Departments of 1 Medicine and 2 Biology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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Stomach smooth muscle (SSM) and left ventricular muscle (LVM) express the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump gene SERCA2. Alternative splicing yields two major isoforms, SERCA2a in LVM and slow twitch muscle and SERCA2b in SSM and most other tissues. The splices have different 3'-untranslated regions (UTR) and also encode proteins that differ slightly in their COOH-terminal domains. SERCA2 transcription rates are similar in the two tissues, yet LVM has a much higher level of SERCA2 mRNA than SSM. To understand the control of SERCA2 RNA expression, we inhibited transcription and showed that the half-life of SERCA2 mRNA is significantly longer (P < 0.05) in primary cultures of LVM cells than in SSM cells. Nuclear SERCA2 mRNA levels were also higher in LVM than in SSM. In vitro decay assays using synthetic RNA corresponding to the 3'-UTR of SERCA2a and -2b showed that nuclear extracts produced a faster decay of SERCA2 RNA than cytoplasmic extracts and that nuclear extracts produced a faster decay of SERCA2b than -2a. This was also true when the full-length native mRNA was used instead of the 3'-UTR RNA, and SERCA2b decay by cytoplasmic extracts was faster for LVM than for SSM. We propose that nuclear decay is an initial step in the control of SERCA2 RNA abundance and that this control is maintained or modulated in the cytoplasm. We discuss how these control mechanisms may be part of a control switch in cardiac development and pathophysiology.
mRNA stability; calcium; adenosinetriphosphatase; transcription; 3'-untranslated region; translation
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INTRODUCTION |
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MOBILIZATION of sarcoplasmic reticulum (SR) Ca2+ is pivotal to cardiac myocyte contractility. Whereas Ca2+ entry through voltage-operated Ca2+ channels and release of Ca2+ from the SR trigger actomyosin contraction, removal of Ca2+ from the cell via the Na+/Ca2+ exchanger, the plasma membrane Ca2+ pump, and its sequestration into the SR are pivotal to the relaxation phase of the contractile cycle (8, 13, 22, 23). Ca2+-Mg2+-ATPases of the SR, termed sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps, are responsible for sequestering Ca2+ into the sarco(endo)plasmic reticulum. Because of their central role in signal transduction, the activity of the SR Ca2+ pumps is controlled by phospholamban, cAMP-dependent protein kinase, and calmodulin kinase (2, 16, 28).
The SR Ca2+ pump protein in heart and stomach smooth muscle is encoded by the SERCA2 gene. SERCA2 pre-mRNA contains 25 exons that can be alternatively spliced to produce two major splice variants (7, 12, 14). Cardiac and slow-twitch muscle cells predominantly express the SERCA2a isoform, whereas most other tissues, including vascular and other smooth muscles, express SERCA2b. All the splice variants contain exons 1-21. As a result, they have a common 5'-untranslated region (UTR) and encode proteins with 993 identical NH2-terminal amino acids. In the SERCA2a splice variant, exon 21 is fused to exon 25. This mRNA encodes a 997-amino acid protein and has a 735-base 3'-UTR. The SERCA2b variant is produced by continuation of exon 21 into exon 22 and polyadenylation at the end of this exon. SERCA2b mRNA encodes a 1,042-amino acid protein and has an 829-base 3'-UTR. Thus SERCA2a and -2b encode proteins differing in their COOH-terminal domains and also have different 3'-UTR. This difference in the 3'-UTR may be important as there are several recent examples showing that 3'-UTR-mediated mRNA decay is a key regulatory mechanism in phenotypic expression (17, 20, 24).
Previously we reported (10, 15) that the SERCA2 protein in left ventricular muscle (LVM) is 100 times more abundant than in stomach smooth muscle (SSM). Although the greater expression of SERCA2 in cardiac tissue is expected, given its need to generate greater force with greater speed, the manner in which this difference in SERCA2 expression is achieved is unknown. Understanding how the expression of SERCA2 is controlled is pivotal to deciphering the SR Ca2+ pump function in heart failure and cardiac diseases. The SERCA2 mRNA level is 15- to 30-fold higher in LVM than in SSM even though the transcription rate of SERCA2 measured with nuclear run-ons does not differ significantly in the two tissues (15). Thus the control of SERCA2 expression is primarily posttranscriptional but pretranslational. Here we show that SERCA2 mRNA is more stable in LVM than in SSM, that nuclear SERCA2 mRNA levels are much higher in LVM than in SSM, and that this difference is mediated by the 3'-UTR and nuclear and cytoplasmic factors.
