A simple <i>in vitro</i> RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of <i>ndh</i> transcripts

Sasaki, Toyokazu; Yukawa, Yasushi; Wakasugi, Tatsuya; Yamada, Kyoji; Sugiura, Masahiro

doi:10.1111/j.1365-313x.2006.02825.x

Cited by 51 publications

(43 citation statements)

References 48 publications

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“…For both sites, mutations introduced into the 215 to 26 region abolished competition for the binding of the editing factor, whereas mutations in the 25 to 21 region had a weaker effect. Consistent with this, mutagenesis of the ndhF RNA substrate showed that purine/pyrimidine exchanges in the 215 to 21 upstream region abolish in vitro editing of the ndhF-1 site (Sasaki et al, 2006).…”

Section: Discussionmentioning

confidence: 53%

A Study of New Arabidopsis Chloroplast RNA Editing Mutants Reveals General Features of Editing Factors and Their Target Sites

et al. 2009

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RNA editing in higher plant organelles results in the conversion of specific cytidine residues to uridine residues in RNA. The recognition of a specific target C site by the editing machinery involves trans-acting factors that bind to the RNA upstream of the C to be edited. In the last few years, analysis of mutants affected in chloroplast biogenesis has identified several pentatricopeptide repeat (PPR) proteins from the PLS subfamily that are essential for the editing of particular RNA transcripts. We selected other genes from the same subfamily and used a reverse genetics approach to identify six new chloroplast editing factors in Arabidopsis thaliana (OTP80, OTP81, OTP82, OTP84, OTP85, and OTP86). These six factors account for nine editing sites not previously assigned to an editing factor and, together with the nine PPR editing proteins previously described, explain more than half of the 34 editing events in Arabidopsis chloroplasts. OTP80, OTP81, OTP85, and OTP86 target only one editing site each, OTP82 two sites, and OTP84 three sites in different transcripts. An analysis of the target sites requiring the five editing factors involved in editing of multiple sites (CRR22, CRR28, CLB19, OTP82, and OTP84) suggests that editing factors can generally distinguish pyrimidines from purines and, at some positions, must be able to recognize specific bases.

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Section: Discussionmentioning

confidence: 53%

A Study of New Arabidopsis Chloroplast RNA Editing Mutants Reveals General Features of Editing Factors and Their Target Sites

et al. 2009

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“…Target Sequences at the RNA-editing Sites-For several editing sites in mitochondria and plastids, in vitro and in organello investigations have identified the cis-elements in the RNA context to occupy the region between 20 or 25 nucleotides upstream (5Ј) and 1 or 3 nucleotides downstream (3Ј) of the edited C (5,6,8,10,11,(43)(44)(45)(46). Therefore, for the sites affected in the mef18 to mef22 mutants, a similar extent of the recognition sequences may be expected.…”

Section: Refs T-dna Insertion Lines T-dna Locationmentioning

confidence: 99%

Reverse Genetic Screening Identifies Five E-class PPR Proteins Involved in RNA Editing in Mitochondria of Arabidopsis thaliana

Takenaka

Verbitskiy

Zehrmann

et al. 2010

Journal of Biological Chemistry

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RNA editing in flowering plant mitochondria post-transcriptionally alters several hundred nucleotides from C to U, mostly in mRNAs. Several factors required for specific RNA-editing events in plant mitochondria and plastids have been identified, all of them PPR proteins of the PLS subclass with a C-terminal E-domain and about half also with an additional DYW domain. Based on this information, we here probe the connection between E-PPR proteins and RNA editing in plant mitochondria. We initiated a reverse genetics screen of T-DNA insertion lines in Arabidopsis thaliana and investigated 58 of the 150 E-PPRcoding genes for a function in RNA editing. Six genes were identified to be involved in mitochondrial RNA editing at specific sites. Homozygous mutants of the five genes MEF18-MEF22 display no gross disturbance in their growth or development patterns, suggesting that the editing sites affected are not crucial at least in the greenhouse. These results show that a considerable percentage of the E-PPR proteins are involved in the functional processing of site-specific RNA editing in plant mitochondria.RNA editing in mitochondria of flowering plants changes 400 -500 selected cytosines to uridines mostly in coding regions of mRNAs and some tRNAs (1-3). In non-flowering plants such as Isoetes species, the number of edited nucleotides is estimated to be more than 1,500 (4), raising the question of how the to-be-edited nucleotides are distinguished from other unaltered moieties.Specific sequence contexts in the pre-mRNA yield the first part of an answer; such cis-elements are required to identify a bona fide editing site as in vivo, in organelle, and in vitro analyses of several mitochondrial RNA-editing sites have shown (5-11). The second part of the answer seems to be provided by the identification of nuclear encoded specificity factors in Arabidopsis thaliana such as MEF1, MEF9, and MEF11 (mitochondrial-editing factor (MEF)2 ), which are required intact for correct editing of specific different sites (12)(13)(14)(15).The present theory is that the cis-elements in the mitochondrial RNA molecules are individually recognized by RNA-binding proteins, which may be these MEF proteins. This model makes several predictions, which need to be tested to correct or substantiate this connection. First, the nuclear-encoded MEF proteins must show an evolutionary diversity and a variation sufficient to recognize and target the more than 400 editing sites in plant mitochondria in, for example, A. thaliana. Secondly, the MEF proteins must be able to bind RNA and do so selectively to specific RNA sequences, the identified cis-elements. Specific RNA binding has been experimentally verified for one of the proteins required for a plastid RNA-editing event, the CRR4 protein (16) in addition to similar proteins involved in other RNA maturation steps (see Refs. 17, 18; for reviews, see Refs. 19,20).In this study, we addressed the first question and initiated an analysis to test if other proteins similar to MEF1, MEF9, and MEF11 are involved i...

