Mitochondrial mRNA stability and polyadenylation during anoxia-induced quiescence in the brine shrimp Artemia franciscana
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
Author for correspondence (e-mail:
shand{at}lsu.edu)
Accepted 11 July 2003
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Summary |
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Key words: brine shrimp, Artemia franciscana, polyadenylation, mitchondria, mRNA stability, anoxia, quiescence, intracellular pH
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Introduction |
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The brine shrimp Artemia franciscana is a useful model species for
evaluating mitochondrial RNA stability. In response to unfavorable
environmental conditions such as anoxia, gastrula-stage embryos of A.
franciscana are able to enter a quiescent state in which metabolism and
development are reversibly downregulated to a profound degree, a state that
can last for up to several years (e.g. Clegg,
1997,
2001
;
Warner and Clegg, 2001
). As
oxygen is removed from the embryos, the pHi (intracellular) falls
from approximately 7.8 to 6.8 after 60-90 min
(Busa et al., 1982
;
Kwast et al., 1995
), and to
about 6.3 overnight (Busa et al.,
1982
). Heat production is reduced by >99.5% with time under
anoxia (Hontoria et al., 1993
;
Hand, 1995
), and ATP
concentrations are also depressed to low levels
(Stocco et al., 1972
;
Rees et al., 1989
). A wide
variety of biochemical and gene-expression changes have been observed to
accompany the quiescent state. For example, nuclear run-on data show an 80%
decrease of 32P-UTP incorporation into RNA after 4 h of anoxic
exposure in vivo, indicating a substantial downregulation of
transcription (van Breukelen et al., 2000); low pHi is a
contributing factor (van Breukelen et al., 2000;
Willsie and Clegg, 2001
).
These and other observations (e.g. Clegg
et al., 1999
; Hand et al.,
2001
) indicate that depressed metabolism and increased
macromolecular stability are important during quiescence.
In response to anoxia, mitochondrial protein synthesis is decreased by 77%
after 30 min, with an 80% decrease at low pH compared to control (aerobic,
high pH) mitochondria (Kwast and Hand,
1996a,b
).
Similarly, exposure of mitochondria in vitro to either acidic pH
(6.4) or anoxia results in large decreases in transcription rates, and
transcriptional initiation is decreased by 50% upon exposure to low pH
(Eads and Hand, 2003
). A
direct effect of O2 on gene expression in the mitochondrion has
thus been demonstrated. Translation in the cytoplasm during entrance to anoxic
exposure is also decreased (Clegg and
Jackson, 1989
; Hofmann and Hand,
1990
,
1994
), while mRNA pools do not
drop (Hofmann and Hand, 1992
).
These observations suggest that message levels are not limiting to
translation. Additionally, dot blots of total RNA from embryos exposed to
anoxia for up to 6 h in vivo demonstrate that the ontogenetic
increase in COXI mRNA is blocked by oxygen deprivation
(Hardewig et al., 1996
).
However, direct evidence that mRNA stability is increased during anoxia has
been lacking. We undertook the present study to determine if mRNA can be
stabilized by anoxia in vitro and if polyadenylation plays a role in
message stability.
To investigate the stability of mitochondrial mRNA we used dot blots of total RNA extracted from isolated mitochondria exposed to conditions designed to reflect those prevalent during anoxia-induced quiescence. We also characterized the poly(A)+ tails of these mRNAs using a reverse transcriptase-polymerase chain reaction (RT-PCR)-based assay. We report here that mRNA stability in A. franciscana mitochondria is responsive to both lowered pH and anoxia per se, and that mRNA species that are undergoing rapid degradation are more polyadenylated, while those being stabilized by anoxia and/or low pH are deadenylated.
