Characterization of SERCA2b Ca2+-Mg2+ ATPase mRNA decay by nuclear proteins

Gene expression is controlled at several levels including mRNA decay. Sarco/endoplasmic reticulum Ca2+-Mg2+-ATPase isoform 2b (SERCA2b) is central to Ca2+ signalling and homeostasis in several tissues. SERCA2b mRNA decay involves interactions between cis-acting elements in its 3'-region and trans-acting nuclear protein factors. In the presence of the protein factors, the synthetic capped and polyadenylated RNA fragment 2b1 (3444-3753) decays faster than other SERCA2b 3'-region fragments. Here we determined the minimum cis-acting destabilizing element in the decay and its interactions with the nuclear protein factors. The in vitro decay required ATP hydrolysis and Mg2+ but not Ca2+. The decay was directional from 3' to 5', and involved a novel 35b GC rich domain designated 2b1-4 corresponding to 3521-3555. The decay of 2b1 RNA was decreased by (a) competition with 2b1-4, (b) mutation of 2b1 to delete 2b1-4, and (c) depleting the extracts of destabilizing trans-acting factors using immobilized 2b1-4. To determine the minimal destabilizing elements 2b1-4 was divided into 7b domains A-E. Deleting AB, BC, CD or DE inactivated the destabilizing cis-acting element but deleting A, B, C, D or E had no effect. In electrophoresis mobility shift assays the nuclear protein extracts retarded the mobility of labeled uncapped 2b1 RNA without a poly A+ tail. A positive co-operativity in the interactions was shown in protein concentration dependence of the shift and in the competition of 2b1-4 in inhibiting the mobility of 2b1 RNA. Based on further experiments, the domain CDE (3535-3555) was sufficient to compete with 2b1 RNA for the protein binding. Consistent with this competition, excess CDE RNA retarded the in vitro decay of 2b1 RNA. Thus the RNA decay required ATP hydrolysis and Mg2+ but not Ca2+, the minimum binding domain was in the sequence 3535-3555, and the decay may involve a multimeric protein complex.     

RNA localization in yeast: moving towards a mechanism

RNA localization is a widely utilized strategy employed by cells to spatially restrict protein function. In Saccharomyces cerevisiae asymmetric sorting of mRNA to the bud has been reported for at least 24 mRNAs. The mechanism by which the mRNAs are trafficked to the bud, illustrated by ASH1 mRNA, involves recognition of cis-acting localization elements present in the mRNA by the RNA-binding protein, She2p. The She2p/mRNA complex subsequently associates with the myosin motor protein, Myo4p, through an adapter, She3p. This ribonucleoprotein complex is transported to the distal tip of the bud along polarized actin cables. While the mechanism by which ASH1 mRNA is anchored at the bud tip is unknown, current data point to a role for translation in this process, and the rate of translation of Ash1p during the transport phase is regulated by the cis-acting localization elements. Subcellular sorting of mRNA in yeast is not limited to the bud; certain mRNAs corresponding to nuclear-encoded mitochondrial proteins are specifically sorted to the proximity of mitochondria. Analogous to ASH1 mRNA localization, mitochondrial sorting requires cis-acting elements present in the mRNA, though trans-acting factors involved with this process remain to be identified. This review aims to discuss mechanistic details of mRNA localization in S. cerevisiae.     

Developmental changes of Ca(2+) handling in mouse ventricular cells from early embryo to adulthood

Transplant of immature cardiomyocytes is recently attracting a great deal of interest as a new experimental strategy for the treatment of failing hearts. Full understanding of normal cardiomyogenesis is essential to make this regenerative therapy feasible. We analyzed the molecular and functional changes of Ca(2+) handling proteins during development of the mouse heart from early embryo at 9.5 days postcoitum (dpc) through adulthood. From the early to the late (18 dpc) embryonic stage, mRNAs estimated by the real time PCR for ryanodine receptor (type 2, RyR2), sarcoplasmic reticulum (SR) Ca(2+) pump (type 2, SERCA2) and phospholamban (PLB) increased by 3-15 fold in the values normalized to GAPDH mRNA, although Na(+)/Ca(2+) exchanger (type 1, NCX1) mRNA was unchanged. After birth, there was a further increase in the mRNAs for RyR2, SERCA2 and PLB by 18-33 fold, but a 50% decrease in NCX1 mRNA. The protein levels of RyR2, SERCA2, PLB and NCX1, which were normalized to total protein, showed qualitatively parallel developmental changes. L-type Ca(2+) channel currents (I(Ca-L)) were increased during the development (1.3-fold at 18 dpc, 2.2-fold at adult stage, vs. 9.5 dpc). At 9.5 dpc, the Ca(2+) transient was, unlike adulthood, unaffected by the SR blockers, ryanodine (5 microM) and thapsigargin (2 microM), and also by a blocker of the Ca(2+) entry via Na(+)/Ca(2+) exchanger, KB-R 7943 (1 microM). The Ca(2+) transient was abolished after application of nisoldipine (5 microM). These results indicate that activator Ca(2+) for contraction in the early embryonic stage depends almost entirely on I(Ca-L).     

Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins

The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.     