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“…These mutant IDS cDNAs were selected on the basis of the type of mutation and its location: (i) a stop codon mutation (p.W12X), located in the signal peptide, 4 codons downstream of the p.R8X (c.22C>T) mutation, and resulting from a G>A transition at position c.35; (ii) a premature stop codon (p.L279X) located in exon 6 resulting from a micro-deletion (c.836delT) and (iii) a missense mutation (p.C432R) located in exon 9 and caused by the transition c.1294T>C. No trace of wild-type IDS sequence was evident in cDNA derived from these three patients. To address further the possibility of experimental artefacts in the analysis of material derived from pts #1, #2 and #3, the results obtained by classical sequencing [Sanger et al, 1977] were corroborated both by pyrosequencing [Ronaghi et al, 1998;Sodhi et al, 2005] and single-nucleotide primer extension assay (SNuPE) [Sasaki et al, 2006]. Pyrosequencing, which employs a luciferasebased enzyme reaction to monitor DNA/cDNA synthesis in real time, served to confirm that transcripts bearing the wild-type IDS sequence were indeed present among cloned RT-PCR products in all three patients in addition to the expected IDS transcripts bearing the mutant sequences (Figure 2).…”

Section: Mutational Analysis Of the Ids Gene And Characterization Of mentioning

confidence: 99%

“…Single nucleotide primer extension analysis (SNuPE) is a method for allelic transcript discrimination that requires only a 1-bp difference between alleles [Sasaki et al, 2006]. RT-PCR products, obtained using the forward primer 1U (5'-GCTGCGGCGGCGGCTGCTAACTGCG-3'), located in the transcribed region upstream of exon 1, and the reverse primer, 3AL (5'-GGTCACATAGCCATTCTCCTTG-3'), located within exon 3, were purified using a Montage PCR centrifugal filter device (Millipore Corporation, Bedford, MA, USA).…”

Section: C) Single Nucleotide Primer Extension Analysismentioning

confidence: 99%

Enigmatic In Vivo iduronate-2-sulfatase (IDS) mutant transcript correction to wild-type in Hunter syndrome

et al. 2010

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Sequence analysis of the X-linked iduronate-2-sulfatase (IDS) gene in two Hunter syndrome patients revealed a lack of concordance between IDS genomic DNA and cDNA. These individuals were found to be hemizygous respectively for a nonsense mutation [c.22C>T;p.R8X] and a frameshift micro-insertion [c.10insT;p.P4Sfs] in their genomic DNA. However, both wildtype and mutant IDS sequences were evident upon cDNA analysis. Similar discrepant results were also obtained in a third unrelated patient carrying the same p.R8X mutation. Since both p.R8X mutations were inherited from carrier mothers, somatic mosaicism could be excluded. Although the presence of wild-type IDS mRNA-transcripts was confirmed in all three patients by restriction enzyme digestion, clone sequencing, pyrosequencing and single nucleotide primer extension (SNuPE), no wild-type IDS genomic sequence was detectable. The relative abundance of wild-type and mutation-bearing IDS-transcripts in different tissues was quantified by SNuPE. Although IDS transcript levels, as measured by real-time PCR, were reduced (51-71% normal) in these patients, some wild-type IDS protein was detectable by western blotting. Various possible explanations for these unprecedented findings (e.g. accidental contamination, artefactual in vitro nucleotide misincorporation, malsegregation of an extra maternal X-chromosome) were explored and experimentally excluded. PCR-based discriminant assay and segregation analysis of a linked IDS polymorphism (rs1141608) also served to exclude the presence of IDS cDNA derived from the maternal wild-type chromosome. Although it remains to be formally demonstrated by direct experimentation, the intriguing possibility arises that we have observed the in vivo correction of heritable gene lesions at the RNA level operating via a correction mechanism akin to RNA-editing.

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A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts

Cited by 51 publications

References 48 publications

A Study of New Arabidopsis Chloroplast RNA Editing Mutants Reveals General Features of Editing Factors and Their Target Sites

A Study of New Arabidopsis Chloroplast RNA Editing Mutants Reveals General Features of Editing Factors and Their Target Sites

Reverse Genetic Screening Identifies Five E-class PPR Proteins Involved in RNA Editing in Mitochondria of Arabidopsis thaliana

Enigmatic In Vivo iduronate-2-sulfatase (IDS) mutant transcript correction to wild-type in Hunter syndrome

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