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By mediating the coupled movement of Na, K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 The Na-K-Cl cotransporter (NKCC) operates in conjunction with the Na pump, a K channel, and a Cl channel to carry out transepithelial salt movement. In secretory epithelia, the importance of this system has been recently underscored by the discovery that mutations in the structure of the Cl-channel protein (CFT7R) comprise the defect in cystic fibrosis (1, 2). In an absorptive epithelium, the thick ascending limb of the loop of Henle in the mammalian kidney, the Na-K-Cl cotransporter is the target of the potent "loop diuretic" drugs furosemide and bumetanide (cf. ref. 3).The rectal gland of the dogfish shark is a model saltsecreting epithelium and is among the richest known sources of the Na-K-Cl cotransport protein (4); it has also been the subject of recent investigations of CFTR, K channels, and Na pumps (5-8). In the secretory cell, cotransporter activity appears to be regulated by the level ofintracellular Cl-through a process involving direct phosphorylation of the transport protein (9,10).We have previously prepared monoclonal antibodies to the shark rectal gland cotransporter, a 195-kDa glycosylated protein with a core molecular mass of -135 kDa (11). Here we report the cloning of a cDNA encoding the secretory Na-K-Cl cotransporter using these antibodies to screen a shark cDNA expression library. § A full-length cDNA has been used to direct the expression of the shark cotransporter in a mammalian cell line-the expressed protein is found to retain the low affinity for loop diuretics characteristic of the native shark protein. It is also shown that the expressed cotransporter is subject to regulation in response to ionic concentration changes in the foreign cells. MATERIALS AND METHODSIsolation of cDNA and Sequence Analysis. A cDNA library was prepared in AZAP II from 20 ,ug of poly(A)+-enriched shark (Squalus acanthias) rectal gland RNA using oligo(dT) and random primers (Stratagene custom library). Filter transfers of the plated library were screened using a mixture of antibodies J3 and J7, which recognize epitopes in the C-terminal half of the transport protein (11). Three overlapping clones (3B, 9C, 24A) were isolated in two rounds of plaque purification. An additional clone (16-2) was obtained by rescreening the library with the 1.4-kb EcoRI/EcoRI fr...

Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788–1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22–24-kD domain.

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RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-x S 5 splice site (5 ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-x L . Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-x S 5 ss selection, leading to decreased Bcl-x S isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5 ss; however, it was not required when a strong consensus 5 ss was present. In support of a role for RBM25 in modulating the selection of a 5 ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-x S 5 ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.Alternative splicing is a regulatory mechanism that allows eukaryotes to generate numerous protein isoforms, often with diverse biological functions, from a single gene (2,26,49). Pre-mRNA splicing takes place within the spliceosome, which is assembled stepwise by the addition of small nuclear ribonucleoprotein particles (snRNP) and numerous accessory nonsnRNP splicing factors (23, 31). The excision of introns and the joining of exons depend on the recognition and usage of 5Ј splice sites (5Ј ss) and 3Ј ss by the splicing machinery (21, 33). The commitment complex forms when the 5Ј ss is recognized by U1 snRNA base pairing and stabilized by U1 snRNP while the 3Ј ss is recognized by the U2 auxiliary factor (U2AF) through U2 snRNA base pairing with the branch point. Subsequently, the U4/5/6 tri-snRNP is incorporated into the complex and the U1 snRNA base paired at the 5Ј ss is replaced by U6 snRNA. These processes result in a fully assembled spliceosome that supports a series of rearrangements via RNA-RNA and RNA-protein interactions and activates the catalytic steps of cleavage, exon joining, and intron release (2, 26).However, the splice site signals that define the 5Ј ss and 3Ј ss are often degenerate. How and when they are used are believed to be modulated by a combinational interplay of positive (splicing enhancers) and negative (splicing silencers) cis elements and trans-acting factors (2, 26), forming the basis of alternative splicing. The splicing regulatory (SR) proteins (18, 41) and the heterogeneous nuclear ribonucleoproteins (hnRNPs) (11,46) bind with specificity to pre-mRNA (20).The SR proteins generally bind to enhancer elements through the RNA-binding domain and activate splicing at...