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Materials and methods |
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Mitochondrial isolation and in organello
treatments
Mitochondria were isolated from gastrula-stage Artemia franciscana
embryos and their functional integrity was assessed by respirometry as
previously described (Eads and Hand,
1999). In organello treatments (low pH, anoxia, low pH
and anoxia, and 1.5 mmol l-1 exogenous ATP under anoxia) and
controls (normoxic, high pH) were performed on mitochondria from the same
preparation. Following differential centrifugation, the final mitochondrial
pellet was resuspended in 2 ml fortified homogenization buffer (FHB)
containing: 500 mmol l-1 sucrose, 120 mmol l-1 KCl, 10
mmol l-1 MES, 10 mmol l-1 Hepes, 10 mmol l-1
KH2PO4, 3 mmol l-1 MgCl2, 1.0 mmol
l-1 EGTA, and 0.5% (w/v) fatty-acid-free bovine serum albumin
(BSA), pH 7.5 (final mitochondrial protein concentration of 18-20 mg
ml-1). For low pH treatment groups, the final mitochondrial pellet
was resuspended in 0.6 ml pH 7.0 FHB. Then a 250 µl portion of the
mitochondrial suspension was added to 600 µl of FHB (pH 5.9), resulting in
a final pH of 6.4. Control mitochondria (250 µl, in pH 7.0 FHB) were added
to 680 µl of FHB (pH 8.3) to give a final pH of 7.8. For the anoxia
studies, the FHB was made without adding the BSA initially to avoid frothing
the mixture and then bubbled vigorously with argon for at least 30 min in a
nitrogen-purged glovebag, which was sufficient to drive off all oxygen as
measured with a Strathkelvin 1302 Clark-type electrode (Glasgow, UK). BSA
powder (0.5% final concentration) was then combined with the anoxic FHB inside
the glovebag, and the mixture was titrated with 1 mol l-1 KOH to
give the appropriate pH in a final 10 ml volume (770 µl for pH 7.9, 320
µl for pH 6.4). FHB was then added to mitochondrial pellets drained of
supernatant after the final centrifugation, and incubated on ice in the purged
glovebag for at least 30 min. The pH of the mitochondrial suspensions was
assessed with a Radiometer electrode (GK2401C) at the beginning and end of the
incubation. All mitochondrial suspensions were preincubated with 100 µg
ml-1 actinomycin D for 10 min on ice prior to use, and then
incubated at room temperature for the experiments. Mitochondrial protein was
quantified according to Peterson
(1977
) using BSA as a
standard.
Assessment of RNA degradation in organello
Mitochondria (500 µl), prepared as described above, were held at room
temperature for the indicated times prior to RNA extraction. For anoxic
studies, mitochondria were transferred to glass vials with screw-tops and
teflon sealing discs and submerged in mineral oil at room temperature.
Immediately after the incubations, each sample was spiked with 200 ng of a 540
bp fragment of exogenous, synthetic hexokinase (HK) RNA from Artemia
franciscana, which was produced using a Megascript kit (Ambion; Austin,
TX, USA). This step controlled for any differences in RNA isolation among
treatments, as well as for any possible RNA degradation during isolation. All
manipulations involving RNA were performed under RNase-free conditions.
Solutions were treated with 0.1% diethylpyrocarbonate (DEPC) prior to
autoclaving (except those containing primary-amine buffers), and glassware was
baked at 250°C for 3 h. Total mitochondrial RNA was isolated by
guanidinium/phenol extraction as described
(Eads and Hand, 1999), DNase
treated, quantified by spectrophotometry and subjected to dot-blot analysis
essentially as described previously
(Hardewig et al., 1996
).
Northern blots were also performed by the method of Hardewig et al.
