Alternative Splicing Region Research Articles

Extracellular matrix proteins in organ transplantation.

Extracellular matrix proteins in organ transplantation.

Alternatively spliced isoforms of the rat eye sodium/calcium+potassium exchanger NCKX1.

We have investigated the structure, function, and expression of the rat eye sodium/calcium+potassium exchanger NCKX1. The sequence of independent rat NCKX1 clones and the analysis of rat eye mRNA by RT-PCR revealed a region of alternative splicing that comprised four exons and encoded a stretch of 113 amino acids near the beginning of the large cytosolic loop. In comparison with other NCKX1 molecules and the rat NCKX2 protein, rat NCKX1 was highly conserved within the hydrophobic regions but was quite divergent in the two large hydrophilic loops. The only exception was the region of the cytosolic loop encoded by the second alternatively spliced exon, which was approximately 60% identical. Similar to bovine, but different from human, rat NCKX1 possessed an acidic motif that was repeated 14 times in the cytoplasmic loop. Analysis of NCKX1 expression in different rat tissues by Northern blot revealed a very high level of expression of a 7-kb transcript in the eye but also lower levels of transcripts of various lengths in other tissues. The recombinant rat NCKX1 protein was tagged in the extracellular loop with the FLAG epitope and expressed in HEK-293 cells. Surface delivery and potassium-dependent sodium/calcium exchange activity were observed for each spliced variant.

Genomic structure and expression profile of LPHH1, a 7TM gene variably expressed in breast cancer cell lines

Gene identification studies, centred on a region of overlapping loss of heterozygosity in breast tumours within band 1p31.1, lead to the characterisation of LPHH1, a novel human 7TM gene. The coding sequence of LPHH1 extends over a 60 kb region and comprises in excess of 28 exons. Alternative splicing occurs minimally at five positions, four of which are within the coding sequence. The fifth region of alternative splicing occurs at the extreme 5′ end of the transcript. A clear tissue specific bias in alternative exon selection is observed to some degree at all five positions, including the extreme 5′ region, which raises the possibility of multiple and perhaps tissue specific promoters. One such putative promoter region, which appears to be utilised predominantly in breast cancer cells, has been identified. LPHH1 is highly evolutionarily conserved, with the simplest (19 exon) gene product being 95% identical between human and rat. Comparison of the alternatively spliced exons between three species, where data are available, has so far revealed 100% identity in the encoded peptide sequences, suggesting conservation of a functional aspect of this splicing. Gene expression has been observed in all tissues and cell lines tested, with the exception of lymphoblastoid and multiple myeloma lines, where there appears to be only a very low level of transcription. LPHH1 also appears to be downregulated in human bone marrow. These data are consistent with a role for the gene products in adhesion-mediated signalling. Analysis of a panel of breast tumour cell lines revealed that a number apparently overexpressed the gene whilst others showed very low levels of transcription. In one case, the overexpression correlated with a low level increase in gene copy number in the tumour line. In addition to differences in the overall levels of expression, LPHH1 mRNAs were alternatively spliced to varying degrees with shifts in the major gene product to truncated or altered forms in some lines. No somatic LPHH1 mutations were detected through sequence analysis of four primary breast tumours that showed loss of the adjacent 1p31.1 marker D1S207.

Participation of dynamin in the biogenesis of cytoplasmic vesicles.