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EXPERIMENTAL METHODS |
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Stability in cultured cells.
Cells from LVM and SSM of 2-day-old rabbits were isolated by
collagenase digestion as previously described (15). LVM
cells were then plated in DMEM (Invitrogen) with 20% fetal bovine
serum at a density of 1 × 107 cells/10-cm dish. After
24 h, cells were rinsed with phosphate-buffered saline (PBS; in
mM: 137 NaCl, 2.7 KCl, 8 Na2HPO4, and 1.5 KH2PO4, pH 7.4) and either harvested or treated
with the transcription inhibitor
5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB, 70 µM; Sigma-Aldrich) in fresh DMEM with 10% fetal bovine serum for
various times (21). DRB treatment was stopped at the
appropriate time by rinsing with PBS and trypsinizing as for the
control cells. Transcription was inhibited in cultured SSM cells as
described for the LVM cultures with the following modification: the
cells were plated at a density of 1 × 106 cells/10-cm
plate for 7 days before treatment with DRB.
Characterization of cultured cells.
Cells were isolated and cultured as described in Stability in
cultured cells. For LVM cultures, cells were fixed after
24 h with 1% formaldehyde at 4°C. Similarly, SSM cells were
fixed after 7 days in culture. Cells were washed with 100 mM glycine to
remove excess formaldehyde before permeabilization with 3% Triton
X-100 at 23°C for 30 min. Cells were washed with PBS and blocked with
1% fetal bovine serum-PBS for 1 h at 37°C. Immunostaining was
performed at 37°C for 1 h with either of the following primary antibodies: MF20 supernatant (Developmental Studies Hybridoma Bank
developed under the sponsorship of the National Institute of Child
Health and Human Development and maintained by the University of Iowa),
used to detect the muscle-specific myosin heavy chain, or smooth
muscle-specific -actin (Sigma-Aldrich). Cells were then washed three
times with PBS and incubated with FITC-conjugated anti-rabbit or
anti-mouse secondary antibodies for MF-20 and smooth muscle
-actin,
respectively, at 37°C for 1 h. Cells were then washed five times
with PBS before fluorescent imaging with a Zeiss LSM510 microscope at
488 nm.
Total RNA isolation and estimation. All the buffers used for RNA were prepared in diethyl pyrocarbonate (DEPC)-treated water. Total RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's instructions and estimated routinely as absorbance at 260 nm (A260). Ribosomal RNA was estimated by dot blot analysis or after Northern blotting with a biotinylated 18S ribosomal RNA probe and poly A+ RNA by using a biotinylated (dT)20 probe as described previously (15). Because yeast tRNA was included as a carrier in the analysis of SERCA2 RNA stability in cultured cells, ribosomal RNA was considered as the total cellular RNA for estimation.
Isolation of nuclear and cytoplasmic RNA.
Cytoplasmic and nuclear fractions were isolated essentially by using a
previously reported protocol (4, 25). LVM and SMM tissues
were dissected as previously described in sterile PBS and placed in 2.5 ml of ice-cold buffer MA per heart or stomach (in mM: 10 Tris · HCl pH 7.4, 10 NaCl, and 10 MgCl2)
containing 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl
fluoride (PMSF), and 10 U/ml RNAguard (Amersham Pharmacia)
(15). The tissues were homogenized with a Polytron PT20
for 12 s (at position 4.5). The nonionic detergent Nonidet P-40
was added to the homogenate to a final concentration of 0.5%. The
solution was mixed gently by inverting the tubes several times,
incubated on ice for 15 min, and then centrifuged at 500 g
for 5 min to obtain a nuclear pellet and a supernatant. The supernatant
was centrifuged at 14,000 g for 10 min, the pellet was
discarded, and the resulting supernatant was retained as the
cytoplasmic fraction. The nuclear pellet was resuspended with a
Teflon-glass homogenizer in buffer MA containing 0.5%
Nonidet P-40, filtered through a nylon mesh (size 320), and centrifuged
at 500 g for 5 min to obtain a crude nuclear fraction. Nuclei were purified further as described previously (15).