(1996
) to assess quality of
RNA preparations and specificity of our probes (see
Fig. 1). For dot blots, 5 µg
of each mitochondrial RNA sample was combined with 10-20 µg of yeast RNA in
a final volume of 98 µl of denaturing solution (80% deionized formamide,
3.7% formaldehyde, 20 mmol l-1 phosphate, pH 7.0). Synthetic RNA
for each of the mRNA species to be quantified was prepared from linearized
plasmids containing the inserts, and known quantities of synthetic RNA from 10
pg to 20 ng were added to 10 µg yeast RNA in denaturing solution. Samples
of yeast RNA (25 ng) served as a negative control. These mixtures were
denatured at 70°C for 10 min, quick-chilled on ice, and then dilute
loading dye was added to each sample prior to application. Gene Screen Plus
nylon membranes were blotted and washed according to manufacturer's
recommendations on a Minifold-II manifold from Schleicher and Schuell (Keene,
NH, USA). After aspiration, the membranes were thoroughly dried and
UV-crosslinked, which markedly improved the signal strength. The membranes
were prehybridized in 5x SSPE (20x SSPE: 3.6 mol l-1
NaCl, 200 mmol l-1 sodium phosphate buffer, 20 mmol l-1
EDTA, pH 7.2) with 4% SDS at 65°C for at least 2 h prior to addition of
gel-purified PCR probes, which were synthesized from mitochondrial genes of
A. franciscana cloned into pGemT vectors as described previously
(Eads and Hand, 1999
;
Hardewig et al., 1996
). Probes
(25 ng) were radiolabeled using [
32P]-ATP (3.7 GBq
ml-1, 162.2 GBq mmol-1; NEN/Perkin Elmer) using a Hi
Prime Kit (Roche, Indianapolis, IN, USA) to a specific activity
>108 d.p.m. µg-1. Following hybridization at
65°C for 36-48 h, membranes were washed twice for 15 min each in 2x
SSPE plus 0.1% SDS at 65°C, and twice at room temperature in 0.2x
SSPE plus 0.1% SDS for 10 min. The blots were visualized using a Typhoon
(Molecular Dynamics, Sunnyvale, CA, USA). ImageQuant v. 1.1 by
Molecular Dynamics was used for quantitation of radioactivity, and Statview by
SAS (Cary, NC, USA) was used for statistical analyses; P<0.05 was
considered significant.
|
In order to assess the effectiveness of actinomycin D in inhibiting
mitochondrial transcription during incubations to measure RNA degradation,
transcriptional run-on assays were performed in organello under
identical incubation conditions, except that 1.85 MBq 32P-UTP and 5
µmol l-1 UTP (specific activity 17.65 GBq mol-1) were
added at the beginning of the assays. Incorporation of radiolabel over time
was quantified with a filter-based assay, followed by TCA precipitation and
scintillation counting as previously described
(Eads and Hand, 1999).
Measurement of 3' polyadenylation in mitochondrial RNA
The size of the poly(A)+-tail (PAT) of the same mRNAs measured
by dot blots (ATP synthase subunit 6, cytochrome c oxidase subunit I,
cytochrome b, NADH dehydrogenase subunit 1) were assessed essentially
as described by (Salles et al.,
1999). Briefly, 500 ng of RNA isolated from the degradation
experiments described above were used for cDNA synthesis with AMV-RT and an
oligo-d(T)-anchor primer
(5'-GTTCCACCTCTTTTGGTT(T)15-3'). The RNA and primer
(
200 ng) were combined in 6 µl of water, denatured at 65°C for 5
min and transferred to 42°C. The mixture was incubated with a final
concentration of 1x AMV-RT buffer, 0.5 mmol l-1 dNTPs, and 20
units of AMV-RT in a final volume of 20 µl and incubated at 42°C. After
1 h the reverse transcriptase was heat inactivated at 65°C for 20 min, and
the undiluted reaction used as template for PCR. The anchor primer [minus the
d(T) moiety] and a message-specific primer (0.1 µmol l-1) were
used in 50 µl reactions containing 1 µl template, 1x PCR buffer
(10 mmol l-1 Tris, 50 mmol l-1 KCl, 0.1% Triton X-100),
1.5 mmol l-1 MgCl2, 0.2 µmol l-1 dNTPs,
and 2.5 units Taq polymerase (Promega). The message-specific primers used
were: ATP6, 5'-TTTATAGTAATATCTTTCTGA; COXI,
5'-GTATTTGAGAGGCCATGATC; CYB, 5'-GCCAACATTTCTATTTGATGA; ND1,
5'-AAGATTTTGGGTTACATTCAG. Cycling conditions for PCR were: 94°C, 45
s; primer annealing temperature, 1.5 min; 72°C, 2 min; repeated 30 times.
Control reactions were run in parallel, including: RNA minus reverse
transcriptase; PCR reactions with no cDNA template or no primer; and
restriction digestions with no restriction enzyme.
