Transcriptional initiation under conditions of anoxia-induced quiescence in mitochondria from Artemia franciscana embryos
1 Department of Biological Sciences, Louisiana State University, Baton
Rouge, LA 70803, USA
2 Department of Environmental, Population and Organismic Biology, University
of Colorado, Boulder, CO 80303-0334, USA
* Present address: Department of Zoology, University of Wisconsin, Madison, WI
53706, USA
Author for correspondence (e-mail:
shand{at}lsu.edu)
Accepted 28 October 2002
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Summary |
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Key words: anoxia, transcriptional initiation, mitochondria, Artemia franciscana, brine shrimp, pH, hypometabolism, ribonuclease protection assay, DNA footprinting, gene expression
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Introduction |
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During the first hour of anoxic exposure, intracellular pH (pHi) in A.
franciscana embryos drops by approximately one full unit, from 7.8 to
6.8, and embryos respond with a rapid and profound arrest of both anabolic and
catabolic processes that are triggered in part by this drop
(Clegg, 2001;
Hand, 1998
). For example,
run-on assays using isolated nuclei from A. franciscana embryos
demonstrated a decrease in transcription of over 80% in response to anoxic
incubation and by 55% under artificial acidification
(van Breukelen et al., 2000
).
The current study used isolated mitochondria in a similar fashion to measure
transcription, with the added experimental use of an anoxic treatment in
vitro based upon the ability of mitochondria to re-initiate transcription
de novo. The use of nuclear run-on assays to provide a `snapshot' of
transcription at the time of nuclear isolation is predicated on the
observation that new initiation of transcription does not occur in
vitro (e.g. Stallcup et al.,
1978
). For isolated mitochondria, however, it has been
demonstrated that transcription initiation can occur in isolated organelles
de novo; i.e. radioisotope incorporation is not strictly a reflection
of transcriptional activity in vivo but, to a large extent, reflects
incubation conditions in vitro
(Gaines and Attardi, 1984
). In
fact, over half of the measured transcriptional activity in mitochondria of
HeLa cells seemed to result from initiation
(Gaines and Attardi, 1984
),
and so it was of interest to us to measure new initiation in our assay system.
We therefore developed a ribonuclease protection assay (RPA) to measure
[
-32P]UTP incorporation in upstream and downstream portions
of the 12S rRNA, which in A. franciscana mitochondria lies
immediately downstream of the major heavy strand (H strand) transcriptional
promoter (Carrodeguas and Vallejo,
1997
). Our results establish not only that new initiation
contributes substantially to transcription in isolated mitochondria but also
that at low pH (6.4) this activity is largely inhibited.
Regulation of mitochondrial transcription could logically be expected at
the level of initiation, but information regarding this process in A.
franciscana is lacking. In all metazoan mitochondria studied to date,
transcriptional initiation occurs in an area typically 1 kb known as the
control region (Taanman,
1999
). Previous studies with isolated mitochondria have
demonstrated the utility of DNA footprinting analysis for documenting
proteinDNA interactions correlated with changes in transcription,
specifically at transcriptional promoters in the control region (Ghivizzani et
al., 1993
,
1994
;
Cantatore et al., 1995
;
Micol et al., 1997
;
Enríquez et al., 1999
).
We therefore chose to examine promoter occupancy to test the hypothesis that
reductions in de novo initiation at low pH were caused by polymerase
or transcription factor absence by using methylation interference and primer
extension analysis. Our results indicate that promoter occupancy does not
change with in vitro incubation conditions, so decreased
re-initiation at acidic pH is not due to a lack of DNAprotein
interactions but to other possible mechanisms such as covalent
modification.