The pellet was resuspended with a pipette in buffer MB (in
mM: 2,200 sucrose, 10 Tris · HCl pH 7.4, and 10 MgCl2) containing 1 mM PMSF, 2.8 mM DTT, and 14 U/ml
RNAguard and centrifuged at 110,000 g in a 41 Ti rotor for
60 min at 4°C. The pellet was resuspended in 5 vols of buffer
MA and centrifuged at 1,000 g for 20 min. The final
pellet was resuspended in 5 ml of buffer MA. To determine cytoplasmic contamination of nuclear fractions, the samples were boiled
in a reducing SDS sample buffer for 2 min and analyzed by 12%
polyacrylamide-SDS gel electrophoresis followed by Western blotting
with an anti--tubulin antibody (Sigma-Aldrich) as a cytoplasmic
marker (11). Detection was performed with Amersham Pharmacia ECL detection reagents. To determine nuclear contamination of
the cytoplasmic fractions, the histone contents were determined by
ELISA with an anti-histone H1 primary antibody (AE-4, Santa Cruz
Biotechnology) and an anti-mouse IgG conjugated to alkaline phosphatase
secondary antibody (Sigma-Aldrich). Linearity of the assays was
determined in initial experiments by using several concentrations of
the nuclear extracts. The histone contents were then compared by using
1- and 2-µg protein extracts for cardiac and stomach samples, respectively.
RNase protection assays. RNase protection assays (RPAs) were performed with the RPAIII kit, and probes for these assays were synthesized with the MaxiScript kit (both from Ambion). Templates for transcription were constructed by PCR. SP6 and T7 RNA polymerase sites were included in each product to allow transcription of the antisense strand by SP6 and sense RNA by T7. The SERCA2 riboprobe used did not distinguish between SERCA2a and -2b. It corresponded to 3064 to 3370 bp of the rabbit SERCA2 cDNA and was flanked by the primers SR2U3064 (5'-CTG CTG CGT GGT GGT TCA TT-3') and SR2D3370 (5'-GTG ATC TGG AAG ATA AGC GGC A-3'). The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe used was from 242 to 389 of the rabbit G3PDH sequence and was flanked by the primers G3PDH242up (5'-CAC GGT CAA GGC TGA GAA C-3') and G3PDH389dn (5'-CTT CTC CAT GGT GGT GAA-3').
Transcription reactions for all RPA probes were set up according to Ambion's instructions with [Decay of synthetic 3'-UTR RNA.
In vitro decay assays were conducted as described elsewhere
(5). The full-length 3'-UTR of SERCA2a and -2b were
amplified, respectively, by PCR from pBR322 and pBSSK+ plasmids
containing the full-length SERCA2 sequences. For the SERCA2a 3'-UTR,
the following primers were used: T7SR2a3444up (5'-ATT ACG ACT CAC TAT
AGA TTC TCA TGG ACG AGA C-3') and SP6PBR3639dn (5'-ATT TAG GTG
ACA CTA TAG ACT GAT GAA GCC ATA CC-3'). Similarly, for
the SERCA2b 3'-UTR, the primers were: T7SR2b3485up (5'-ATT ACG ACT CAC
TAT AGT CTC ATG GAC GAG ACT CTC AAG-3') and SP6BSSR2bdn (5'-ATT TAG GTG
ACA CTA TAG GCT CTA GAA CTA GTG GA-3'). RNA containing the 3'-UTR, an
m7G(5')ppp(5')G cap analog (Amersham Pharmacia), and a
40-base-long poly A+ tail were then transcribed from
purified PCR products with the SP6 MaxiScript kit and
[-32P]CTP (800 Ci/mmol, Amersham Pharmacia).
