Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing.

We here report the existence of 6 additional isoforms of the NMDA receptor generated via alternative splicing by molecular analysis of cDNA clones isolated from a rat forebrain cDNA library. These isoforms possess the structures with an insertion at the extracellular amino-terminal region or deletions at two different extracellular carboxyl-terminal regions, or those formed by combinations of the above insertion and deletions. One of the deletions results in the generation of a new carboxyl-terminal sequence. All these isoforms possess the ability to induce electrophysiological responses to NMDA and respond to various antagonists selective to the NMDA receptor in the Xenopus oocyte expression system. In addition, a truncated form of the NMDA receptor also exists that contains only the extreme amino-terminal sequence of this protein molecule. These data indicate that the NMDA receptor consists of heterogeneous molecules that differ in the extracellular sequence of the amino- and carboxyl-terminal regions.     

VAMP-1 has a highly variable C-terminus generated by alternative splicing.

VAMP-1 (synaptobrevin1) is one of the key proteins in the SNARE complex which is involved in regulated exocytosis. Recently, Isenmann et al. (1998, Mol. Biol. Cell 9, 1649-1660) showed the extreme C-terminal region of VAMP-1A and 1B to be involved in subcellular targeting of the isoforms. Four new splice variants (VAMP-1C to F) were identified in addition to the previously published variants VAMP-1A and VAMP-1B. Interestingly, the four new isoforms also have variable sequences only at the extreme C-terminus. This suggests that the C-terminal region has an important function for VAMP-1 and vesicle targeting. All six variants were a result of alternative splicing that linked exons 1-4 which encode the conserved region of VAMP-1 with one of the exons 5A to 5F that encodes the highly variable extreme C-terminus. Exon (5A-E) encode C-termini of two to five amino acid residues, whereas exon 5F encoded a long C-terminal amino acid extension. The splice variants were differentially expressed in human brain, kidney, and inflammatory cells.     

Two messenger RNA isoforms of the gonadotrophin-releasing hormone receptor,generated by alternative splicing and/or promoter usage,are differentially expressed in rainbow trout gonads during gametogenesis

The recent cloning of a gonadotrophin-releasing hormone receptor (GnRH-R) cDNA from rainbow trout showed that it contains several in-frame ATG codons, one of which, ATG2, corresponds to that found in other species. However, an upstream codon, ATG1, could give rise to a protein with a larger extracellular domain. Using S1 nuclease assay and a method combining primer extension and RACE-PCR, we characterized a second population of mRNA, termed mRNA-2, with a distinct 5'untranslated region and lacking ATG1. The genomic origin of the two mRNAs was determined by establishing the complete gene structure, which shows, for the first time in a vertebrate species that an alternative splicing and promoter usage generate two GnRH-R mRNA variants whose 5' extremities are encoded by two different exons. The analysis of the tissue distribution indicated that mRNA-2 presents a broader pattern of expression and is detected at higher levels than mRNA-1. Interestingly, it was found that those two mRNAs are differentially expressed in male and female gonads during gametogenesis. In particular, the variations of mRNA-1 levels parallel those of sGnRH expression during spermatogenesis, indicating that tissue-specific processing of the GnRH-R mRNA may underlie the effects of GnRH as a paracrine/autocrine regulator of gonadal functions.     

Cytoplasmic Drosha activity generated by alternative splicing

RNase III enzyme Drosha interacts with DGCR8 to form the Microprocessor, initiating canonical microRNA (miRNA) maturation in the nucleus. Here, we re-evaluated where Drosha functions in cells using Drosha and/or DGCR8 knock out (KO) cells and cleavage reporters. Interestingly, a truncated Drosha mutant located exclusively in the cytoplasm cleaved pri-miRNA effectively in a DGCR8-dependent manner. In addition, we demonstrated that in vitro generated pri-miRNAs when transfected into cells could be processed to mature miRNAs in the cytoplasm. These results indicate the existence of cytoplasmic Drosha (c-Drosha) activity. Although a subset of endogenous pri-miRNAs become enriched in the cytoplasm of Drosha KO cells, it remains unclear whether pri-miRNA processing is the main function of c-Drosha. We identified two novel in-frame Drosha isoforms generated by alternative splicing in both HEK293T and HeLa cells. One isoform loses the putative nuclear localization signal, generating c-Drosha. Further analysis indicated that the c-Drosha isoform is abundant in multiple cell lines, dramatically variable among different human tissues and upregulated in multiple tumors, suggesting that c-Drosha plays a unique role in gene regulation. Our results reveal a new layer of regulation on the miRNA pathway and provide novel insights into the ever-evolving functions of Drosha.     