PCR products were then restriction digested with appropriate enzymes to verify specificity and improve resolution of PAT length. Restriction enzymes were chosen using Webgene at SUNY Geneseo Biology (URL: www.Darwin.bio.geneseo.edu/~yin/webgene/RE.html). The enzymes were: for ATP6 products, EcoRI; for COXI and CYB, VspI; for NDI, HinDIII. Following digestion, products were phenol-chloroform extracted, ethanol precipitated, washed in 70% ethanol and resuspended in 6 µl 100% formamide. Samples were electrophoresed in non-denaturing 6% polyacrylamide at 100 V, stained with 0.5 µg ml-1 ethidium bromide, and visualized on a Molecular Dynamics Typhoon (Amersham Biosciences, Piscataway, NJ, USA) using a 532 nm excitation laser and a 610BP30 emission filter. To quantify polyadenylation, each polyadenylated band in a lane was normalized to the upstream (non-polyadenylated) band in that lane created by digestion. Fluorescence intensity in the lower band was divided by the fluorescence in the upper, nonpolyadenylated band using ImageQuant software. This approach accounted for loading differences between lanes and made direct lane-to-lane comparisons possible.
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Results |
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These patterns contrast with control values that showed significant
increases in message levels during the first hour of incubation (data not
shown), followed by a steep decline from 1-6 h. Calculations of degradation
rate constant (k) and transcript half-life (t1/2)
(Table 1) indicated that marked
stabilization relative to controls occurred for ATP6, where
t1/2 increased by more than 23-fold, from 48.2 to 1113
min. CYB and COXI displayed an increased half-life of 7.5-fold and 7.0-fold,
respectively, relative to controls. ND1 displayed the greatest stabilization
among all four mRNAs under conditions of anoxia plus low pH. From 1 h to 6 h
during the incubation, ND1 levels did not change; the half-life and decay
constants were therefore not calculated. To measure the contribution of RNA
synthesis to the measured RNA levels under these experimental conditions,
transcriptional run-on assays were performed
(Fig. 2). As anticipated, the
actinomycin D-inhibited incorporation was very low compared to the rates
measured under optimized conditions (e.g. all nucleotides added and
inhibitor-free conditions; Eads and Hand,
1999) (not shown). From 1 h to 6 h incubation, no RNA synthesis
was measurable under any of the experimental conditions
(Fig. 2), and it is important
to note that this period was used for calculating message decay rates and
half-lives.
|
|
Anoxia and low pH can independently stabilize mitochondrial mRNA
To evaluate the contributions of low pH (pH 6.4) and anoxia independently,
dot blots and run-on assays were performed on mitochondria incubated under
either anoxia or low pH. As shown in Fig.
3 and Table 1, both
anoxia and pH 6.4 had stabilizing effects on RNA levels. For example, during
treatment with anoxia, no significant change was seen in mRNA levels of any of
the four measured mRNA species (Fig.
3; open squares). Increases in transcript half-life reflected the
lack of change (Table 1). The
most dramatic extension occurred in CYB, from 67.5 min to 1085 min (a 16-fold
increase), while COXI and ND1 increased by 3.5-fold and ATP6 by 2.5-fold.
Taken together, these data suggest a downregulation of degradation of these
mRNAs under anoxia alone.
|
The effect of low pH, which is a hallmark of anoxic quiescence in A. franciscana embryos, was similar to the anoxic effect. As depicted in Fig. 3 (filled squares), levels of all four mRNA species at low pH were unchanged (P>0.05) from the first hour of incubation to the 6 h time point. The increase in half-life ranged from 1.2 times for COXI to 7.9 times for ND1, with CYB and ATP6 each increased by 3.9 times (Table 1). These data demonstrate that RNA degradation is depressed at low pH.
Exogenous ATP does not increase the degradation of mitochondrial
mRNA
In response to anoxia, A. franciscana embryos undergo a net
hydrolysis of ATP (Stocco et al.,
1972). Although matrix ATP levels in vivo are unknown,
anoxia promotes a decline in ATP in isolated mitochondria
(Kwast and Hand, 1996a
). RNA
decay pathways appear to require ATP, either as a cofactor, such as in the
bacterial degradosome (cf.
