized in vivo are edited by С to U changes and dinucleotide insertions. The site of adjacent С to U changes is cleaved when nascent RNA is annealed to ssDNA corresponding to edited sequence, but fully protected by the unedited sequence. The opposite result is observed at sites of dinucleotide insertion, indicating that these two types of editing occur at different times in vivo.

Citation: Gott J, Visomirski-Robic L. 1998. RNA Editing in Mitochondria, p 395-411. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch22
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Figure 4

Coupling of transcription and editing in . Editing by С insertion (indicated by C's) is significantly reduced when RNAs are synthesized in the presence of low levels of CTP in isolated mitochondria. Once unedited RNA is made, it remains unedited even when incubated under “editing” conditions. RNA synthesized downstream of unedited regions is edited under these conditions. See for data and experimental details. Horizontal lines, RNA made in vivo; hatched bars, RNA made during pulse-labeling; solid bars, RNA made during chase.

Citation: Gott J, Visomirski-Robic L. 1998. RNA Editing in Mitochondria, p 395-411. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch22
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Figure 5

Location of potential -acting signals in editing. Precedents for roles in other processes for each of these regions are indicated (see references in text).

Citation: Gott J, Visomirski-Robic L. 1998. RNA Editing in Mitochondria, p 395-411. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch22
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Tables

Generic image for table
Table 1

Characterized editing events in mitochondria

Citation: Gott J, Visomirski-Robic L. 1998. RNA Editing in Mitochondria, p 395-411. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch22

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