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Materials and methods |
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For low pH treatments, 250 µl of mitochondria were added to 600 µl of FHB (pH 5.9), resulting in a final pH of 6.35, while another 250 µl of mitochondria were added to 680 µl of FHB at pH 8.3 to give a final pH of 7.88. The pH was measured with a Radiometer electrode (model GK2401C) at the beginning and end of the incubation. For in vitro anoxia, the FHB was made without adding the BSA initially to prevent frothing of the buffer during gassing. The mixture was vigorously bubbled with argon in a nitrogen-purged glovebag for more than 30 min, which was sufficient to drive off any oxygen measurable with a Strathkelvin model 1302 polarographic oxygen electrode (defined here as nominally anoxic). The FHB was then added to 0.5% BSA (final concentration) that had equilibrated in a nitrogen-purged glovebag, and 1 mol l-1 KOH was added to give the appropriate pH (770 µl for pH 7.88, 320 µl for pH 6.35 in 10 ml total). This FHB was then added to the mitochondrial pellets, which had been drained of supernatant after the final centrifugation step, and allowed to equilibrate at 0°C in the purged glovebag for at least 30 min. The mitochondria were then incubated on ice for 30 min before the start of the assays. All anoxic assays were performed in their entirety inside the nitrogen-purged glovebag.
The effect of anoxia on mitochondrial transcription
To test the effect of anoxia on mitochondrial run-on transcription assays,
we exposed both the embryos (in vivo) and their isolated mitochondria
(in vitro) to anoxic incubations. This approach was taken to ensure
that incubation conditions reflected as closely as possible the intracellular
milieu of an animal undergoing anoxic exposure. We therefore performed all
steps of mitochondrial isolation anoxically and at low temperature. After
dechorionation and an 8 h developmental incubation, embryos were placed in
nitrogen-bubbled 0.25 mol l-1 NaCl for 4 h. Embryos were then
chilled on ice, transferred anaerobically to a nitrogen-purged glovebox,
blotted dry, and 10 g of tissue was then homogenized with homogenization
buffer made anoxic as described above. All subsequent steps of the isolation
procedure were performed in the glovebox, except for centrifugation runs,
which were performed after transferring the preparation into gas-impermeable
centrifuge tubes with screw-caps (Oak Ridge 3119-0500). The isolated
mitochondria were then stored on ice in anoxic FHB at the appropriate pH (6.4
or 7.9) until use. It is appropriate to note that mitochondria stored on ice
for 9 h after isolation showed no decrease in transcription relative to
mitochondria assayed 45 min after the same isolation (data not shown).
Transcriptional run-on assays were performed essentially as described
previously (Eads and Hand,
1999). Briefly, assays were initiated by adding 50 µmol
l-1 each of ATP, CTP and GTP, 100 µmol l-1 UTP and
3.7 MBq [
-32P]UTP (222 TBq mmol-1, 370 MBq
ml-1; Perkin-Elmer, Wellesley, MA, USA) to 80 µl of mitochondria
diluted with FHB and 10 units of RNasin to a final volume of 210 µl.
Assuming a negligible contribution of endogenous UTP from the mitochondrial
preparation, the final specific radioactivity of UTP was 176.5 GBq
mol-1. The transcription reaction was allowed to proceed at
30°C, and 30 µl aliquots were applied to glass-fiber filters (GF/C;
Whatman) at the indicated time-points. Transcription was measured as the
incorporation of [
-32P]UTP into trichloroacetic
acid-insoluble RNA counted by liquid scintillation
(Eads and Hand, 1999
).
Mitochondrial protein content was measured by a modified Lowry assay
(Peterson, 1977
). Statistical
analysis was carried out using the unpaired Student's t-test or
analysis of variance (ANOVA) using Statview software (SAS Inc., Cary, NC,
USA). Values are presented as means ± S.E.M.
Measurement of transcriptional initiation for mitochondria with a
nuclease protection assay
The contribution of in vitro (or de novo) transcriptional
initiation to the run-on assays described in Eads and Hand
(1999) were measured using a
methodology adapted from Gaines and Attardi
(1984
). The main site for
heavy-chain transcription initiation in A. franciscana mitochondria
is immediately upstream (5') from the 12S rRNA (Carroduegas and Vallejo,
1997), so 5' and 3' fragments of this gene were chosen for
measuring new initiation (see Fig.
1 for a schematic rationale). Primers were chosen to PCR-amplify a
fragment of approximately 300 bp at the 5' end and 110 bp at the
3' end of the 12S rRNA (Table
1) using A. franciscana mtDNA. These fragments were
gel-purified and ligated into pGem-T vectors (Promega, Madison, WI, USA)
according to the manufacturer's instructions. Orientation of inserts was
established by restriction digest; restriction sites were chosen using the
program available from SUNY Geneseo Biology at
http://darwin.bio.geneseo.edu/~yin/WebGene/RE.htm.