A nucleolin-binding 3' untranslated region element stabilizes beta-globin mRNA in vivo

The normal expression of human beta globin is critically dependent upon the constitutively high stability of its encoding mRNA. Unlike with alpha-globin mRNA, the specific cis-acting determinants and trans-acting factors that participate in stabilizing beta-globin mRNA are poorly described. The current work uses a linker-scanning strategy to identify a previously unknown determinant of mRNA stability within the beta-globin 3' untranslated region (3'UTR). The new determinant is positioned on an mRNA half-stem opposite a pyrimidine-rich sequence targeted by alphaCP/hnRNP-E, a factor that plays a critical role in stabilizing human alpha-globin mRNA. Mutations within the new determinant destabilize beta-globin mRNA in intact cells while also ablating its 3'UTR-specific interaction with the polyfunctional RNA-binding factor nucleolin. We speculate that 3'UTR-bound nucleolin enhances mRNA stability by optimizing alphaCP access to its functional binding site. This model is favored by in vitro evidence that alphaCP binding is enhanced both by cis-acting stem-destabilizing mutations and by the trans-acting effects of supplemental nucleolin. These studies suggest a mechanism for beta-globin mRNA stability that is related to, but distinct from, the mechanism that stabilizes human alpha-globin mRNA.     

Folding of A+U-rich RNA elements modulates AUF1 binding. Potential roles in regulation of mRNA turnover

In mammals, A+U-rich elements (AREs) are potent cis-acting determinants of rapid cytoplasmic mRNA turnover. Recognition of these sequences by AUF1 is associated with acceleration of mRNA decay, likely involving recruitment or assembly of multi-subunit trans-acting complexes. Previously, we demonstrated that AUF1 deletion mutants formed tetramers on U-rich RNA substrates by sequential addition of protein dimers (Wilson, G. M., Sun, Y., Lu, H., and Brewer, G. (1999) J. Biol. Chem. 274, 33374-33381). Here, we show that binding of the full-length p37 isoform of AUF1 to these RNAs proceeds via a similar mechanism, allowing delineation of equilibrium binding constants for both stages of tetramer assembly. However, association of AUF1 with the ARE from tumor necrosis factor (TNFalpha) mRNA was significantly inhibited by magnesium ions. Further fluorescence and hydrodynamic experiments indicated that Mg(2+) induced or stabilized a conformational change in the TNFalpha ARE. Based on the solution of parameters describing both the protein-RNA and Mg(2+)-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg(2+) and AUF1 binding to the TNFalpha ARE. These studies provide the first evidence that some AREs may adopt higher order RNA structures that regulate their interaction with trans-acting factors and indicate that mRNA structural remodeling has the potential to modulate the turnover rates of some ARE-containing mRNAs.     

mRNA stability: in trans-it.

The regulation of mRNA stability is an important step in the control of gene expression. Characterization of the mechanisms involved in the turnover of individual mRNAs has identified a requirement for specific cis-acting sequences and trans-acting factors, as well as an involvement of the translation apparatus. In the past year, significant progress has been made in the identification of trans-acting factors by both biochemical and genetic approaches. This review summarizes that progress and promotes the notion that the ribosome itself should also be considered as a trans-acting component of the mRNA decay machinery.     

Compartmentalized expression of three novel sarco/endoplasmic reticulum Ca2+ATPase 3 isoforms including the switch to ER stress, SERCA3f, in non-failing and failing human heart

The human sarco/endoplasmic reticulum (ER) Ca(2+)ATPase 3 (SERCA3) gene gives rise to SERCA3a-3f isoforms, the latter inducing ER stress in vitro. Here, we first demonstrated the co-expression of SERCA3a, -3d and -3f proteins in the heart. Evidence for endogenous proteins was obtained by using isoform-specific antibodies including a new SERCA3d-specific antibody, and either Western blotting of protein lysates or immunoprecipitation of membrane proteins. An immunolocalization study of both left ventricle tissue and isolated cardiomyocytes showed a distinct compartmentalization of the SERCA3 isoforms, as a uniform distribution of SERCA3a was detected while -3d and -3f isoforms were observed around the nucleus and in close vicinity of plasma membrane, respectively. Second, we studied their expressions in failing hearts including mixed (MCM) (n=1) and idiopathic dilated (IDCM) cardiomyopathies (n=4). Compared with controls (n=5), similar expressions of SERCA3a and -3d mRNAs were observed in all patients. In contrast, SERCA3f mRNA was found to be up-regulated in failing hearts (125+/-7%). Remarkably, overexpression of SERCA3f paralleled an increase in ER stress markers including processing of X-box-binding protein-1 (XBP-1) mRNA (176+/-24%), and expression of XBP-1 protein and glucose-regulated protein (GRP)78 (232+/-21%). These findings revisit the human heart's Ca(2+)ATPase system and indicate that SERCA3f may account for the mechanism of ER stress in vivo in heart failure.     