Unincorporated low-molecular-weight materials were removed with
Ultrafree-DA DNA extractor columns (Millipore). The amount of RNA was
determined as described for the RPA probes. LVM or SSM tissue extracts
were prepared as previously described in MOPS buffer [in mM: 10 MOPS-NaOH (pH 7.2), 200 sodium chloride, 2.5 magnesium acetate] with 2 mM DTT, 1 mM PMSF, and 2 µg/ml each of pepstatin and leupeptin (Sigma
Aldrich) (1). The tissue was homogenized with a Polytron
PT20 set at 4.5 for 12 s before centrifugation at 25,000 g for 20 min. The supernatant was centrifuged at 10,000 g for 30 min at 4°C. The resulting supernatant was taken
to be the cytoplasmic fraction. The nuclear pellet was resuspended with
a Teflon-glass homogenizer in 2.5 ml of MOPS buffer per heart or
stomach, filtered through nylon mesh into a new tube, and centrifuged
at 1,000 g for 10 min. The supernatant was removed, and the
pellet was resuspended in ~1 ml of MOPS buffer and disrupted for
5 × 12 s with the Polytron PT20. This homogenization step
was sufficient to disrupt nuclei, and hence high-ionic strength buffers
were avoided to preserve integrity of the proteins. The suspension was
left on ice, vortexing every 5 min for 20 min. This suspension was then
centrifuged at 10,000 g for 30 min at 4°C, and the
supernatant was taken as the nuclear fraction. Typically, 0.05 ng/µl
of 32P-labeled RNA was incubated with 0.25 µg/µl of
cell-free extracts from LVM or SSM for between 0 and 60 min at 37°C
in MOPS buffer containing 1 mM ATP, 0.1 mM spermine, 2 mM DTT, and 1 U/µl SUPERasin (Ambion). Yeast tRNA (10 µg) was added as a carrier,
and RNA was deproteinated by phenol-chloroform extraction followed by
ethanol precipitation. Precipitated samples were then analyzed by
acrylamide-urea gel electrophoresis as described in RNase
protection assays.
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RESULTS |
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We characterized cells in primary cultures from LVM and SSM, examined the stability of SERCA2 mRNA in them, determined the abundance of SERCA2 in nuclear and cytoplasmic extracts, and then investigated the effects of nuclear and cytoplasmic proteins on the decay of synthetic SERCA2a and -2b 3'-UTR RNA and the full-length native SERCA2 mRNA.
SERCA2 mRNA is more stable in LVM than in SSM. Our first goal was to determine whether mRNA stability is a major determinant in the discrepancy between SERCA2 transcription levels and mRNA levels in LVM and SSM. We determined the SERCA2 mRNA stability by monitoring its levels in cells after inhibiting transcription. We used DRB (70 µM), which inhibits transcription effectively at 50-100 µM (21). To confirm the effectiveness of DRB in inhibiting transcription, we determined incorporation of [3H]uridine into the primary cultures of LVM and SSM over a period of 4 h. DRB inhibited [3H]uridine incorporation by 89 ± 3% in the LVM cultures and by 83 ± 2% in the SSM cultures. Thus inhibition of [3H]uridine incorporation was similar in the LVM and SSM cell cultures (P > 0.05).
We also determined whether the cells in primary cultures retained their phenotypes. We used the anti-sarcomeric protein antibody MF20 for the LVM cells and anti-smooth muscle actin antibody for the SSM cells. LVM cells, when stained within 6 h of plating, reacted with the anti-MF20 antibody in immunocytochemistry (not shown). Even after 24 h in culture, the cells reacted with this antibody. In five bright-field images from two preparations of LVM cells, there were 167 cells, of which 153 reacted with the anti-MF20 antibody. This corresponds to 92% of the cells being muscle cells and 8% being other cell types or dead cells (Fig. 1, A and B). However, LVM cells in culture for 7 days without DRB lost the reactivity to MF20 (not shown). Therefore, in subsequent experiments, the LVM cells were cultured for only 24 h and then treated for up to 24 h with DRB. SSM is known to react with the anti-smooth muscle actin antibody. After 7 days in culture, all the SSM cells examined in five fields from two preparations were positive (Fig. 1, C and D). As additional negative controls, we also examined the reaction of MF20 with the SSM cells and of anti-smooth muscle actin with the LVM cells. As expected, no reaction was observed in either of the negative controls.
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Nuclear abundance of SERCA2 mRNA is greater in LVM than in SSM. Next we asked whether nuclei from LVM contain higher levels of SERCA2 mRNA than nuclei from SSM or the difference occurs only in the cytoplasm. To answer this question, we first characterized the cellular fractions and then measured the SERCA2 mRNA levels.
Figure 3 shows the distribution of total, ribosomal, and poly A+ RNA in the nuclear and cytoplasmic fractions isolated from LVM and SSM. The total amount of RNA obtained per animal for LVM (411 ± 57 µg) and SSM (502 ± 51 µg) was comparable (Fig. 3A). Nuclear RNA comprised ~10% in either tissue. In SSM, the ribosomal RNA content in the nuclear fraction (37 ± 9%) was significantly less than in the cytoplasmic fraction (68 ± 6%) (P < 0.05). This distribution was similar in LVM (Fig. 3B). Similarly, the poly A+ RNA content of the nuclear fraction was also lower than the cytoplasmic fraction in both tissues (Fig. 3C). As further confirmation of the purity of the nuclear and cytoplasmic fractions we determined the protein levels of histone H1 (nuclear marker) and
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In vitro decay of synthetic 3'-UTR of SERCA2 mRNA is isoform and
tissue specific.