The clones yielding antisense RNA were used for synthesis reactions, and
antisense RNA was produced with Ambion's Megascript kit (Ambion, Austin, TX,
USA). These unlabeled antisense riboprobes were used in the nuclease
protection assays described below.
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Mitochondria were given in organello treatments as outlined
earlier. However, for treatment with anoxia in vivo, hydrated embryos
were dechorionated, given an 8 h developmental incubation and then transferred
to media bubbled with 100% nitrogen for 4 h. Embryos were blotted dry,
homogenized aerobically, and mitochondria were isolated as described
previously (Eads and Hand,
1999). To start the assay, 180 µl of mitochondria in FHB were
incubated at 30°C with 50 µmol l-1 each of rATP, rCTP and
rGTP, 10 units of RNasin, 70 mmol l-1 dithiothreitol and 3.7 MBq of
[
-32P]UTP in a final volume of 200 µl. After 30 min, 1 ml
of a lysis buffer, containing 4.5 mol l-1 guanidinium thiocyanate,
50 mmol l-1 EDTA, 25 mmol l-1 Tris-HCl, 100 mmol
l-1 ß-mercaptoethanol, 0.2% antifoam A (Sigma) and 2%
N-laurelsarcosine, was added to the mitochondrial mixture. After
thorough vortexing, the sample was centrifuged at 10 000 g
(4°C) for 5 min, and an equal volume of ice-cold 100% isopropanol was
added to the supernatant. The sample was chilled at -20°C for 2 h,
centrifuged at 14 000 g for 30 min (4°C) and washed with
70% isopropanol. Due to low specific activity of the labeled RNA, the entire
nucleic acid pellet (approximately 108 d.p.m.) was taken up in a
final volume of 30 µl of hybridization buffer, which contained 5 mol
l-1 guanidinium thiocyanate, 5 mmol l-1 EDTA (pH 7.0)
and 3-5 µg unlabeled antisense RNA. The hybridization reaction was
incubated for at least 6 h at 37°C. Control reactions containing no
unlabeled probe or no labeled sample were run in parallel.
Due to the large amounts of labeled, unhybridized RNA, the samples were divided into three aliquots for nuclease digestion. Each 10 µl aliquot was added to 240 µl of buffer containing 450 mmol l-1 NaCl, 25 mmol l-1 Tris (pH 8.0) and 5 mmol l-1 EDTA with 15 µl of nuclease cocktail (Ambion) and incubated at 37°C for 2 h. A control with no nuclease cocktail was also incubated in parallel. 250 µl of K buffer, containing 0.3% sarkosyl, 20 µg proteinase K and 1 mmol l-1 CaCl2 (final concentrations), was then added and incubated for 1 h at 37°C. The samples were then phenolchloroform extracted twice (phenol pH 4.0), extracted once with chloroformisoamyl alcohol (24:1) and ethanol precipitated with 10 µg yeast tRNA. Pellets were washed in 90% ethanol, dried and solubilized in 8 µl 100% deionized formamide. At this point, pellets from the same hybridization were generally recombined, although in some cases they were run separately. After denaturing for 1 min at 80°C and quick chilling, samples were run with 1 µl loading dyes (6x; Promega) in 8% polyacrylamide/8 mol l-1 urea gels in 1x TBE buffer (89 mmol l-1 Tris base, 89 mmol l-1 boric acid, 2 mmol l-1 EDTA) at 600-700 V for approximately 1 h. Gels were dried and imaged using a Molecular Dynamics phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA). Molecular size markers (Roche, Indianapolis, IN, USA) were run in parallel and separately stained in ethidium bromide for size determination.