Dynamin is a 100-kDa GTPase that has been implicated in endocytosis. To extend our understanding of its cellular functions, we have microinjected specific affinity-purified anti-dynamin antibodies into cultured mammalian epithelial cells. Using this approach, dynamin function can be inhibited specifically and rapidly in single cells. Effects of microinjected inhibitory antibodies on distinct endocytic processes and plasmalemmal morphology were then assayed by fluorescence microscopy (FM) and ultrastructural analysis. Micro-injected antibodies inhibit the clathrin-mediated endocytosis of fluorophore-labeled transferrin and cause a marked invagination of the plasma membrane. Many of these long plasmalemmal invaginations had clathrin-coated pits along their cytoplasmic surface. A number of distinct noncoated pits resembling plasmalemmal caveolae also accumulated in anti-dynamin antibody-injected cells. Further, the cellular uptake of cholera toxin B, which normally occurs by the internalization of caveolae, was inhibited in these cells. In support of these observations, immunoisolation techniques, double-label immuno-FM, and immunoelectron microscopy (immuno-EM) provided biochemical and morphological evidence that dynamin associates with plasmalemmal caveolae. Together, these observations indicate that dynamin mediates scission from the plasma membrane of both clathrin-coated pits and caveolae during distinct endocytic processes. These results demonstrate that dynamin isoforms are involved in an additional endocytic process that is distinct from clathrin-mediated endocytosis and provide significant insights into the molecular mechanisms governing the GTP-mediated internalization of caveolae. Evidence is provided demonstrating that dynamin isoforms have a differential distribution in mammalian cells. Targeting information for these isoforms is provided at least in part by regions of alternative splicing. Thus, the different dynamin isoforms may be localized to distinct cellular compartments but provide a similar scission function during the biogenesis of nascent cytoplasmic vesicles.

Brain and muscle express a unique alternative transcript of alphaII spectrin.

Alternative splicing of pre-mRNA transcripts of alpha and beta spectrin has emerged as an important generator of diversity in this gene family, yet the functional consequences and extent of this diversity remains unknown. We have cloned and characterized full-length alphaII spectrin cDNA from human fetal brain (GenBank and ). On the basis of the predicted amino acid sequence, 11 amino acid substitutions, presumably representing polymorphisms, have been identified that distinguish this alphaII spectrin from human lung fibroblast alphaII spectrin. In addition, human fetal brain spectrin displays a novel five amino acid insertion in repeat 15 that arises from alternative mRNA splicing and that distinguishes this spectrin from lung fibroblast alphaII++ spectrin. This discovery, together with two previously identified regions of alternative mRNA splicing in alphaII spectrin suggest that as many as eight different splice forms of the mature protein might exist if all combinations (at inserts 1, 2, and 3) of alternative mRNA splicing are utilized. To assess this possibility, the tissue distribution of alternative exon usage was investigated by semiquantitative PCR with intron-jumping primer sets. Tissues examined were from mouse and included heart, kidney, lung, liver, thymus, spleen, brain, ovary, testis, and skeletal muscle, as well as mouse embryonic tissue. Transcripts both with and without insert 1, representing a 60 bp insertion within alphaII spectrin repeat 10, were identified in all tissues. In contrast, transcripts with insert 2, the novel 15 bp insertion reported here, were only expressed in brain, heart, skeletal muscle, and embryonic tissue. In all tissues examined only transcripts positive for insert 3, an 18 bp insertion in repeat 21, were amplified, even under conditions in which a 30% level of insert 3 negative transcript could be easily detected in artificially prepared control samples. All combinations of insert 1 and insert 2 were identified together in individual transcripts, verifying at least four distinct isoforms of alphaII spectrin. These have been named alphaIISigma1 through alphaIISigma4, in accord with current spectrin naming conventions. Dynamic molecular modeling of the 15th repeat unit incorporating insert 2 predicts that the spliced sequence forms a loop between helices A and B, and suggests that this insert might constitute a novel protein interaction site. The presence of this sequence in alphaIISigma3 and alphaIISigma4 spectrin suggests a specialized and heretofore unanticipated function for the 15th repeat of this molecule.