Grunberg-Manago, 1999
), or for
hydrolysis, as in a mitochondrially localized 3'-5' exonuclease
characterized in yeast, which increases activity in response to elevated NTP
levels (Min and Zassenhaus,
1993
). For these reasons, we hypothesized that the lowered ATP
levels under anoxia might be responsible in part for the increased half-lives
of mRNA observed under this condition, which proved to be incorrect. As shown
in Fig. 4 and
Table 1, the addition of 1.5
mmol l-1 ATP under anoxia had no effect, or in some cases slightly
decreased degradation rates relative to anoxia alone. These trends are
reflected by increases in half-lives, from 1.9- and 3.7 fold-increases for
COXI and CYB, respectively, to 14.9- and 17 fold-increases for ATP6 and ND1
(Table 1).
|
Mitochondrial mRNA polyadenylation can be reproducibly
quantified
Patterns of gene-specific polyadenylation of mitochondrial mRNAs were
quantified using a PCR-based assay referred to as RACE-PAT
(Salles et al., 1999) coupled
with ethidium bromide fluorescence of electrophoresed products. The
reproducibility of the assay was quantified by performing the assays in
triplicate and calculating the coefficient of variation for the amount of PAT
in each lane (Table 2). Three
mitochondrial RNA preparations were used as template for the cDNA reactions,
except under conditions of anoxia plus low pH, where two preparations were
used. The results of the experiment demonstrated that the quantification of
PAT for COXI is quite robust, with the coefficient of variaton (CV) ranging
from 3.4 to 10.2 (Table 2). The
assay was repeated using ATP6, and the CV ranged from 5.6 to 12.3.
Representative gels of RACE-PAT products are shown in
Fig. 5. PAT lengths were
calculated by subtracting non-PAT portions of the DNA products (i.e. the
distance between the restriction site and the 3' end of the coding
region; also subtracting the anchor primer minus the d(T) moiety). Our
analyses led us to conclude that evaluating prominent PAT bands for changes
was more revealing than choosing size ranges in some cases, hence band sizes
being compared vary from gene to gene. PAT length ranged from approximately
300 nucleotides to almost zero, or non-adenylated (e.g.
Fig. 5, lanes B5, C6).
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The same trends were also seen for COXI and ND1, which showed an increase from 1-6 h in polyadenylation of controls for all size-classes except the longest ND1 PAT (Fig. 6B,C). Experimental treatments generally reduced the amounts of PAT accumulated over time for these transcripts, although the results were not significant relative to control amounts (Fig. 6B,C). Decreases in PAT amounts during the assay were evident for all experimental treatments with ND1, by twofold and greater (Fig. 6C). Only the PAT of 130-190 bp under conditions of anoxia plus low pH and a band of 70-120 bp under conditions of low pH showed an increase (1- to 1.5-fold). For COXI, under control conditions the largest increase in PAT amounts was seen in the smallest size class (15-25 bp; 12-fold increase), although longer PATs accumulated as well (Fig. 6B). Also with COXI, when PAT amounts at 6 h were compared between experimental treatments and controls, significant differences were seen for all treatments (Fig. 7). Thus both metrics we used to quantify the amount of PAT in our assays showed some significant increases in polyadenylated fragments under control conditions and a decrease under experimental treatments (Figs 6, 7).
For ATP6, the experimental treatments had less apparent effect on PAT amounts relative to controls, when analyzed as a change in PAT amounts over time (Fig. 6D). Anoxia plus 1.5 mmol l-1 ATP had no effect, with identical amounts of PAT present in both bands at 1 and 6 h, and low pH slightly increased PAT amounts above control levels (Fig. 6D). However, when analyzed as the total amount of PAT after the 6 h incubation, control levels were significantly greater than those for experimental treatments, with the exception of anoxia plus ATP, where only a modest difference was noted (Fig. 7). Our measurements of polyadenylation of selected mitochondrial messages indicate in general that amounts of PAT are decreased in the experimental treatments relative to controls. Thus, increased polyadenylation is associated with message instability, and decreased PAT amounts are correlated with stability of message.
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Discussion |
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Previous work in this laboratory and others has shown that a downregulation
of gene expression occurs in A. franciscana embryos during quiescence
(reviewed by Hand, 1998). For
example, nuclei isolated from embryos exposed to 4 h of anoxia in
vivo decrease incorporation of [
-32P]-UTP by >79%
relative to controls, with a further decrease to 88% of control values after
24 h of anoxic incubation (van Breukelen et al., 2000). The acidification of
pHi during anoxia accounts for over half of the effect seen in
vitro (van Breukelen et al., 2000). In addition, isolated mitochondria
show a marked decrease in [
-32P]-UTP incorporation rate
either under anoxia (89%) or at pH 6.4 (74%;
Eads and Hand, 2003
).