Footprinting of mitochondrial DNA by methylation interference
Isolated, intact mitochondria from aerobically incubated embryos were given
in organello treatments as described above and subjected to
dimethylsulfide (DMS) methylation. Briefly, approximately 4 µg
mitochondrial protein were incubated with 50 µmol l-1 rNTPs (200
µl final volume) in FHB at 30°C for 20 min. Fresh DMS (2% in water;
Acros Chemical, Geel, Belgium) was added to a final concentration of 0.05-0.2%
and incubated for 3 min at 30°C. Some aliquots received no DMS in
organello but were used for DMS treatment in vitro after
extraction of DNA (`naked' DNA). 0.5 ml of ice-cold phosphate-buffered saline
(PBS: 137 mmol l-1 NaCl, 2.7 mmol l-1 KCl, 4.3 mmol
l-1 Na2PO4, 1.4 mmol l-1
KH2PO4) was then added, and samples were vortexed and
centrifuged at 10 000 g (4°C) for 1 min. The wash was repeated
twice with 0.9 ml PBS, and the mitochondrial pellet was resuspended in 400
µl of buffer containing 10 mmol l-1 Tris (pH 7.5), 0.2 mol
l-1 NaCl, 0.1% sodium dodecyl sulfate (SDS) and 0.1 mg
ml-1 proteinase K. Samples were vortexed and incubated at room
temperature for 1 h, followed by extraction with an equal volume of phenol (pH
7.9). The aqueous phase was extracted twice with phenolchloroform and
twice with chloroform-isoamyl alcohol. 50 µl of a DMS stop buffer
containing 1.5 mol l-1 sodium acetate (pH 7.0), 1 mol
l-1 ß-mercaptoethanol and 0.1 mg ml-1 yeast tRNA
was then added to the supernatant, followed by addition of 625 µl of
ice-cold 100% isopropanol. After 2 h at -20°C, the samples were
centrifuged at 14 000 g for 30 min (4°C) and washed in 70%
isopropanol. The pellet was resuspended in 100 µl of 1x TE buffer (10
mmol l-1 Tris, pH 7.5, 1 mmol l-1 EDTA), reprecipitated
with ethanol and resuspended in 100 µl of 1 mol l-1 piperidine
(Acros Chemical).
Control DNA given no DMS in organello was resuspended in 100 µl of water instead of 1 mol l-1 piperidine. Aliquots of 20-40 µg nucleic acid were incubated in 200 µl of 1x TE buffer and 0.05-0.2% DMS (final concentrations) at 37°C for 2 min, then added to 50 µl of DMS stop buffer. Samples were added to 780 µl of ice-cold 100% ethanol, held at -20°C for 2 h and centrifuged at 14 000 g for 30 min. After washing in 70% ethanol, pellets were solubilized in 1 mol l-1 piperidine.
All samples (in organello-treated and naked DNA) were incubated at
90°C for 30 min, then quick-chilled at -80°C for 15 min before drying
in a vacuum concentrator. Pellets were resuspended in 300 µl of water and
dried twice more to remove residual piperidine. Piperidine-cleaved samples
were used as template in primer-extension assays using Taq polymerase
(Promega). Briefly, oligonucleotide primers (10-20 pmol) were 5'
end-labeled using T4 polynucleotide kinase (New England Biotech, Beverly, MA,
USA) and 1.85-3.0 MBq [-32P]ATP (222 TBq mmol-1,
370 MBq ml-1; Perkin Elmer), purified over G-25 Sephadex
spin-columns, phenol-chloroform extracted and ethanol precipitated with 2.5
µg yeast RNA. For primer annealing and extension reactions, 0.5-1 µg of
DNA was used in 50 µl reactions containing 1x PCR buffer (Taq
polymerase A; Promega), 0.1 mmol l-1 dNTPs, 1.5 mmol l-1
MgCl2, 5 units of Taq and 1 pmol of oligonucleotide primer (see
Table 1 for primer
identification). Samples were covered with mineral oil and cycled 15-20 times
[94°C for 1 min, primer annealing temperature (49-62°C) for 1.5 min,
72°C for 2 min] using an MJ Research thermal cycler (MJ Research, Waltham,
MA, USA).
Samples were phenol-chloroform extracted and ethanol precipitated in the
presence of 20 mmol l-1 sodium acetate and 10 µg yeast RNA,
centrifuged at 14,000 g for 30 min (4°C) and washed with 70%
ethanol. After drying, pellets were resuspended in 2 µl of 100% deionized
formamide plus 1 µl loading dye, denatured at 95°C for 2 min, quick
chilled and electrophoresed on 8% polyacrylamide/7 mol l-1 urea
sequencing gels. Gels were run at 1800 V, 50 W and 30 mA until xylene cyanol
had reached the bottom of the gel. They were then dried and exposed on a
Phoshorimager cassette (Molecular Dynamics). Bands were quantified using
ImageQuant software (Molecular Dynamics), and lanes were normalized using
bands outside the footprinted region to account for loading differences.