Regulation of Calcyclin (S100A6) Binding by Alternative Splicing in the N-terminal Regulatory Domain of Annexin XI Isoforms

Annexin XI is a Ca2+/phospholipid-binding protein that interacts with a member of S100 protein family, calcyclin (S100A6), in a Ca2+-dependent manner. There are two isoforms of annexin XI, annexin XI-A and -B, generated by alternative splicing in the N-terminal regulatory domain. To determine the role of the alternative splicing region in the calcyclin-binding, we identified and characterized its calcyclin binding site. Experiments with glutathione S-transferase fusion proteins with N-terminal sites of annexin XI-A showed the calcyclin binding site to be in residues Gln49-Thr62 of rabbit annexin XI-A, which contains part of the splicing region. A synthesized peptide corresponding to Tyr43-Thr62 of annexin XI-A inhibited the interaction of annexin XI with calcyclin in liposome co-pelleting assay. The calcyclin binding site possesses a hydrophobic residue cluster conserved among S100 binding sites of annexin I and II. Recombinant annexin XI isoforms were expressed in Sf9 cells using a baculovirus expression system. In contrast to annexin XI-A, it was found that annexin XI-B protein could not bind to calcyclin by the liposome co-pelleting assay. In Sf9 cells coexpressing calcyclin with annexin XI isoforms, the calcyclin binding was observed only for annexin XI-A isoform. These results indicate that the calcyclin binding ability of annexin XI is an annexin XI-A isoform-specific character, suggesting that annexin XI isoforms might play distinct roles in cells through each alternative splicing regions.

Molecular Cloning of a Novel Potassium-dependent Sodium-Calcium Exchanger from Rat Brain

We have isolated a novel cDNA clone from rat cerebral cortex encoding a protein of 670 amino acids (NCKX2) that has significant similarity to the 1199-amino acid-long Na/Ca-K exchanger of bovine rod outer segment (NCKX1). NCKX2 transcripts are 10.5 kilobase pairs in length and are expressed abundantly in neurons throughout the brain and with much lower abundance in selected other tissues. The predicted topology of the rat NCKX2 protein is very similar to that of bovine NCKX1, beginning with a solitary transmembrane segment (M0), which is removed as a "signal peptide" in bovine NCKX1, an extracellular loop, a cluster of five transmembrane spanning segments (M1 to M5), a long cytoplasmic loop, and a final hydrophobic cluster (M6 to M11). Within the hydrophobic clusters, rat NCKX2 shares 80% identity and 91% similarity with bovine NCKX1. The two larger hydrophilic loops are much shorter in NCKX2 than in NCKX1, accounting largely for the difference in length between the two proteins, and are dissimilar in sequence except for a 32-amino acid stretch with 69% identity in the cytosolic loop. NCKX2 was epitope-tagged in the extracellular domain and was shown to be expressed at the surface of transfected HEK cells. Analysis of NCKX2 function by fluorescent imaging of fura-2-loaded transfected cells demonstrated that NCKX2 is a potassium-dependent sodium/calcium exchanger.

Isoform-Specific Intracellular Vesicle Formation by Recombinant Annexin XI-A in Sf9 Cells

Annexins are a group of structurally related proteins that bind phospholipids in a Ca2+-dependent manner and have the ability to self-aggregate and to promote vesicle aggregation and membrane fusion. Two isoforms of annexin XI, termed XI-A and XI-B, were previously identified by screening a bovine chondrocyte cDNA library. But little is known about differences in their biological function. In the present study, we therefore examined the results of expression of the two proteins inSpodoptera frugiperda(Sf9) insect cells, and in mammalian COS-7 cells. Annexin XI isoforms were expressed in Sf9 cells using a baculovirus expression system. Recombinant annexin XI-A but not XI-B caused formation of spherical “annexin XI-associated vesicles,” in the cytoplasm of Sf9 cells. Furthermore, indirect immnocytofluorescence studies showed similar phenomenon, that of local aggregation, with transfected annexin XI-A in COS-7 cells, whereas annexin XI-B remained diffusely distributed throughout the cytoplasm. Since annexin XI isoforms differ in amino acid sequence only in the alternative splicing region of the N-terminal domain, these findings suggest that this domain has distinct biological significance in terms of aggregation and vesicle formation.