Furthermore, new transcriptional initiation during the assay was shown to
decrease dramatically at pH 6.4, from 77% at pH 7.9 to 31% at pH 6.4
(Eads and Hand, 2003
). The
decrease in transcription seen under anoxia is mirrored by the blockage of the
ontogenetic increase in both actin and COXI mRNA by anoxia and aerobic
acidosis in vivo (Hardewig et
al., 1996
). These observations point to a sharp decrease in
anabolic activity for these reactions, coupled to decreased degradation of RNA
pools, to preserve stores during prolonged quiescence.
In the context of the current study, it is appropriate to note that protein
synthesis and degradation studies also support the hypothesis that gene
expression is arrested under anoxia, and that the pHi drop plays a
significant role (reviewed by Hand,
1997). Mitochondrial protein synthesis is decreased under
conditions of low pH and anoxia (Kwast and Hand,
1996a
,b
),
and under anoxia the half-life of cytochrome c oxidase protein is
increased by 77-fold (Anchordoguy et al.,
1993
). By analogy, we expected to see an increase in mRNA
half-life under anoxia due to a decrease in degradation, which was indeed the
case, although not to the same extent as estimated for protein. Because
mitochondrial mRNA levels remain virtually unchanged during the period when
translation is arrested (the present study;
Hardewig et al., 1996
), it
seems clear in A. franciscana that message limitation during anoxia
is not responsible for the rapid depression of mitochondrial protein
synthesis.
Taken together, these data show that both pH and anoxia are able to
influence mitochondrial RNA metabolism independently of one another, which may
indicate that the influence of these two factors are mediated by separate
mechanisms. Distinct signaling cascades or different target processes could be
used to affect the arrest of RNA metabolism. For example, it has been shown
that transcription initiation is sensitive to low pH but not anoxia
(Eads and Hand, 2003). On the
other hand, our data showing that conditions of anoxia and low pH combined
have an effect on mRNA half-life similar to that of either treatment alone may
indicate a common target. One parsimonious explanation is that the acidic
pHi during anoxia is a general inhibitor, while there are specific
O2-sensitive processes that also influence gene expression.
Identifying the cis- and trans-elements of RNA metabolism in
mitochondria is important for understanding the regulatory mechanisms of gene
expression.
Although the role of nucleo-cytoplasmic polyadenylation in gene expression
has been studied extensively in developing organisms (cf.
Bashirulla et al., 2001;
Richter, 1999
), there are
little data available on the role of the PAT in mitochondrial RNA metabolism.
In both Xenopus sp. and Drosophila sp., changes in PAT
length of cytoplasmic message are known to signal a variety of important gene
expression events, including mRNA localization, translation/silencing and
degradation (Richter, 1999
),
while very little is known about these processes in animal mitochondria
(reviewed by Taanman, 1999
;
Scheffler, 1999
). Although
poly(A)+ length has been examined in parallel with mRNA half-life
in murine mitochondria, only ribosomally bound mRNAs were used in the analysis
(Avadhani, 1979
). Two classes
of mRNA with distinct half-lives were present on the ribosomes and these mRNAs
possessed PATs of 55-75 nucleotides
(Avadhani, 1979
). However, any
inference about the relationship between mRNA half-life and PAT length in this
case is confounded with translation. Given that only 10% of mitochondrial mRNA
may be bound by ribosomes (Scheffler,
1999
), it may be misleading to use only ribosomally bound mRNA in
examining the relationship between PAT length and message half-life.
A report of mRNA degradation in trypanosome mitochondria documents the
presence of a UTP-dependent mechanism that preferentially and rapidly degrades
poly(A)+ RNA, as well as a UTP-independent process that does not
depend on a PAT (Militello and Read,
2000). Considering the unusual features of trypanosome
mitochondrial transcription involving UTP such as editing and 3'
uridylation, it is not clear that the UTP-dependent pathway of degradation is
representative of mitochondrial RNA metabolism in general. However, the rapid
degradation of polyadenylated mRNAs in mitochondria described here for A.
franciscana embryos may reflect a more general phenomenon. Similar
reports exist for plant mitochondria
(Gagliardi and Leaver, 1999
;
Kuhn et al., 2001
),
chloroplasts (Hayes et al.,
1999
; Schuster et al.,
1999
), and bacteria (Sarkar,
1997
; Blum et al.,
1999
; Carpousis et al.,
1999
). Thus it appears that polyadenylation has an opposite effect
on mRNA stability in mitochondria to that seen in the cytoplasmic compartment
of eukaryotic cells.