Following the convention used in other reports on in organello
footprinting (Cantatore et al.,
1995; Roberti, 1999), bands showing >30% difference were
considered footprinted. In this study, we designate the bases visualized for
the in organello DMS-treated mitochondria as `overmethylated' or
`undermethylated' by using naked DNA as the reference. Either result can
indicate the presence of proteins in organello, as a consequence of
protein-induced changes in DNA topology that allow more or less access to
methylating agent.
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Results |
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In order to explore the effect of low pH independent of the effect of anoxia, we repeated the low pH assay under normoxia. The results shown in Fig. 2C reveal that overall [32P]UTP incorporation by mitochondria incubated at pH 6.4 is significantly lower (59% decrease; P<0.02) than in mitochondria incubated at pH 7.9. Additionally, transcription rate is decreased by 74% to 574 nmol UTP mg protein-1 h-1 (Table 2). Mitochondria held at pH 6.3 for 1 h and then assayed for transcription showed no difference in [32P]UTP incorporation relative to mitochondria held at pH 7.0 or 7.9 for 1 h and then assayed similarly (data not shown). Thus, exposure to low pH in vitro for this time period apparently does not promote irreversible effects on mitochondrial transcription.
Transcriptional initiation decreases under low pH in
organello
Ribonuclease protection assays of mitochondrial RNA labeled in
organello showed that under control conditions (normoxia, pH 7.9), in
vitro transcriptional initiation contributes approximately 77% to the
measured [32P]UTP incorporation
(Fig. 1;
Table 3). This value is
indistinguishable from new initiation under conditions of anoxia at pH 7.9
(78.8%). Exposure of intact embryos to anoxia in vivo followed by
normoxic isolation and incubation did not promote a significant difference in
new initiation relative to controls (Table
3). However, a major depression is seen in the proportion of
transcription due to new initiation under conditions of low pH (pH 6.4),
either aerobically or anoxically (32%;
Table 3). In contrast to the
original method developed by Gaines and Attardi
(1984), we used both 5'
and 3' antisense probes in the same incubations rather than separately,
and therefore controlled a major source of error. Standard errors were in the
order of
11% (Table 3).
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Identification of protein-DNA contacts in the H-strand and L-strand
promoter regions
The technique of methylation interference and primer extension was used to
map regions of mtDNA bound by proteins, typically polymerases and
transcription factors (see Ghivizzani et
al., 1994; Micol et al.,
1997
; Enríquez et al.,
1999
). Using this method, the presence of proteins was deduced
along regular intervals in the control region of the mitochondrial genome
upstream of the 12S rRNA, which previously had been indirectly inferred
(Carrodeguas and Vallejo, 1997
)
to contain promoters for H-strand transcriptional initiation (see
Fig. 3 for a schematic
representation). As shown in Fig.
3, the primers HSP 3, HSP 4 and HSP 5 (see
Table 1) revealed differences
in methylation between control (naked) DNA and DNA methylated in
organello, consistent with protein binding that increased or decreased
the availability of methylation sites on the DNA. As shown in Figs
4,5,6,7,
we found differences in methylation at a number of sites in the following
genomic regions: 12030-12065, 14125-14138, 14220-14240, 14270-14300,
14335-14355 and 15740-15760. Primers HSP 1 and HSP 2, designed to probe the
region immediately upstream from the 12S rRNA (12S rRNA starts at 14000; see
Table 1 for primer
identification), were initially used. A previous report of transcriptional
initiation in A. franciscana provided evidence that this area
probably contained a strong promoter
(Carrodeguas and Vallejo,
1997
). Protein binding was located in the region 75-95 bp upstream
(5') to the 12S rRNA using HSP 5 (Figs
6C,
7C). Specifically, bases 14074,
14080, 14083, 14087 and 14093 (numbering after
Valverde et al., 1994a
) were
all undermethylated in organello. We were unable to find interference
patterns further downstream, in an area predicted to contain the strong
H-strand promoter (Fig. 4A,B). Several primers were used to amplify both strands and detect any methylation
differences, but these experiments did not reveal protein binding (data not
shown).
