Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria

With the expansion of the RNA world, antisense strategies have become widespread to manipulate nuclear gene expression but organelle genetic systems have remained aside. The present work opens the field to mitochondria. We demonstrate that customized RNAs expressed from a nuclear transgene and drive...

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Bibliographic Details
Published in:Nucleic acids research 2011-11, Vol.39 (21), p.9262-9274
Main Authors: Val, Romain, Wyszko, Eliza, Valentin, Clarisse, Szymanski, Maciej, Cosset, Anne, Alioua, Malek, Dreher, Theo W., Barciszewski, Jan, Dietrich, André
Format: Article
Language:English
Subjects:
Arabidopsis - genetics
Arabidopsis - metabolism
Arabidopsis thaliana
Base Sequence
Biochemistry, Molecular Biology
Cellular Biology
Chlamydomonas
Genetic Engineering
Life Sciences
Mitochondria - metabolism
Molecular Biology
Molecular Sequence Data
Nicotiana - genetics
Nicotiana - metabolism
RNA - metabolism
RNA Transport
RNA, Catalytic - chemistry
RNA, Catalytic - metabolism
RNA, Mitochondrial
RNA, Transfer - chemistry
RNA, Transfer - metabolism
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Summary:With the expansion of the RNA world, antisense strategies have become widespread to manipulate nuclear gene expression but organelle genetic systems have remained aside. The present work opens the field to mitochondria. We demonstrate that customized RNAs expressed from a nuclear transgene and driven by a transfer RNA-like (tRNA-like) moiety are taken up by mitochondria in plant cells. The process appears to follow the natural tRNA import specificity, suggesting that translocation indeed occurs through the regular tRNA uptake pathway. Upon validation of the strategy with a reporter sequence, we developed a chimeric catalytic RNA composed of a specially designed trans-cleaving hammerhead ribozyme and a tRNA mimic. Organelle import of the chimeric ribozyme and specific target cleavage within mitochondria were demonstrated in transgenic tobacco cell cultures and Arabidopsis thaliana plants, providing the first directed knockdown of a mitochondrial RNA in a multicellular eukaryote. Further observations point to mitochondrial messenger RNA control mechanisms related to the plant developmental stage and culture conditions. Transformation of mitochondria is only accessible in yeast and in the unicellular alga Chlamydomonas. Based on the widespread tRNA import pathway, our data thus make a breakthrough for direct investigation and manipulation of mitochondrial genetics.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkr580