Sarcoplasmic reticulum Ca2+ pump mRNA stability in cardiac and smooth muscle: role of the 3'-untranslated region

Christine M. Misquitta2, James Mwanjewe1, Lin Nie1, and Ashok K. Grover1,2

Departments of 1 Medicine and 2 Biology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL METHODS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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 alpha -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 alpha -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-alpha -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.

To isolate RNA from the cellular fractions, TRIzol was added to the final nuclear resuspension at a ratio of 2:1. Similarly, TRIzol was added to the cytoplasmic supernatant at a ratio of 3:1. RNA was isolated according to the manufacturer's instructions and saved as a pellet until use, when it was suspended in formamide. Total RNA was estimated by A260 with solutions containing equivalent formamide as blanks.

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 [alpha -33P]UTP (2,500 Ci/mmol, Amersham Pharmacia). Probe amounts were quantified by both direct counting and trichloroacetic acid (TCA) precipitation. The probe counts used in each reaction depended on the message being detected. RPA reactions were optimum with 5-6 µg of LVM or 50-60 µg of SSM total RNA for SERCA2 mRNA and 1 µg of LVM or 2 µg of SSM total RNA for G3PDH mRNA. For each reaction, the total RNA amount was adjusted to 80 µg with yeast tRNA and then coprecipitated with the probe. The precipitate was resuspended in 10 µl of Hybridization Buffer III (Ambion) and incubated at 42°C overnight. The RNA was then digested at 37°C for 30 min with a 1:100 dilution of RNase A/T1 mix (Ambion). Samples were analyzed by electrophoresis in a denaturing 6% acrylamide-urea gel (Invitrogen) in a Tris-borate-EDTA buffer. Gels were dried under vacuum and exposed to a PhosphorImager (Molecular Dynamics) overnight for image analysis. All RPAs were determined to be linear over the range of RNA amounts used.

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 [alpha -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.

Total RNA was prepared from LVM and SSM as described in Total RNA isolation and estimation and used for isolating poly A+ RNA with a MicroPoly (A) Pure kit (Ambion) following the instructions of the manufacturer. In vitro decay assays were performed with the poly A+ RNA as described in Decay of synthetic 3'-UTR RNA. Several different protein concentrations were used in initial experiments to optimize reaction conditions. Poly A+ RNA and nuclear extract protein concentrations were 0.2 and 0.1 µg/µl, respectively. After the decay reactions, samples were analyzed in Northern blots as described previously (15). Typically, 1 µg of LVM or 2 µg of SSM poly A+ RNA were loaded per well. The blots were then hybridized with a probe in the conserved region of SERCA2 or with isoform-specific probes from the 3' end. The conserved region probe corresponded to the rabbit SERCA2 cDNA from 514 to 1282 (Entrez sequence X52496) and was labeled with [alpha -32P]dCTP (3,000 Ci/mmol, Amersham Pharmacia) with a Rediprime II random prime labeling kit (Amersham Pharmacia). The isoform-specific probe for SERCA2a corresponded to base positions 3845-4082 of the rabbit sequences and was flanked by the following primers: SR2aN3845up (5'-TGC CTT CAG CTC AAG TA-3') and SR2aN4082dn (5'-TT GCA CCA ACA TCT GTC-3'). Similarly, the SERCA2b-specific probe corresponded to base positions 4117-4404 of the rabbit sequence and was flanked by the primers SR2b4117up (5'-GC ACT GAG CAG AGT CCT GCT AC-3') and SR2b4404dn (5'-GAT TGT GAA GTG CCA CTG AA-3'). Hybridization was performed with these probes at 42°C for >16 h. The blots were washed two times with 0.1× SSC-0.1% SDS for 5 min at room temperature and then two times at 57°C for 30 min. The hybridized band was detected with a PhosphorImager.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Micrographs showing the reaction of cells in primary culture with antibodies. A and B: primary cultures of left ventricular muscle (LVM) cells. C and D: stomach smooth muscle (SSM) cells in primary culture. B and D are bright-field images of the cells, and A and C are fluorescence images showing reaction with MF20 (A) and anti-smooth muscle alpha -actin antibody (C). The following negative controls (not shown) were also carried out: reaction without primary antibody, reaction of SSM with MF20, and LVM with smooth muscle alpha -actin antibody. Bar, 100 µm.

We then examined SERCA2 mRNA levels 0 and 24 h after DRB addition. Total RNA from LVM and SSM cells (6 and 60 µg, respectively) was used in RPAs with the SERCA2 probe that recognizes both SERCA2a and -2b. After 24 h, SERCA2 mRNA was more stable in LVM cells than in SSM cells (Fig. 2A). We further investigated the stability 0, 12, and 24 h after DRB addition and pooled data from three to five preparations (Fig. 2B). The first-order rate constants for the SERCA2 decay in LVM (0.025 ± 0.002 h-1) and SSM (0.0566 ± 0.002 h-1) differed significantly (P < 0.05). Thus SERCA2 mRNA had a significantly (P < 0.05) longer half-life in LVM cells (27 ± 3 h) than in SSM cells (13 ± 0.5 h) in primary cultures. To determine whether this difference in the decay rates was specific to SERCA2 mRNA or was observed with any RNA taken from the two tissues, we examined the stability of 18S rRNA (Fig. 2C). The first-order rate constants for the decay of 18S rRNA in LVM or SSM did not differ significantly from zero or from each other (P > 0.05).


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Fig. 2.   Effect of 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB) treatment. A: PhosphorImage showing sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) mRNA levels after DRB treatment. Primary cultures of neonatal rabbit LVM and SSM were treated with 70 µM DRB for 0 and 24 h as described in EXPERIMENTAL METHODS. Total RNA was analyzed by RNase protection assay (RPA) with the SERCA2 probe. Replicating this experiment 3 times showed that SERCA2 mRNA was more stable in LVM than in SSM (P < 0.05). B: time dependence of SERCA2 mRNA decay in cultured cells. Primary cultures of LVM and SSM were treated with 70 µM DRB. RNA was isolated at the specified times and analyzed as in A. Data are from 3-5 experiments. In each experiment, the amount of SERCA2 at t = 0 h was taken to be 100% and all other values were expressed as its percentage. Values are means ± SE. The rate of first-order decay for SERCA2 mRNA is 0.025 ± 0.002 h-1 in LVM and 0.0566 ± 0.002 h-1 in SSM, differing significantly from each other (P < 0.05). These rates correspond to significantly different half-lives of 27 ± 3 and 13 ± 0.5 h, respectively (P < 0.05). C: time dependence of 18S rRNA decay in cultured cells. Cells were treated as in B, and 18S rRNA levels were examined in Northern blots. The rates of decay for 18S rRNA did not differ significantly from zero or between LVM and SSM (P > 0.05).

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 alpha -tubulin (cytoplasmic marker) (11). Figure 3D shows that, by ELISA, the cytoplasmic fractions of both LVM and SSM did not contain detectable amounts of the nuclear protein. Figure 3E shows that, in Western blots, 20 µg of cytoplasmic protein from LVM or SSM gave a very bright band at 55 kDa for alpha -tubulin but even 80 µg of nuclear protein did not show this band, indicating that there was no detectable cytoplasmic contamination in the nuclear fractions and vice versa.


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Fig. 3.   Characterization of nuclear (Nuc) and cytoplasmic (Cyto) fractions. A: total RNA. B: ribosomal RNA. C: poly A+ RNA. Data are means ± SE of 3 preparations. D: histone H1 distribution. Relative levels of histone/mg protein were determined by taking the reactivity in the SSM nuclei to be 1 in each preparation. The values are means ± SE of 4 preparations. E: Western blots showing alpha -tubulin at 55 kDa. The size of this band was verified with protein molecular weight markers (MBI Fermentas; not shown).

Figure 4A compares the abundance of SERCA2 mRNA in cytoplasmic and nuclear fractions of LVM and SSM. Abundance, when expressed as SERCA2 mRNA per total RNA, is much greater in LVM nuclei than in SSM nuclei (Fig. 4A). SERCA2 abundance in cytoplasm and nuclei of LVM did not differ significantly (P > 0.05). This was also true of SSM. The greater abundance of SERCA2 mRNA in LVM nuclei than SSM nuclei persisted when calculated as SERCA2 mRNA per ribosomal RNA or SERCA2 mRNA per poly A+ RNA. Thus the greater abundance of SERCA2 RNA in LVM compared with SSM originates in the nucleus and is continued in the cytoplasm. In contrast, Fig. 4B shows that the abundance of G3PDH in the nuclei of LVM and SSM is not significantly different.


