The RAD52-like protein ODB1 is required for the efficient excision of two mitochondrial introns spliced via first-step hydrolysis.

José M Gualberto,Kristina Kühn,Monique Le Ret,Barbara Beator

doi:10.1093/nar/gkv540

Abstract

Transcript splicing in plant mitochondria involves numerous nucleus-encoded factors, most of which are of eukaryotic origin. Some of these belong to protein families initially characterised to perform unrelated functions. The RAD52-like ODB1 protein has been reported to have roles in homologous recombination-dependent DNA repair in the nuclear and mitochondrial compartments in Arabidopsis thaliana. We show that it is additionally involved in splicing and facilitates the excision of two cis-spliced group II introns, nad1 intron 2 and nad2 intron 1, in Arabidopsis mitochondria. odb1 mutants lacking detectable amounts of ODB1 protein over-accumulated incompletely spliced nad1 and nad2 transcripts. The two ODB1-dependent introns were both found to splice via first-step hydrolysis and to be released as linear or circular molecules instead of lariats. Our systematic analysis of the structures of excised introns in Arabidopsis mitochondria revealed several other hydrolytically spliced group II introns in addition to nad1 intron 2 and nad2 intron 1, indicating that ODB1 is not a general determinant of the hydrolytic splicing pathway.

Highlights

Higher plant organelle genomes contain several group-II introns originating from mobile ribozymes that invaded these genomes during evolution [1,2,3]
This study reports on the involvement of the Arabidopsis RAD52-like protein ODB1 in the cis-splicing of two specific mitochondrial introns, nad1 intron 2 and nad2 intron 1, both of which are released via a hydrolytic pathway
When subsequently examining the impact of ODB1 loss on mitochondrial gene expression, we detected an alternative nad2 transcript that over-accumulated in odb1–1 mutant plants but was barely seen in the wild type or in odb1–2 (Figure 1A). odb1–2 plants had been found earlier to express reduced amounts of ODB1 protein; in contrast, odb1–1 plants were considered complete loss-of-function mutants, due to lack of detectable levels of ODB1 [37]. nad2 in Arabidopsis is transcribed from two different mitochondrial genome regions

Summary

Introduction

Higher plant organelle genomes contain several group-II introns originating from mobile ribozymes that invaded these genomes during evolution [1,2,3]. Components of the plant mitochondrial splicing machinery have mostly been identified by homology to intronencoded maturases or previously characterised chloroplast splice factors and through analyses of loss-of-function mutants in Arabidopsis thaliana. Other splice factors identified in higher plant mitochondria include DEAD and DExH-box RNA helicases [10,11], which function as RNA chaperones needed to resolve stable inactive secondary structures and promote correct intron folding [3]. Arabidopsis plants lacking these helicases are defective in the splicing of multiple mitochondrial introns.

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Results

Conclusion