The mTERF protein MOC1 terminates mitochondrial DNA transcription in the unicellular green alga Chlamydomonas reinhardtii

Nucleic Acids Research, Jul 2013

The molecular function of mTERFs (mitochondrial transcription termination factors) has so far only been described for metazoan members of the protein family and in animals they control mitochondrial replication, transcription and translation. Cells of photosynthetic eukaryotes harbour chloroplasts and mitochondria, which are in an intense cross-talk that is vital for photosynthesis. Chlamydomonas reinhardtii is a unicellular green alga widely used as a model organism for photosynthesis research and green biotechnology. Among the six nuclear C. reinhardtii mTERF genes is mTERF-like gene of Chlamydomonas (MOC1), whose inactivation alters mitorespiration and interestingly also light-acclimation processes in the chloroplast that favour the enhanced production of biohydrogen. We show here from in vitro studies that MOC1 binds specifically to a sequence within the mitochondrial rRNA-coding module S3, and that a knockout of MOC1 in the mutant stm6 increases read-through transcription at this site, indicating that MOC1 acts as a transcription terminator in vivo. Whereas the level of certain antisense RNA species is higher in stm6, the amount of unprocessed mitochondrial sense transcripts is strongly reduced, demonstrating that a loss of MOC1 causes perturbed mitochondrial DNA (mtDNA) expression. Overall, we provide evidence for the existence of mitochondrial antisense RNAs in C. reinhardtii and show that mTERF-mediated transcription termination is an evolutionary-conserved mechanism occurring in phototrophic protists and metazoans.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://nar.oxfordjournals.org/content/41/13/6553.full.pdf

The mTERF protein MOC1 terminates mitochondrial DNA transcription in the unicellular green alga Chlamydomonas reinhardtii

Lutz Wobbe 1 Peter J. Nixon 0 0 Department of Life Sciences, Faculty of Natural Sciences, Imperial College London , S. Kensington campus, London SW7 2AZ, UK 1 Department of Biology, Algae Biotechnology and Bioenergy-Center for Biotechnology (CeBiTec), Bielefeld University , 33615 Bielefeld, Germany - The molecular function of mTERFs (mitochondrial transcription termination factors) has so far only been described for metazoan members of the protein family and in animals they control mitochondrial replication, transcription and translation. Cells of photosynthetic eukaryotes harbour chloroplasts and mitochondria, which are in an intense cross-talk that is vital for photosynthesis. Chlamydomonas reinhardtii is a unicellular green alga widely used as a model organism for photosynthesis research and green biotechnology. Among the six nuclear C. reinhardtii mTERF genes is mTERF-like gene of Chlamydomonas (MOC1), whose inactivation alters mitorespiration and interestingly also light-acclimation processes in the chloroplast that favour the enhanced production of biohydrogen. We show here from in vitro studies that MOC1 binds specifically to a sequence within the mitochondrial rRNA-coding module S3, and that a knockout of MOC1 in the mutant stm6 increases read-through transcription at this site, indicating that MOC1 acts as a transcription terminator in vivo. Whereas the level of certain antisense RNA species is higher in stm6, the amount of unprocessed mitochondrial sense transcripts is strongly reduced, demonstrating that a loss of MOC1 causes perturbed mitochondrial DNA (mtDNA) expression. Overall, we provide evidence for the existence of mitochondrial antisense RNAs in C. reinhardtii and show that mTERF-mediated transcription termination is an evolutionary-conserved mechanism occurring in phototrophic protists and metazoans. Most of our knowledge about mitochondrial gene expression and its regulation results from research carried out with non-phototrophic organisms, especially mammalian and yeast cells (1,2). In the case of phototrophic eukaryotes, there is an additional level of complexity, as metabolism in the mitochondria needs to be coordinated to that in the chloroplast (3). As yet the molecular mechanisms underpinning the regulation of gene expression in the mitochondria of plant cells, including the unicellular green alga Chlamydomonas reinhardtii, which is an established model organism widely used to study the regulation of nuclear and organelle gene expression, are unclear. Mitochondria of C. reinhardtii retain a small, but information-dense, genome (4), which contains genes encoding eight proteins, including the complex IV subunit 1, five complex I subunits, apocytochrome b of complex III, a reverse transcriptase-like protein and a strain-dependent number of introns (5). The rRNA genes encoding large (L) and small (S) ribosomal rRNAs are split into modules encoding rRNA segments (eight L and four S modules), which are interspersed with one another or protein-coding and tRNA genes (6). All the mitochondrial rRNAs are encoded by the mitochondrial genome of C. reinhardtii (4), but the majority of tRNAs have to be imported from the cytosol (7). In vitro labelling experiments and physical mapping indicated that the mitochondrial DNA (mtDNA) of C. reinhardtii is a linear monomeric molecule (8), but electron microscopy (9) demonstrated that mtDNA preparations contain a small fraction of circular molecules. The telomere structure, known to be decisive for mtDNA architecture (10), of the C. reinhardtii mtDNA is unusual and differs from structures described for other organisms (11). Both strands of the C. reinhardtii mtDNA contain transcription units (1214), and two sequences identified in the intergenic region located between the transcription units might act as a bi-directional promoter (15). Transcription of the C. reinhardtii mtDNA generates long polycistronic transcripts (1214), which are further processed to yield the mature mRNAs. The use of few transcription initiation sites to drive the expression of large transcription units is a feature frequently found in mitochondria of protists (16) and mammals (1), whereas higher plant chondromes represent a more complex system with multiple transcription initiation sites (17). Recent work has identified a nuclear gene of C. reinhardtii termed mTERF-like gene of Chlamydomonas (MOC1), which plays a role in fine-tuning mitochondrial transcription on changes in illumination. Absence of MOC1 in the stm6 mutant causes a pleiotropic phenotype characterized by perturbed mitorespiration (18) and multiple effects on the physiological state of the plastid resulting in light-sensitivity (18,19) and, interestingly, the enhanced production of biohydrogen (20). MOC1 is a member of the mTERF (mitochondrial transcription termination factor) family of transcription factors, which are found in metazoans (21) including higher plants (mono- and dicotyledonous) (22), but which a (...truncated)


This is a preview of a remote PDF: https://nar.oxfordjournals.org/content/41/13/6553.full.pdf

Lutz Wobbe, Peter J. Nixon. The mTERF protein MOC1 terminates mitochondrial DNA transcription in the unicellular green alga Chlamydomonas reinhardtii, Nucleic Acids Research, 2013, pp. 6553-6567, 41/13, DOI: 10.1093/nar/gkt313