Functionally distinct insulin receptors generated by tissue-specific alternative splicing.

Cloning of the insulin receptor cDNA has earlier revealed the existence of two alternative forms of the receptor differing by the presence or absence of 12 amino acids near the C-terminus of the receptor alpha-subunit. This insert has been shown by others to be encoded by a discrete exon, and alternative splicing of this exon leads to tissue-specific expression of two receptor isoforms. We have studied the functional significance of the receptor isoforms and have confirmed that they are generated by alternative splicing. When cDNAs encoding the two forms of the insulin receptors are expressed in Rat 1 cells, the receptor lacking the insert (HIR-A) has a significantly higher affinity for insulin than the receptor with the insert (HIR-B). This difference in affinity is maintained when insulin binding activity is assayed in solution using detergent solubilized, partially purified receptors. These data, combined with the tissue specificity of HIR-A and HIR-B expression, suggest that alternative splicing may result in the modulation of insulin metabolism or responsiveness by different tissues.     

Isoform diversity of phosphorylase kinase alpha and beta subunits generated by alternative RNA splicing

We have sequenced rabbit cDNAs that encode one isoform of the alpha subunit and two isoforms of the beta subunit of phosphorylase kinase, in addition to the single isoform from fast skeletal muscle that has been characterized to date for each subunit. All these isoforms are generated by alternative RNA splicing. The alpha subunit sequence obtained from slow skeletal muscle (soleus) is characterized by an internal deletion of 59 amino acids. This deletion is predominant in mRNA from slow muscle, heart, and uterus and accounts for the smaller alpha subunit variant (alpha') characteristic of phosphorylase kinase purified from slow muscle and heart. The beta subunit mRNA can be differentially spliced at two sites. In all tissues (except skeletal muscle) that were analyzed, an internal segment encoding 28 amino acids of the muscle sequence is replaced by a homologous sequence of identical length, presumably through the use of mutually exclusive exons. In brain and some other tissues, the deduced N-terminal sequence of the beta subunit is also changed. This is achieved by an insertion into the mRNA sequence that interrupts the initial reading frame after 25 codons and starts a new reading frame, encoding a different N terminus of 18 amino acids. This modification probably affects the major regulatory phosphorylation site of the beta subunit.     

Microdiversity of human-plasma-membrane calcium-pump isoform 2 generated by alternative RNA splicing in the N-terminal coding region.

cDNA species covering the entire coding sequence of the human homologue of the rat plasma membrane Ca(2+)-ATPase (PMCA) isoform 2 have been isolated and characterized. The deduced amino acid sequence shows 99% identity with that of the rat protein and can be aligned with the latter without gaps except for one 14-amino-acid-residue insert in the region immediately preceding the putative phospholipid-sensitive domain in the human pump. cDNA clones isolated by anchored polymerase-chain reaction revealed additional microheterogeneity in the same N-terminal PMCA2-coding region. Alternative RNA splicing involving a region of 135 nucleotides generates three types of cDNA. One does not contain any of the 135 bp, and the other two contain 42 bp or the entire 135 bp of the optional sequence. Analysis of genomic DNA indicates that this sequence is encoded by three separate exons of 33, 60 and 42 bp. Although each of these exons could be inserted into the mRNA without changing the reading frame, polymerase-chain amplifications using cDNA libraries from several human tissues show that the 33-bp and the 60-bp exons are never independently used during splicing. The unequal distribution of the splice variants suggests tissue-specific regulation of the alternative-splicing pathways and indicates a functional specialization of the encoded isoform subtypes.     

Two chicken erythrocyte band 3 mRNAs are generated by alternative transcriptional initiation and differential RNA splicing.

The erythrocyte anion transport protein (band 3) mediates two distinct cellular functions: it provides plasma membrane attachment sites for the erythroid cytoskeletal network, and it also functions as the anion transporter between the erythrocyte cytoplasm and extracellular milieu. We previously showed that two chicken band 3 polypeptides are encoded by two different mRNAs with different translation initiation sites. Here we show that these two band 3 mRNAs are transcribed from two separate promoters within a single gene. In addition, the two pre-mRNAs are differentially spliced, leading to fusion with coding exons used in common in the two mRNAs. The chicken erythrocyte band 3 gene is therefore the first example of a gene that has two promoters within a single locus which function equally efficiently in one cell type at the same developmental stage.     

Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing.

Using anchored PCR, three different cDNA isoforms of the mouse retinoic acid receptor beta [mRAR-beta 1, mRAR-beta 2 (formerly mRAR-beta 0) and mRAR-beta 3], generated from the same gene by differential promoter usage and alternative splicing, were isolated. These three isoforms encode RAR proteins with different N-terminal A regions and identical B - F regions. The sequence encoding the first 59 amino acids of the mRAR-beta 3 A region is identical with the entire A region of mRAR-beta 1. However, the sequence of mRAR-beta 3 region A differs from that of mRAR-beta 1 by an additional 27 C-terminal amino acids encoded in an 81 nucleotide-long putative exon which is spliced in between the exons encoding the A and B regions of mRAR-beta 1. Both mRAR-beta 1 and beta 3 cDNAs differ entirely from mRAR-beta 2 in their 5'-untranslated (5'-UTR) and A region coding sequences. This N-terminal variability, in a region which was shown to be important for cell-type specific differential target gene trans-activation by other nuclear receptors, suggests that the three mRAR-beta isoforms may be functionally distinct. The conservation of RAR-beta isoform sequences from mouse to human, as seen by cross-hybridization on Southern blots or DNA sequence analysis, as well as their differential patterns of expression in various mouse tissues, corroborates this view. Additionally, the mRNA analysis data suggest that mRAR-beta 2, whose expression predominates in RA-treated embryonal carcinoma (EC) and embryonic stem (ES) cells, may be important during early stages of development. mRAR-beta 1 and beta 3, on the other hand, which are predominantly expressed in fetal and adult brain, may play some specific role in the development of the central nervous system.     

Identification of three annexin IX isoforms generated by alternative splicing of the carboxyl-terminal exon in silkworm, Bombyx mori.

Annexins (ANXs) are a family of structurally related proteins with Ca(2+)-dependent phospholipid-binding properties. Here we report the cloning of three cDNAs each encoding annexin IX (ANX IX) isoforms from unfertilized eggs of the silkworm, Bombyx mori. The analysis of exon/intron structures showed that the three mRNAs, named ANX IX-A (2300bp), ANX IX-B (1884bp) and ANX IX-C (1409bp), respectively, were generated from a single gene by alternative usage of a 3'-splice site of the last exon. Thus the three isoforms have an identical sequence from amino acid residues 1 to 307 and this region shows approximately 77% identity to Drosophila melanogaster ANX IX. Only amino acid residues 308-324 (A) or 308-323 (B and C), which correspond to the C-terminal tail, are different in the three proteins. A RT-PCR analysis indicated that the three isoforms of silkworm ANX IX were specifically expressed in various larval tissues and development stages. Interestingly, the C-terminal tail in ANXs I, II and V were previously confirmed as a binding region for protein kinase C. Thus generation of the three ANX IX isoforms in the silkworm, that are different from other ANXs, may have a functional significance other than binding to Ca(2+).     

Isoforms of JSAP1 scaffold protein generated through alternative splicing

We have identified four isoforms of c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein that participates in JNK mitogen-activated protein kinase cascades, termed JSAP1a, JSAP1b, JSAP1c, and JSAP1d. The previously identified JSAP1 was renamed JSAP1a to avoid confusion. Analyses of the exon-intron structure of the jsap1 gene indicated that the isoforms are generated through alternative splicing involving exons 5 and 6. The mRNA expression levels of the JSAP1 isoforms differed among the mouse tissues examined. We also investigated the region of JSAP1 responsible for its interaction with JNK, and found that the JNK-binding domain is located between aa residues 201 and 217 in JSAP1a, which is encoded by part of exon 6. As all the JSAP1 isoforms contain this binding domain, we examined the binding affinity of the JSAP1 isoforms for JNK1, JNK2, and JNK3. JSAP1c and JSAP1d, which contain a 31-aa sequence not present in JSAP1a or JSAP1b, had a lower binding affinity for the JNKs, especially JNK3. These results suggest that JSAP1c and JSAP1d may attenuate the scaffolding activity of JSAP1a and/or JSAP1b in JNK cascades, especially the JNK3 cascades.