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Although a footprint was not localized within 75 bp of the 12S rRNA near
the putative stronger promoter (14 000-14 075), the area from 14 125-14 355
showed four discrete footprinted areas in a region thought to contain a weaker
H-strand promoter. Methylation differences are depicted in Figs
6B and
7B at sites 14 269, 14 281, 14
291, 14 297, 14 304, 14 335, 14 344, 14 354 and 14 357 that could indicate
protein binding immediately upstream of the putative H-strand promoter (at 14
250; Carrodeguas and Vallejo,
1997). Also, the region from 14 200 to 14 240 shows several highly
undermethylated guanines relative to naked DNA (Figs
6A,
7A), and from 14 120 to 14 173
there is marked overmethylation of both adenines and guanines
(Fig. 6A). The size of these
footprints is compatible with the typical footprint of mitochondrial
transcription factor A (Ghivizzani et al.,
1994
; Cantatore et al.,
1995
). The region between 15 739 and 15 763 corresponds to a
sequence hypothesized to contain the L-strand promoter
(Carrodeguas and Vallejo, 1997
)
and has overmethylated residues at 15 739, 15 743 and 15 747 and
undermethylations at 15 752 and 15 787 (Figs
4D,
5B). The area between 12 030
and 12 065 contains a sequence in the tRNAleu gene believed to bind
the mitochondrial transcription factor TERM (previously known as mTERF;
Valverde et al., 1994b
) and
has several methylation differences (Figs
4C,
5A). To our knowledge, this is
the first evidence that the 12 030-12 065 sequence is protein-bound in A.
franciscana. The identity of this protein awaits a direct
demonstration.
Interestingly, experimental incubations in organello that reduced the frequency of de novo initiation did not cause changes in footprinting patterns relative to the controls (Figs 4, 6). Based on these observations, we speculate that the depressed initiation seen at low pH could be triggered by processes such as covalent modification or proteinprotein assembly at promoter sites that might not be detected by methylation interference.
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Discussion |
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The energetic cost of even low levels of mitochondrial transcription during
long-term anoxia is likely to be unsustainable for A. franciscana
embryos. DNA replication and transcription are estimated to require
approximately 10% of the energy budget of a mammalian cell (Buttgereit and
Brand, 1996; Rolfe and Browne, 1995), a value that may be a bit high for
gastrula-stage embryos of A. franciscana, because little DNA
replication occurs across pre-emergence development (Olson and Clegg, 1977).
The 89% decrease elicited in mitochondrial transcription rate in our study
does not constitute full arrest, which could reflect imperfect simulations of
in vivo conditions in our assays. The addition of optimal levels of
exogenous NTPs during the assays (Eads and
Hand, 1999) could represent, for example, ATP concentrations that
are above physiological concentrations, given that anoxic A.
franciscana embryos experience a decline of over 80% in ATP during the
first hour of exposure (Stocco et al.,
1972
; Anchordoguy and Hand,
1994
). Also, the increased stability of mRNA under conditions of
both anoxia and low pH (B. D. Eads and S. C. Hand, unpublished data) implies
that transcripts should accumulate faster in mitochondria incubated under
these conditions than under normoxia and pH 7.9, which would artificially
underestimate the degree of transcriptional depression.
Gaines and Attardi (1984)
noted a decrease in the relative labeling of certain RNAs in isolated
mitochondria across a pH range similar to the one reported in this study.
These authors speculated that changes in RNA stability and/or processing
occurred at low pH in a way that appeared to be transcript-specific, such that
fewer long transcripts and more short transcripts accumulated. Our study of
mRNA decay (B. D. Eads and S. C. Hand, unpublished data) under the same
conditions examined in the present study indicated that both anoxia and low pH
are able to extend half-life to varying and mRNA-specific degrees, although we
did not evaluate rRNA stability in that work. Further investigation on the
pH-based regulation of rRNA metabolism in A. franciscana mitochondria
is likely to be a fruitful avenue for studying transcription in these
animals.
