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Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis
Published on: May 14, 2020
A mitochondrial rRNA dimethyladenosine methyltransferase in Arabidopsis
Uwe Richter1, Kristina Kühn, Sachiko Okada
1Institut für Biologie/Genetik, Humboldt-Universität, Chausseestr. 117, 10115 Berlin, Germany.
Abstract:
S-adenosyl-L-methionine-dependent rRNA dimethylases mediate the methylation of two conserved adenosines near the 3' end of the rRNA in the small ribosomal subunits of bacteria, archaea and eukaryotes. Proteins related to this family of dimethylases play an essential role as transcription factors (mtTFBs) in fungal and animal mitochondria. Human mitochondrial rRNA is methylated and human mitochondria contain two related mtTFBs, one proposed to act as rRNA dimethylase, the other as transcription factor. The nuclear genome of Arabidopsis thaliana encodes three dimethylase/mtTFB-like proteins, one of which, Dim1B, is shown here to be imported into mitochondria. Transcription initiation by mitochondrial RNA polymerases appears not to be stimulated by Dim1B in vitro. In line with this finding, phylogenetic analyses revealed Dim1B to be more closely related to a group of eukaryotic non-mitochondrial rRNA dimethylases (Dim1s) than to fungal and animal mtTFBs. We found that Dim1B was capable of substituting the E. coli rRNA dimethylase activity of KsgA. Moreover, we observed methylation of the conserved adenines in the 18S rRNA of Arabidopsis mitochondria; this modification was not detectable in a mutant lacking Dim1B. These data provide evidence: (i) for rRNA methylation in Arabidopsis mitochondria; and (ii) that Dim1B is the enzyme catalyzing this process.
Insights
Arabidopsis mitochondria contain S-adenosyl-L-methionine-dependent rRNA dimethylases. The protein Dim1B is imported into mitochondria and catalyzes the methylation of conserved adenosines in the 18S rRNA.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- S-adenosyl-L-methionine-dependent rRNA dimethylases modify conserved adenosines in small ribosomal subunits across domains of life.
- Related proteins function as mitochondrial transcription factors (mtTFBs) in fungi and animals, while human mitochondria possess both rRNA dimethylase and mtTFB-related proteins.
Purpose of the Study:
- To investigate the function of Arabidopsis thaliana nuclear-encoded dimethylase/mtTFB-like proteins in mitochondria.
- To determine if Dim1B is involved in mitochondrial rRNA methylation in Arabidopsis.
Main Methods:
- Phylogenetic analysis to determine evolutionary relationships.
- In vitro transcription assays to assess Dim1B's effect on mitochondrial RNA polymerase.
- Complementation assay using E. coli KsgA.
- Analysis of 18S rRNA methylation in wild-type and Dim1B-deficient Arabidopsis mitochondria.
Main Results:
- Dim1B is imported into Arabidopsis mitochondria.
- Dim1B does not stimulate mitochondrial transcription initiation in vitro.
- Dim1B functionally complements the E. coli KsgA rRNA dimethylase.
- Arabidopsis mitochondria exhibit methylation of conserved adenosines in 18S rRNA, a modification absent in Dim1B mutants.
Conclusions:
- Arabidopsis mitochondria undergo rRNA methylation.
- Dim1B is the enzyme responsible for catalyzing this mitochondrial rRNA methylation process.
- Dim1B is phylogenetically closer to non-mitochondrial rRNA dimethylases than to mtTFBs.
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