Regucalcin (RGN/SMP30) alters agonist- and thapsigargin-induced cytosolic [Ca2+] transients in cells by increasing SERCA Ca(2+)ATPase levels

Regucalcin (RGN), also reported as senescence marker protein-30 (SMP30), plays a role in Ca(2+) homeostasis by modulating a number of Ca(2+)-dependent proteins. RGN also plays a cyto-protective role and its decrease is linked to age-related diseases and cell death. This study shows that RGN reduces agonist (histamine)-induced Ca(2+) transients in RGN(+) transfected COS-7 cells (RGN(+)) and also increases their Ca(2+) storage capacity. These observations are explained by RGN(+) cells having increased mRNA and protein expression levels of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA). Therefore down-regulation of RGN expression may contribute to characteristics of age-dependent Ca(2+) homeostasis dis-regulation, by decreasing SERCA levels.     

Peroxynitrite resistance of sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery endothelium and smooth muscle

We examined the effects of peroxynitrite pre-treatment on sarco/endoplasmic reticulum Ca(2+) (SERCA) pump in pig coronary artery smooth muscle and endothelium. In saponin-permeabilized cells, smooth muscle showed much greater rates of the SERCA Ca(2+) pump-dependent (45)Ca(2+) uptake/mg protein than did the endothelial cells. Peroxynitrite treatment of cells inhibited the SERCA pump more severely in smooth muscle cells than in endothelial cells. To determine implications of this observation, we next examined the effect of the SERCA pump inhibitor cyclopiazonic acid (CPA) on intracellular Ca(2+) concentration of intact cultured cells. CPA produced cytosolic Ca(2+) transients in cultured endothelial and smooth muscle cells. Pre-treatment with peroxynitrite (200 microM) inhibited the Ca(2+) transients in the smooth muscle but not in the endothelial cells. CPA contracts de-endothelialized artery rings and relaxes precontracted arteries with intact endothelium. Peroxynitrite (250 microM) pre-treatment inhibited contraction in the de-endothelialized artery rings, but not the endothelium-dependent relaxation. Thus, endothelial cells appear to be more resistant than smooth muscle to the effects of peroxynitrite at the levels of SERCA pump activity, CPA-induced Ca(2+) transients in cultured cells, and the effects of CPA on contractility. The greater resistance of endothelium to peroxynitrite may play a protective role in pathological conditions such as ischemia-reperfusion when excess free radicals are produced.     

SECIS-SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy

Selenocysteine incorporation at UGA codons requires cis-acting mRNA secondary structures and several specialized trans-acting factors. The latter include a selenocysteine-specific tRNA, an elongation factor specific for this tRNA and a SECIS-binding protein, SBP2, which recruits the elongation factor to the selenoprotein mRNA. Overexpression of selenoprotein mRNAs in transfected cells results in inefficient selenocysteine incorporation due to limitation of one or more of these factors. Using a transfection-based competition assay employing overexpression of selenoprotein mRNAs to compete for selenoprotein synthesis, we investigated the ability of the trans-acting factors to overcome competition and restore selenocysteine incorporation. We report that co-expression of SBP2 overcomes the limitation produced by selenoprotein mRNA overexpression, whereas selenocysteyl-tRNA and the selenocysteine-specific elongation factor do not. Competition studies indicate that once bound to SECIS elements, SBP2 does not readily exchange between them. Finally, we show that SBP2 preferentially stimulates incorporation directed by the seleno protein P and phospholipid hydroperoxide glutathione peroxidase SECIS elements over those of other selenoproteins. The mechanistic implications of these findings for the hierarchy of selenoprotein synthesis and nonsense-mediated decay are discussed.     

The mechanism of agonist induced Ca2+ signalling in intact endothelial cells studied confocally in in situ arteries

In endothelial cells there remain uncertainties in the details of how Ca(2+) signals are generated and maintained, especially in intact preparations. In particular the role of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), in contributing to the components of agonist-induced signals is unclear. The aim of this work was to increase understanding of the detailed mechanism of Ca(2+) signalling in endothelial cells using real time confocal imaging of Fluo-4 loaded intact rat tail arteries in response to muscarinic stimulation. In particular we have focused on the role of SERCA, and its interplay with capacitative Ca(2+) entry (CCE) and ER Ca(2+) release and uptake. We have determined its contribution to the Ca(2+) signal and how it varies with different physiological stimuli, including single and repeated carbachol applications and brief and prolonged exposures. In agreement with previous work, carbachol stimulated a rise in intracellular Ca(2+) in the endothelial cells, consisting of a rapid initial phase, then a plateau upon which oscillations of Ca(2+) were superimposed, followed by a decline to basal Ca(2+) levels upon carbachol removal. Our data support the following conclusions: (i) the size (amplitude and duration) of the Ca(2+) spike and early oscillations are limited by SERCA activity, thus both are increased if SERCA is inhibited. (ii) SERCA activity is such that brief applications of carbachol do not trigger CCE, presumably because the fall in luminal Ca(2+) is not sufficient to trigger it. However, longer applications sufficient to deplete the ER or even partial SERCA inhibition stimulate CCE. (iii) Ca(2+) entry occurs via STIM-mediated CCE and SERCA contributes to the cessation of CCE. In conclusion our data show how SERCA function is crucial to shaping endothelial cell Ca signals and its dynamic interplay with both CCE and ER Ca releases.