Studies of nuclear transcriptional events during periods of oxygen
limitation have received attention in mammalian systems during hypoxia and
have revealed a fundamental role for the transcription factor HIF-1
(Semenza, 1998). The
transcriptional responses of anoxia-tolerant vertebrates such as the wood frog
Rana sylvatica and the crucian carp Carassius carassius have
also been studied. Both anoxia and freezing induce an increase in five
mitochondrial tRNAs in the wood frog (Cai
and Storey, 1997
), although the physiological significance remains
obscure. Likewise, crucian carp do not decrease RNA synthesis in response to
long-term anoxia, even though protein synthesis is downregulated
(Smith et al., 1999
). Anoxia
tolerance has been studied in numerous invertebrates (reviewed in
Grieshaber et al., 1994
), but
data regarding transcriptional changes have only recently begun to accumulate.
For example, a number of genes are differentially expressed during exposure of
Drosophila melanogaster to anoxia
(Haddad, 2000
), but this
species possesses poor anoxia tolerance overall. Upregulation of several genes
has been observed in the yeast Saccharomyces cerevisiae during anoxia
(reviewed in Zitomer and Lowry,
1992
; Kwast et al.,
1998
). In the above cases, differential gene expression may serve
to re-program metabolism for survival under oxygen limitation. Especially in
vertebrates, tolerance is generally limited to a few days or weeks unless
temperature is lowered. In A. franciscana, by comparison, the ability
to survive anoxia at room temperature for years (cf.
Clegg, 1997
) apparently
requires such deep metabolic depression that differential gene expression is
no longer energetically feasible.
Isolated mitochondria exhibit a substantial degree of local
(versus nuclear-mediated) control over transcription that can be
exploited to study mechanisms of initiation. This feature is quite useful in
the present context because transcriptional studies of A. franciscana
embryos cannot be performed in vivo due to a virtually impermeable
cyst wall. The downregulation of initiation by pH is a mechanism that promotes
a 67% decrease in transcription in mitochondria isolated from A.
franciscana and hypothetically may be responsible in vivo for
low mitochondrial transcription during anoxia-induced quiescence. Other, as
yet undiscovered, mechanisms may operate in concert to depress fully this step
in gene expression. In vitro initiation of mitochondrial
transcription has been known for some time
(Gaines and Attardi, 1984),
but its effect during in organello assays has generally been ignored.
Studies of in organello transcription have focused on the acute
influence of exogenous factors such as ATP
(Gaines et al., 1987
;
Enríquez et al., 1996
),
thyroid hormone (Enríquez et al.,
1999
) or mitochondrial transcription factor A (TFAM, formerly
mTFA; Montoya et al., 1997
) on
overall UTP incorporation or on mtDNAprotein interactions as they
relate to transcription. These studies and others
(Eads and Hand, 1999
;
Micol et al., 1997
) indicate a
substantial degree of autonomy regarding mitochondrial transcription when
removed from nuclear inputs. The possibility remains that nucleo-cytoplasmic
regulation may impact endogenous mitochondrial control, particularly over
extended time periods. However, faithful reproduction of in vivo
patterns is a hallmark of transcription in isolated mitochondria
(Gaines and Attardi, 1984
;
Enríquez et al., 1996
),
and the acute and profound effect of in vitro treatments underscores
the importance of autonomous regulation.
To examine the decreased initiation at low pH, the approach selected was to
footprint transcriptional promoter regions in isolated mitochondria by
methylation interference. Footprinting in mitochondria is, in principle,
similar to nuclear footprinting, although there is a key difference.
Mitochondrial transcription proteins are, in general, much smaller than their
nuclear counterparts (e.g. the 105 kDa mtRNA polymerase of A.
franciscana is comparable with a single subunit of RNA polymerase II),
reflected by smaller regions of DNA contact and less striking differences in
footprint patterns (see Ghivizzani et al.,
1994; Cantatore et al.,
1995
; Micol et al.,
1997
; Enríquez et al.,
1999
; Roberti et al.,
1999
). The agreement between footprinting in organello
and in vivo (Ghivizzanni et al., 1994;
Micol et al., 1997
) indicates
that this technique can be used reliably to localize transcriptionally
important proteins. Previous reports have correlated differences in promoter
occupancy of the mitochondrial H strand with changes in transcription
(Micol et al., 1997
;
Enríquez et al., 1999
).
For example, in HeLa cells, mRNA and rRNA production responded differentially
to exogenous ATP (Gaines et al.,
1987
), and this response was directly correlated with changes in
protein binding in the promoter region of the H strand
(Micol et al., 1997
).
