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
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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)