We used an in vitro assay to monitor the effects of LVM and SSM
cell-free extracts on RNA decay. In initial experiments, we used the
transcribed sense strand of G3PDH RNA to ensure that there was no
nonspecific RNase activity in our preparation. After incubation of the
G3PDH RNA with 5 µg of protein for 60 min, the G3PDH RNA was 104 ± 5% of the initial RNA amount with total cell-free extract of LVM
and 89 ± 5% with that of SSM. Thus the extracts did not produce
any significant decay of the G3PDH RNA. We then used capped RNA
containing the 3'-UTR of SERCA2a or -2b with a 40-base poly
A+ tail to monitor the decay in cell-free cytoplasmic or
nuclear extracts. Figure 5 compares the
decay patterns of SERCA2a 3'-UTR and SERCA2b 3'-UTR with LVM and SSM
extracts (5 µg protein) over a period of 60 min in three to five
experiments. Figure 6 shows the
first-order rate constants for the decay. In all extracts examined, the
decay rate constants of SERCA2a did not vary significantly and were
much lower than those for the SERCA2b 3'-UTR. An exception was the
decay in the presence of SSM cytoplasmic extract, where both SERCA2a
and -2b appeared to be equally stable (Figs. 5 and 6). Thus, in the
nuclear extracts of either LVM or SSM, synthetic SERCA2a 3'-UTR RNA is
significantly more stable than SERCA2b 3'-UTR RNA.
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DISCUSSION |
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Our results show that SERCA2 mRNA is more stable in LVM than in SSM, that the nuclear abundance of SERCA2 mRNA is substantially higher in LVM than in SSM, and that in in vitro decay assays with nuclear extracts, the synthetic 3'-UTR RNA for SERCA2a is more stable than SERCA2b. This section focuses on methodological issues and the possible role of the 3'-UTR in SERCA2 expression in cardiac diseases.
SERCA2 mRNA in primary cultures of LVM cells was more stable than in SSM cells. This is consistent with a much longer half-life reported for SERCA2 in rat LVM cells in one study than that of vascular smooth muscle reported in another (19, 27). In control experiments, DRB treatment inhibited [3H]uridine incorporation but had no effect on the integrity of 18S ribosomal RNA. Thus the differences in the decay rate of SERCA2 in LVM and SSM do not reflect a degradation of all cellular RNA. Whereas it is possible that transcriptional inhibitors by themselves may affect mRNA stability (6), our results are consistent with other studies on SERCA2.
There are no previous reports to compare cytoplasmic vs. nuclear
distribution of SERCA2 mRNA. We were concerned about possible contamination of the isolated nuclear fraction with cytoplasm and about
the method of normalizing our data. The isolated nuclei contain a lower
proportion of ribosomal and poly A+ RNA than the cytoplasm
and no detectable amounts of the cytoplasmic marker -tubulin,
indicating that it is unlikely that the greater abundance of
SERCA2 mRNA in the LVM nuclei than in the SSM nuclei results from such
a contamination. Furthermore, this difference in the LVM vs. SSM nuclei
persists when the data are normalized with respect to ribosomal or poly
A+ RNA rather than the total RNA. In contrast, G3PDH mRNA
does not show this trend. The finding that the difference in SERCA2
mRNA levels between the two tissues was found at the nuclear level, before export to the cytoplasm had taken place, indicates that the
nucleus is the initial site of control of SERCA2 expression.