Similarly, upregulation of mitochondrial transcription promoted in
organello by thyroid hormone was reflected by changes in the pattern of
footprinting in the H-strand promoter
(Enríquez et al.,
1999
). Thus, we expected to find a difference in footprinting
patterns between incubations at low and high pH that would correlate with the
decrease in transcriptional initiation. The inability to document such an
association has several possible explanations.
Although models of transcriptional initiation exist for mitochondria of
several species (see Tracy and Stern,
1995), it has not been shown whether A. franciscana
mitochondira require a DNA-binding protein such as a TFAM homolog, a
dissociable specificity factor like mitochondrial transcription factor B
(mTFB; inferred from Santiago and Vallejo,
1998
) or both. Our data are consistent with a role for TFAM in
A. franciscana mitochondria. Mitochondria from rat liver
(Cantatore et al., 1995
) and
human placenta (Ghivizzani et al.,
1994
) show footprinting in regions corresponding to TFAM-binding
domains, including transcriptional promoter regions, and TFAM remains bound to
promoters during mitochondrial transcription
(Taanman, 1999
). The protein
binding we documented in A. franciscana occurred at intervals similar
to those in the studies just mentioned, and the location of footprints in the
control region spans the area containing two putative H-strand promoters.
However, because these patterns did not change across experimental
incubations, the acute effect of pH that we observed on transcriptional
initiation is compatible with covalent modification. While no sequence data is
available for the A. franciscana mtRNA polymerase, phosphorylation of
the enzyme or a specificity protein could control initiation, by analogy to
the Pol II carboxy-terminal domain (Uptain
et al., 1997
). Several residues in the conserved carboxy-terminal
domains of TFAM are potential sites of phosphorylation (Goto, 2001) as
well.
Interestingly, we were unable to detect a footprint in the stronger
H-strand promoter of the A. franciscana mtDNA
(Carrodeguas and Vallejo,
1997), while the weaker H-strand and L-strand promoters did
indicate protein binding. Perhaps only a single H-strand promoter exists. In
mapping transcription start sites, Carrodeguas and Vallejo
(1997
) used in vitro
capping of mitochondrial RNA, a process that does not work equally well for
all RNAs (cf. Levens et al.,
1981
). A 5' leader sequence for the 12S rRNA that had
initiated from the `weaker' promoter at position 14250 would be expected under
the single promoter hypothesis. The leader could be processed
co-transcriptionally to yield an uncapped or rapidly degraded 5'
fragment and a nascent, capped rRNA. Alternatively, it is possible that
transcriptional initiation at the strong H-strand promoter does not depend on
TFAM binding but rather on an mTFB-type protein. In this case, a footprint
would not be expected because mTFB associates with mtRNA polymerase rather
than binding directly to DNA like TFAM does (cf.
Prieto-Martín et al.,
2001
). However, because reconstituted transcription depends
critically on high levels of TFAM as well as mTFB
(Falkenberg et al., 2002
), we
consider the latter scenario unlikely.
Finally, the footprint found of mTERF in the tRNAleu gene at
positions within and near a conserved sequence for rDNA transcription
(Valverde et al., 1994b)
indicates that in organello this sequence is bound by the termination
factor. Presumably, regulated binding at this site in transcriptionally active
mitochondria controls the formation of mRNAs in the H strand (see
Fig. 4). Our study shows no
differences among in organello treatments regarding mTERF occupancy,
similar to previous work in HeLa cells reporting no effect of ATP
concentration or ethidium bromide (Micol
et al., 1997
).
In summary, anoxia and low pH are able to decrease mitochondrial transcription rate to 11% of controls, consistent with metabolic arrest by A. franciscana embryos during anoxia. De novo initiation contributes significantly to transcription in isolated mitochondria, and at low pH the input of new initiation decreases by over 50%. This depression of initiation is potentially a significant regulator of RNA synthesis in vivo in mitochondria of A. franciscana embryos. Several sites of protein binding were discovered that correspond to the transcription termination region downstream of the ribosomal RNAs. Protein binding was also observed in areas of the control region previously indicated to contain transcriptional promoters. No differences were found in patterns of methylation interference based on treatments of the isolated mitochondria such as incubation at low pH. Thus, the regulation of transcriptional initiation in A. franciscana mitochondria apparently involves other mechanisms in addition to proteinDNA binding.
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