Molecular cloning of human cardiac troponin T isoforms: Expression in developing and failing heart

Troponin T, which links the troponin complex to tropomyosin, is found as multiple isoforms in the hearts of many animal species. Changes in isoform composition have been correlated with variation in myofilament sensitivity to calcium. In order to determine the origin of diversity of the cardiac troponin T (cTnT) isoforms indicated by existing protein data, we have determined the sequences and patterns of expression of mRNAs encoding troponin T in fetal and adult heart and those present in adult heart in end-stage failure. Three main regions of alternative splicing within the cTnT coding region were identified using reverse-transcriptase polymerase chain reaction (RT-PCR). Alternatively spliced RNAs are developmentally regulated and some of the fetal forms are expressed in adult failing heart. The molecular structure of the spliced regions was determined from cloned cDNAs and RT-PCR products. In the 5' region of the mRNA, isoforms are generated by the inclusion or exclusion of 15-, 3- and 27-nucleotide (nt) sequences and by the inclusion or exclusion of a separate 3-nt sequence. In the 3' region of the mRNA, alternative splicing involves a 9-nt sequence which can be present in full, in part or not at all. A further splicing site was identified in the central region involving a 234-nt sequence and resulting in rare but detectable mRNAs. This work demonstrates the complexity of cTnT RNA composition in human heart and provides the information necessary to address the function of cTnT isoforms in contraction.

Identification of the alternative spliced form of the α2/δ subunit of voltage sensitive Ca 2+ channels expressed in PC12 cells

The α2/δ subunit of voltage sensitive Ca 2+ channels expressed in PC12 has been cloned and partially sequenced. The message observed in Northern blot analysis displays a 7.5 kb transcript, identical in size to mRNA of rabbit skeletal muscle and rat brain. the nucleotide sequence of the cloned α2 subunit of the PC12 specific cDNA is >99% identical to rat brain sequence and 85% to skeletal muscle. Reverse-transcriptase-polymerase chain reaction (RT-PCR) of the alternative splicing region identifies two deleted regions of 57 bp and 21 bp in PC12 expressed α2/δ transcript. The alternative variant α 2e of α2/δ subunit which is expressed in PC12 cells was previously identified in human embryonic kidney (HEK293) cells. RT-PCR analysis show two different sized alternative PCR fragments in rat lung and none in rat spleen, kidney and intestine. Antibodies prepared against a 19 amino acid peptide within the alternative spliced region effectively inhibits [ 3H]dopamine release in PC12 cells. This implies that the alternatively spliced region is positioned extracellularly and is involved in regulation of the L-type Ca 2+ channel-mediated transmitter release.

Tissue-specific expression of Na(+)-Ca2+ exchanger isoforms.

The sodium-calcium exchanger (NCE) plays a critical role in diverse processes in many different tissues including heart, nerve, and kidney. Surprisingly, the NCE is encoded by a single gene. We have isolated and sequenced a rat renal NCE clone, denoted F1, that was identical to previous rat NCEs, except for two unique sequences: one in the 5'-untranslated region and the other at a site of alternative splicing in the coding sequence. To explore these regions further, we examined NCE transcripts in several tissues using "rapid amplification of cDNA 5'-ends" and polymerase chain reaction amplification. Three species were identified each with a different 5'-end exon spliced to a common NCE core at nucleotide -34 in the 5'-untranslated region. Based on Northern analysis, each of these species had a unique tissue distribution. Whereas the F1 5'-end variant was abundantly expressed only in kidney, a second variant was expressed mostly in heart, and the third variant was expressed ubiquitously elsewhere. Investigation of the region of alternative splicing in the coding sequence also revealed tissue-specific expression of five major species. These findings indicate that the NCE expression is controlled and regulated under the influence of different promoters in a tissue-specific fashion. Therefore, we propose that the structural complexity of the single NCE gene allows it to respond independently to the unique demands of different environments.