The in vitro decay of SERCA2 3'-UTR RNA was substantially faster than the decay of SERCA2 mRNA in cultured cells. The exact ratios of the nuclear factors to the RNA in the cells are not known, and the matter is further complicated by the possibility that in the cells several species of RNA may bind to the same protein factors whereas in vitro there was only one species of RNA. Results of the in vitro decay assays indicate that the decay of SERCA2 3'-UTR RNA is primarily nuclear and that the SERCA2a 3'-UTR RNA is more stable than the SERCA2b 3'-UTR RNA. In addition, the SERCA2b 3'-UTR RNA is more stable in SSM cytoplasmic extracts than in the LVM cytoplasmic extracts, indicating that the decay is not only sequence dependent but also tissue specific. In vitro decay of SERCA2b mRNA by SSM nuclear extracts was greater than with the SSM cytoplasmic extracts (Figs. 5 and 6), indicating the mediation of nuclear factors in the decay. Because in most extracts the decay of SERCA2b mRNA is faster than that of SERCA2a and because stomach expresses mostly SERCA2b, these data are consistent with a faster SERCA2 mRNA decay in SSM than in LVM cultured cells observed in Fig. 2. Finally, SERCA2 decay patterns in LVM and SSM extracts cannot be ascribed to nonspecific nucleases because there are differences between the decay rates of SERCA2a and -2b and because these extracts did not affect the stability of G3PDH RNA. Therefore, we propose that even though the transcription rates of SERCA2 in LVM and SSM are similar, differences in decay rates of SERCA2a and -2b by nuclear factors initiate the difference in the SERCA2 abundance between the two tissues. Once initiated, the SERCA2 mRNA levels may be maintained by tissue-specific cytoplasmic factors. However, our observations do not exclude the roles of transcriptional pause, differences in SERCA2 RNA residence time in nuclei, poly A+ tail length, or nuclear export and cytoplasmic factors.
Because SERCA2a and -2b contain different 3'-UTR sequences, it is
possible that this difference contributes to the mRNA stability. The
3'-UTR contributes to the control of stability of mRNA encoding -adrenergic receptors, ANG II receptors, nitric oxide (NO)
synthases, cyclooxygenase-2, endothelium growth factor, globin,
elastin, proteins involved in cell cycle regulation, oncogenes,
cytokines, and lymphokines (17, 20, 26). The
3'-UTR of these mRNA contain AU-rich elements with specific
cis-acting determinants that bind either stabilizing or
destabilizing trans-acting factors. Both SERCA2 sequences
(7, 9, 12) contain an AU-rich 3'-UTR. The AU content
is 57.5% in SERCA2a and 60.5% in SERCA2b. SERCA2b contains sequences
such as AUUUA (at 4060 and 4342) and GUUUG (at 4188). SERCA2a contains
AUUUA (at 4160) and GUUUG (at 3716, 3765, 3919, and 4173), but it also
contains additional consensus sequences such as UUAUUUAUA (at 3925) and
UUAUUUAAU (at 4044) for possible binding to trans-acting
factors. However, whether these or any other sequences are key to
SERCA2a and -2b mRNA stability and what factors bind them to control
their expression remains to be determined.
The decay of individual mRNA species may not only be tissue dependent
but may also change in certain diseases. For example, in congestive
heart failure there is a decrease in the expression of both
-adrenergic receptor protein and mRNA. The 3'-UTR of
1-adrenergic receptor mRNA, along with the
trans-acting factors AUF1, HuR, and heterogeneous nuclear
ribonucleoprotein (hnRNP)-A1, may mediate mRNA stability (3, 17,
18). In rats with myocardial infarction due to
congestive heart failure, ventricular SERCA2 mRNA and protein levels
decrease (29). The mechanisms underlying these changes in
SERCA2 protein and mRNA levels may involve regulation of the mRNA
stability. The 3'-UTR of
1-adrenergic receptor mRNA and
SERCA2a both contain the nonamer sequences that bind to AUF1 and HuR.
It remains to be determined whether, in heart failure, the same switch
alters expression of multiple messages such as adrenergic receptors and SERCA2.
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ACKNOWLEDGEMENTS |
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We thank Drs. Gerald Wilson and Gary Brewer (Robert Wood Johnson Medical School, Piscataway, NJ) for their suggestion on the nuclear vs. cytoplasmic distribution experiment, D. Mack for the result on DRB inhibition of transcription, F. Zaib for help with the ELISA, Sue E. Samson for technical assistance, and Dr. E. S. Werstiuk for critical comments on the manuscript.
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FOOTNOTES |
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This work was funded by a grant from Canadian Institute of Health Research (CIHR) (MOP53256). C. M. Misquitta received a Doctoral Scholarship from CIHR, and A. K. Grover received a Career Investigator Award from the Heart & Stroke Foundation of Ontario.
Address for reprint requests and other correspondence: A. K. Grover, Dept. of Medicine, HSC 4N41, McMaster Univ., 1200 Main St. West, Hamilton, ON, Canada L8N 3Z5 (E-mail: groverak{at}mcmaster.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 18, 2002;10.1152/ajpcell.00527.2001
Received 5 November 2001; accepted in final form 8 April 2002.
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