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Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria.

ABSTRACT: 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.

SUBMITTER: Val R

PROVIDER: S-EPMC3241634 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria.

Val Romain R Wyszko Eliza E Valentin Clarisse C Szymanski Maciej M Cosset Anne A Alioua Malek M Dreher Theo W TW Barciszewski Jan J Dietrich André A

Nucleic acids research 20110717 21

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 ...[more]

PMID: 21768127

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Project description:Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFNγ) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFNγ polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2-/- mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.

Dataset Information

Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria.

Publications

Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria.

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