A number of premature translation termination mutations (nonsense mutations) have been described in the human a-and (8-globin genes. Studies on mRNA isolated from patients with IP-thalassemia have shown that for both the (3-17 and the (8-39 mutations less than normal levels of (8-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human (3-globin mRNA.) In vitro studies using the cloned (1-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. We have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, we have studied the effect of five nonsense mutations and two missense mutations on the expression of human (3-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.Many of the steps in /-globin gene expression have been well-studied. Globin mRNA is transcribed from DNA, spliced, polyadenylylated, and then translated into protein in the cytoplasm. Less is known about the determinants of nuclear and cytoplasmic globin mRNA stability and about nuclear-cytoplasmic transport. The /3-39 gene causes thalassemia because it encodes a truncated nonfunctional globin protein chain. (The codon at which the mutation occurs designates the name of the mutation.) Although the nonsense mutation at codon 39 affects translation, studies from our laboratory and others suggest that this mutation has an additional unexpected effect on mRNA metabolism (1-8).

Background Human heart failure (HF) is associated with decreased cardiac voltage-gated Na+ channel current (encoded by SCN5A), and the changes have been implicated in the increased risk of sudden death in HF. Nevertheless, the mechanism of SCN5A downregulation is unclear. A number of human diseases are associated with alternative mRNA splicing, which has received comparatively little attention in the study of cardiac disease. Splicing factor expression profiles during human HF and a specific splicing pathway for SCN5A regulation were explored in this paper. Methods and Results Gene array comparisons between normal human and heart failure tissues demonstrated that 17 splicing factors, associated with all major spliceosome components, were upregulated. Two of these splicing factors, RBM25 and LUC7L3, were elevated in human heart failure tissue and mediated truncation of SCN5A mRNA in both Jurkat cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). RBM25/LUC7L3-mediated abnormal SCN5A mRNA splicing reduced Na+ channel current 91.1 ± 9.3% to a range known to cause sudden death. Overexpression of either splicing factor resulted in an increase in truncated mRNA and a concomitant decrease in the full-length SCN5A transcript. Conclusions Of the 17 mRNA splicing factors upregulated in HF, RBM25 and LUC7L3 were sufficient to explain the increase in truncated forms and the reduction in full length Na+ channel transcript. Since the reduction in channels was in the range known to be associated with sudden death, interruption of this abnormal mRNA processing may reduce arrhythmic risk in heart failure.

The inclusion of exon 16 in the mature protein 4.1R messenger RNA (mRNA) is a critical event in red blood cell membrane biogenesis. It occurs during late erythroid development and results in inclusion of the 10-kd domain needed for stabilization of the spectrin/actin lattice. In this study, an experimental model was established in murine erythroleukemia cells that reproduces the endogenous exon 16 splicing patterns from a transfected minigene. Exon 16 was excluded in predifferentiated and predominantly included after induction. This suggests that the minigene contained exon and abutting intronic sequences sufficient for splicing regulation. A systematic analysis of the cis-acting regulatory sequences that reside within the exon and flanking introns was performed. Results showed that (1) the upstream intron of 4.1R pre-mRNA is required for exon recognition and it displays 2 enhancer elements, a distal element acting in differentiating cells and a proximal constitutive enhancer that resides within the 25 nucleotides preceding the acceptor site; (2) the exon itself contains a strong constitutive splicing silencer; (3) the exon has a weak 5 splice site; and (4) the downstream intron contains at least 2 splicing enhancer elements acting in differentiating cells, a proximal element at the vicinity of the 5 splice site, and a distal element containing 3 copies of the UGCAUG motif. These results suggest that the interplay between negative and positive elements may determine the inclusion or exclusion of exon 16 IntroductionProtein 4.1R is a critical 80-kd cytoskeletal protein found in circulating red blood cells (RBCs). It mediates the formation and maintenance of spectrin/actin complex and anchors the cytoskeleton to the overlying lipid bilayer. 1 Human 4.1R is encoded by a single genomic locus over 200 kb in length, 2 and is expressed as multiple isoforms resulting from complex alternative premessenger RNA (pre-mRNA) splicing pathways. Previous studies have shown that inclusion of a 21-amino acid sequence motif at the N-terminus of the 10-kd spectrin/actin-binding (SAB) domain is required to promote cytoskeletal junctional complex stability. 3,4 Genomic cloning of both the mouse and human genes confirmed that the 63-nucleotide (nt) motif is encoded by an individual exon, exon 16. 2,5 The splicing of this exon is therefore regulated in a cassette fashion. Exon 16 is omitted from much of the 4.1R mRNA of pre-erythroid cells but is included in most of the mRNA produced in late erythroid cells (Figure 1). 6,7 Splicing of exon 16 is thus highly regulated in a differentiation stage-specific manner, and this regulated event is critical for production of 4.1R isoforms that sustain the function of 4.1R in circulating RBCs.Alternative splicing of pre-mRNA is a fundamental mechanism for regulating eukaryotic gene expression. 8 In many cases, alternative RNA splicing contributes to developmentally regulated and cell type-specific patterns of gene expression. Although a great deal of information is available concerning the genera...