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Fig. 4.   Estimation of nuclear and cytoplasmic SERCA2 RNA by RPA. A: abundance of SERCA2 mRNA in cytoplasmic and nuclear extracts. All values were obtained as PhosphorImages as in Fig. 2A. In each preparation, the abundance of SERCA2 mRNA was calculated as the amount of SERCA2 mRNA per total RNA. The value for the abundance of the cytoplasmic SSM was taken as 1, and all the other values are expressed relative to this value. Data shown are means ± SE of 3 preparations. B: abundance of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA in cytoplasmic and nuclear extracts. Computations were performed as in A.

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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Fig. 5.   In vitro decay curves for synthetic SERCA2a and -2b 3'-untranslated regions (UTR) in cytoplasmic and nuclear extracts. A: LVM cytoplasmic. B: LVM nuclear. C: SSM cytoplasmic. D: SSM nuclear. For each experiment, the relative intensity at t = 0 min was taken as 100% and the mean relative intensity for other time points was calculated. Values shown are means ± SE of 3-5 experiments.



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Fig. 6.   Comparison of in vitro decay rates of synthetic SERCA2a and -2b 3'-UTR. Instead of taking the mean values as in Fig. 5, decay rates were determined as slopes of ln(%RNA remaining) vs. time for individual experiments. Values shown are means ± SE of 3-5 experiments. The decay rates for SERCA2b mRNA rates were significantly greater than those for SERCA2a for nuclear extracts from LVM and SSM and cytoplasmic extracts from LVM (P < 0.05) but not for cytoplasmic extracts from SSM (P > 0.05). The decay of SERCA2a did not differ significantly between nuclear and cytoplasmic extracts (P > 0.05). SERCA2b decay did not differ significantly between nuclear and cytoplasmic extracts from LVM (P > 0.05), but it was greater with the nuclear extracts than with the cytoplasmic extracts from SSM (P < 0.05).

We then investigated the in vitro decay of full-length SERCA2a and -2b mRNA by LVM nuclear extract to verify the prediction from Fig. 5B that these factors would decay the SERCA2b mRNA more rapidly than SERCA2a. We used LVM and SSM poly A+ RNA as sources of SERCA2a and -2b mRNA, respectively. First, we examined the SERCA2 mRNA decay by Northern blotting with a probe at the 5' end of the coding region. The decay curves (log RNA vs. time) are shown in Fig. 7A. They appear to be nonlinear, but the nonlinearity may be attributed to the low concentrations of SERCA2 RNA giving signals fairly close to the background. Incubation at the same nuclear extract protein concentration resulted in faster decay of SERCA2b RNA than of SERCA2a (Fig. 7A). Because LVM and SSM express mostly SERCA2a and -2b mRNA, respectively, but they also express small amounts of the other isoform, we verified the results of this experiment with isoform-specific probes. PhosphorImages from this experiment (Fig. 7B) and their analysis (Fig. 7C) show that the crude nuclear protein extract caused a more rapid decay of the SERCA2b isoform than of SERCA2a. Thus the in vitro decay experiments using the full-length mRNA gave results predicted from those using synthetic RNA fragments.


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Fig. 7.   In vitro decay of full-length SERCA2a and -2b mRNA by LVM nuclear extracts. A: analysis with a probe that recognizes both SERCA2a and -2b. Poly A+ RNA from LVM and SSM was incubated with the LVM nuclear extracts as in Fig. 5. After the decay reaction the RNA was analyzed by Northern blotting with a probe in the 5'-end of the coding region that is conserved in SERCA2a and -2b. For each experiment, the relative intensity at t = 0 min was taken as 100% and the mean relative intensity for other time points was calculated. Values shown are means ± SE of 2 experiments. The values for 15, 30, and 60 min differed significantly (P < 0.05) between LVM and SSM but not the value for 5 min (P > 0.05). B: PhosphorImages of Northern blotting with isoform-selective probes in the 3'-UTR. Incubation was carried out as in A but only for 0 or 5 min. C: analysis of decay with isoform-specific probes. For each experiment, the relative intensity at t = 0 min was taken as 100% and the mean relative intensity for other time points was calculated. The values for SERCA2a and -2b at 5 min differed significantly from each other (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 beta -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 beta -adrenergic receptor protein and mRNA. The 3'-UTR of beta 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 beta 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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