Mechanisms for altering mRNA stability during oxygen limitation that do not
involve polyadenylation have received attention for a few selected mRNA
species in mammalian cells (reviewed by
Bunn and Poyton, 1996). For
instance, the increased stability of mRNAs for erythropoietin and vascular
endothelial growth factor during hypoxia has been documented, and appears to
be due in part to the activity of 3'-UTR binding proteins that protect
the mRNAs from degradation (McGary et al.,
1997
; Claffey et al.,
1998
). Several specific RNA-binding activities have also been
documented for metabolic enzymes, including glutamate dehydrogenase and
catalase (Hentze, 1994
),
glyceraldehyde-3-phosphate dehydrogenase
(Nagy and Rigby, 1994
) and
lactate dehydrogenase (Pioli et al.,
2002
). Similarly, ribosomal proteins from chloroplasts are able to
stabilize ribosome-free mRNAs (Nakamura et
al., 2001
). However, Artemia mitochondrial genes lack UTR
binding sites, which are the apparent basis of the activities described above,
and argues against this scenario. An alternative mechanism for stabilizing
mRNA, the lack of ATP as a substrate for nuclease activity, is also unlikely
to be operating in A. franciscana because exogenous addition of 1.5
mmol l-1 ATP did not increase message degradation rate
(Fig. 4).
It is appropriate to speculate as to why the relationship between
polyadenylation and decreased stability of mRNA in prokaryotes/organelles is
counter to that observed for the nucleo-cytoplasmic compartment. In the
eukaryotic cytoplasm, PATs are associated with mature, fully processed
messages and are generally required for successful translation, although the
role of the PAT in transcript degradation and translation is complex
(Mitchell and Tollervey,
2000). Addition of a PAT is not required in Artemia to
create a complete stop codon for most genes
(Valverde et al., 1994
), and
is likely not required for translation. In fact, recent unpublished data for
A. franciscana embryos (S. Hand, B. Eads, D. Jones) indicate that
ribosomal RNA in the mitochondrion is polyadenylated while that in the
cytoplasmic compartment is not, thus implying that mitochondrial
polyadenylation is not being used as a translation signal. Others have noted
the possibility that the PAT plays a more general role in RNA metabolism in
bacteria (Li et al., 1998
).
The lower overall levels of polyadenylation in bacteria and many organelles
than in the eukaryotic cytoplasm further indicates that PATs may not be
required for successful translation. Rather, in these less complicated
systems, polyadenylation of mitochondrial message might provide an organizing
center for nuclease(s) to begin digesting the 3' end. Recent work in
potato mitochondria (Gagliardi et al.,
2001
) provides support for the idea that 3' polyadenylation
promotes 3'-5' degradation. While differences in the mechanism of
degradation may exist between plant and animal mitochondria on one hand, and
chloroplasts and eubacteria on the other, the relationship between elevated
polyadenylation and increased degradation appears to have been conserved
between these groups (Gagliardi et al.,
2001
). Animal mitochondria typically contain very little
non-coding DNA and have extremely compacted coding regions, while chloroplasts
and plant mitochondria are generally much larger and contain genes not found
in animal mtDNA, relatively more noncoding DNA, and intergenic spacers and
UTRs that could serve as cis-processing signals
(Scheffler, 1999
). Perhaps
nuclear genes have subsumed roles in animal mitochondria that remain
mitochondrially encoded in other groups. The machinery responsible for
organellar RNA degradation is generally unknown, although some mitochondrially
localized nucleases have been isolated and characterized in yeast (Margossian
and Butow, 1997; Min and Zassenhaus,
1993
) and in various other organisms (see
Morales et al., 1992
; Alfonzo
et al., 1998; Puranam and Attardi,
2001
). Certainly the involvement of polyadenylation in
mitochondrial mRNA turnover and translation is only beginning to be
understood.
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Acknowledgments |
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Footnotes |
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References |
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