Cardiac L-type Ca 2+ channel triggers transmitter release in PC 12 cells

Among the various voltage-sensitive Ca 2+ channels present in PC 12 cells are the dihydropyridine (DHP)-sensitive L-channel, the ω-conotoxin (ω-CgTx)-sensitive N-channel, and an atypical ω-CgTx/DHP-insensitive Ca 2+ channel. Depolarization-evoked Ca 2+ entry and [ 3H]dopamine release is mediated by L-type Ca 2+ channels determined by the use of Ca 2+ channel antagonists, and a single protein of 250 kDa is recognized by L-type-specific antibodies. Screening of a PC12 cDNA library revealed two types of Ca 2+channels which were identified by partial sequencing. A pc12-L clone displayed virtually identical sequence homology to the cardiac L-type channel. The identical sequence homology of the single alternative splicing region confirmed clone pc12-L as the rbC-I transcript, a cardiac-neuronal α1 subunit expressed in rat brain. Clone pc12-N displayed identical sequence homology to rbB-I, a neuronal al subunit of the N-type Ca 2+ channel expressed in rat brain; Northern blot analysis identified RNA of a size similar to that previously described for rat brain.

Tenascin mRNA isoforms in the developing mouse brain.

The extracellular matrix glycoprotein tenascin is expressed in the developing mouse cerebellum as a group of four protein species of different molecular weights. The difference is most likely due to alternative splicing which is known to occur in tenascin mRNA within the region of the fibronectin type III repeats. In order to systematically analyze tenascin mRNA isoforms that would account for this heterogeneity, tenascin splice variants were isolated from mouse brain by the polymerase chain reaction (PCR). In agreement with Northern blot analysis, amplification by PCR revealed a general decrease in tenascin mRNA expression during development from embryonic and early postnatal to adult stages. This decrease was more pronounced for isoforms of high molecular weight compared to those of low molecular weight. In accord with the observations at the protein level, four splice variants were found to be predominantly expressed, containing insertions of either six, five, or one fibronectin type III repeat, or comprising no insertion. In addition, a minor splice variant with an insertion of four fibronectin type III repeats was isolated. Three of the isolated mRNA splice variants have not yet been described for mouse tenascin. Among them, an isoform containing six alternatively spliced repeats was found to include a novel fibronectin type III repeat. The sequence of this repeat displays 96.7% similarity to a corresponding type III repeat in human tenascin, revealing a strict evolutionary conservation between tenascin molecules from different species in the region of alternative splicing. Southern blot analysis of the amplified mRNA isoforms showed that the novel mouse type III repeat is confined to splice variants with an insertion of six fibronectin type III repeats. Furthermore, in situ hybridization on sections from mouse embryos indicated that tenascin-specific mRNAs containing the novel type III repeat are predominantly expressed in the central nervous system.

Alternative 5C actin transcripts are localized in different patterns during Drosophila embryogenesis

The Drosophila actin gene located at cytogenetic position 5C forms at least 9 and perhaps as many as 15 different transcripts with the use of alternative transcriptional start points, differential splicing, and different regions of cleavage/polyadenylation. Each transcript contains one of two alternative 5' exons. We have subcloned unique recombinant DNA probes specific for each separate 5' exon and for three polyadenylation regions into vectors containing T3 and T7 promoters. Single stranded, tritium-labeled RNA probes were generated in vitro from these constructs. These probes have been hybridized in situ to RNA transcripts present in tissue sections from Drosophila embryos. The results of these experiments indicate that transcripts homologous to the two separate 5' exon-specific probes accumulate in strikingly different patterns during Drosophila development. Thus the incorporation of a particular 5' exon into a transcript is correlated with tissue-specific localization of that transcript. In contrast, probes for each of the three polyadenylation regions do not detect any tissue-specific localization of transcripts.

Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells.

To study the alternative splicing of fibronectin during embryogenesis and oncogenic transformation, we isolated cDNA clones of chicken fibronectin. The partial amino acid sequence deduced from sequencing of these clones showed that, overall, chicken fibronectin is approximately 80% identical with mammalian fibronectins. However, two of the three known regions of alternative splicing differed from this average. The V region was significantly more divergent, and RNA from embryonic chicken liver showed a pattern of V exon splicing which was distinct from that seen in human or rat fibronectins. In contrast, the EIIIB segment was very highly conserved (96%). As in mammals, this segment and another (EIIIA) were alternatively spliced in a cell-type-specific fashion. EIIIA+ and EIIIB+ species were almost absent in liver but predominated in total embryo RNA at all times from 2.5 to 11 days postfertilization. We also examined the possible contributions of fibronectin splicing and integrin receptor expression to the loss of fibronectin on oncogenic transformation. We detected little change in fibronectin splicing, other than a slight increase in representation of EIIIB+ species in fibroblasts after transformation by Rous sarcoma virus. It was also established that the overall reduction in fibronectin mRNA level observed after transformation was not accompanied by a decrease in integrin mRNA levels, indicating that fibronectin and integrin receptors are not coordinately regulated at the transcriptional level.

Identification of a third region of cell-specific alternative splicing in human fibronectin mRNA.

We describe here a third region of variability in human fibronectin (FN) due to alternative RNA splicing. Two other positions of alternative splicing have been reported previously (ED and IIICS). The third region involves a 273-nucleotide exon encoding exactly one 91-amino acid repeat of type III homology, located between the DNA- and the cell-binding domains of FN, which is either included in or excluded from FN mRNA. The two mRNA variants arising by an exon-skipping mechanism are present in cells known to synthesize the cellular form of FN. However, liver cells, which are the source of plasma FN, produce only messengers without the extra type III sequence. Therefore, the region described here resembles, both structurally and functionally, the previously described ED (for extra domain) region, located toward the C terminus of the molecule, between the cell- and heparin- (hep 2) binding domains. We conclude that both the extra type III repeat (named EDII) and ED represent sequences restricted to cellular FN. Combination of all the possible patterns of splicing in the three regions described to date may generate up to 20 distinct FN polypeptides from a single gene.

Multiple sites of alternative splicing of the rat fibronectin gene transcript.

We describe analyses of the structure and expression of the rat fibronectin gene with particular attention to the 40-kb stretch from the center of the gene which encodes 17 type-III repeating units. Each repeat is precisely separated from its neighbors by introns and most are encoded by pairs of exons. Three repeats are encoded precisely by single exons and two of these (EIIIA and EIIIB) are alternatively spliced in a cell type-specific fashion. A third site of alternative splicing (EIIIB) reported here is similar in expression to the previously described EIIIA segment. Both are excluded from mRNA in liver cells and are, therefore, absent from plasma fibronectin. These two alternative splices, plus a third one (V) reported previously, can occur in all possible combinations giving 12 fibronectin mRNAs from a single gene. These splicing variations account for most but not all of the known fibronectin subunit variants. We report investigations designed to detect other regions of alternative splicing. We also show that the pattern of alternative splicing is somewhat altered on oncogenic transformation.

Cell-type-specific fibronectin subunits generated by alternative splicing.

Multiple fibronectin mRNAs arise by alternative splicing of the primary transcript of a single gene. We describe analyses of the contribution of this alternative splicing to fibronectin subunit heterogeneity in three different cell types using antisera directed against specific segments of fibronectin. beta-galactosidase-fibronectin fusion proteins produced with the lambda gt11 bacterial expression vector were used as immunogens. One region of alternative splicing accounts for differences in subunit size, while a second contributes to differences between the fibronectins present in blood plasma and in fibroblastic cells. We also show, however, that these two regions of alternative splicing do not account for all detectable subunits. We have also used these segment-specific antisera to show that blood platelets contain a spectrum of fibronectin subunits distinct from that found in blood plasma.