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PubMed2005

ASePCR: alternative splicing electronic RT-PCR in multiple tissues and organs.

RT-PCR is one of the most powerful and direct methods to detect transcript variants due to alternative splicing (AS) that increase transcript diversity significantly in vertebrates. ASePCR is an efficient web-based application that emulates RT-PCR in various tissues. It estimates the amplicon size for a given primer pair based on the transcript models identified by the reverse e-PCR program of the NCBI. The tissue specificity of each PCR band is deduced from the tissue information of expressed sequence tag (EST) sequences compatible with each transcript structure. The output page shows PCR bands like a gel electrophoresis in various tissues. Each band in the output picture represents a putative isoform that could happen in a tissue-specific manner. It also shows the EST alignment and tissue information in the genome browser. Furthermore, the user can compare the AS patterns of orthologous genes in other species. The ASePCR, available at http://genome.ewha.ac.kr/ASePCR/, supports the transcriptome models of the RefSeq, Ensembl, ECgene and AceView for human, mouse, rat and chicken genomes. It will be a valuable web resource to explore the transcriptome diversity associated with different tissues and organs in multiple species.

Algorithms↗
PubMed2003

Alternative splicing of the human cyclin D-binding Myb-like protein (hDMP1) yields a truncated protein isoform that alters macrophage differentiation patterns.

We have cloned two novel, alternatively spliced messages of human cyclin D-binding Myb-like protein (hDMP1). The known, full-length protein has been named hDMP1alpha and the new isoforms, hDMP1beta and hDMP1gamma. The hDMP1alpha, -beta, and -gamma splice variants have unique expression patterns in normal hematopoietic cells; hDMP1beta mRNA transcripts are strongly expressed in quiescent CD34+ cells and freshly isolated peripheral blood leukocytes, as compared with hDMP1alpha. In contrast, activated T-cells and developing myeloid cells, macrophages, and granulocytes express low levels of hDMP1beta transcripts, and hDMP1gamma is ubiquitously and weakly expressed. Mouse Dmp1 has been shown to activate CD13/aminopeptidase N (APN) and p19ARF gene expression via binding to canonical DNA recognition sites in the respective promoters. Assessment of CD13/APN promoter responsiveness demonstrated that hDMP1alpha but not hDMP1beta and -gamma, is a transcriptional activator. Furthermore, hDMP1beta was found to inhibit the CD13/APN promoter transactivation ability of hDMP1alpha. Stable, ectopic expression of hDMP1beta and, to a lesser extent hDMP1gamma, reduced endogenous cell surface levels of CD13/APN in U937 cells. Moreover, stable, ectopic expression of hDMP1beta altered phorbol 12-myristate 13-acetate-induced terminal differentiation of U937 cells to macrophages and resulted in maintenance of proliferation. These results demonstrate that hDMP1beta antagonizes hDMP1alpha activity and suggest that cellular functions of hDMP1 may be regulated by cellular hDMP1 isoform levels.

Alternative Splicing↗
PubMed1999

Characterization of the expression of the alternative splicing of the ED-A, ED-B and V regions of fibronectin mRNA in bovine ovarian follicles and corpora lutea.

Fibronectin is an extracellular matrix glycoprotein. Alternative splicing of fibronectin mRNA within three specific regions, the extra domains (ED) A and B and the variable (V) or IIICS region, result in the production of different isoforms of fibronectin. These isoforms differentially regulate tissue developmental processes, such as those occurring during follicular and luteal development. The specific isoforms of fibronectin present in follicles and corpora lutea have not been identified. To identify these, primers for reverse transcription polymerase chain reaction (RT-PCR) were designed to the known bovine amino acid sequence of exons flanking the ED-A, ED-B and V regions. PCR products from bovine fetal liver cDNA were determined to be bovine fibronectin by the correct product size and DNA sequence homology to other species, and to the known bovine amino acid sequence. Bovine ovarian follicles (0.5-9 mm diameter) and corpora lutea (cyclic, early to late mid-luteal phase) were shown to express ED-A+, ED-A-, ED-B+, ED-B-, V+ and V fibronectin isoforms, similar to the liver, lung and kidney of fetuses, but generally not of adult animals. Thus follicles and corpora lutea express isoforms of fibronectin usually expressed in developing tissues.

Alternative Splicing↗
PubMed2002

An alternatively spliced form of rodent alpha-synuclein forms intracellular inclusions in vitro: role of the carboxy-terminus in alpha-synuclein aggregation.

In the rat, the -synuclein gene is alternatively spliced and exists in three forms, rat synuclein 1 (rSYN1), synuclein 2 (rSYN2) and synuclein 3. rSYN2 cDNA encodes a 149 amino acid protein that is homologous to rSYN1 and human -synuclein for the first 100 amino acids, but is divergent for the 49 amino acid carboxy-terminal region. We demonstrate here that rSYN2 forms small aggregates throughout the cytoplasm when overexpressed in human H4 cells, whereas rSYN1 expression is diffuse. Inhibition of the proteasome promotes the formation of larger, cytoplasmic rSYN2 inclusions in transfected cells. Although a survey of the available databases suggests that there is no human splice form equivalent of rSYN2, thus arguing against a direct role in Lewy body formation and Parkinson's disease, these data nonetheless suggest that modifications of the carboxy-terminal region of -synuclein predispose it to inclusion formation.

Alternative Splicing↗
PubMed1996

Directing alternative splicing: cast and scenarios.

Recent progress in the study of alternative RNA splicing indicates that the interaction of RNA-binding proteins with specific target elements modulates splice site recognition and spliceosome assembly. The identity of splicing signals, the presence of modulating elements and differences in the distribution of RNA-binding proteins are key determinants involved in the tissue-specific regulation of splice site selection.

Alternative Splicing↗
PubMed2001

Novel alternatively spliced mRNA (1c) of the protein kinase A RIα subunit is implicated in haploid germ cell specific expression.

By using 5' RACE on rat testis cDNA we identified three alternatively spliced mRNAs of the RIalpha subunit of cAMP-dependent protein kinase that differed in their 5' untranslated regions. Two of these 5'-regions showed similarity with the human RIalpha exons 1a and 1b, while the third (1c) constituted a novel mRNA splice variant. Northern blot analysis showed that the 1c mRNA was specifically expressed in testis and only in postmeiotic germ cells. In contrast, the RIalpha 1b and RIalpha 1a mRNAs were present both in premeiotic germ cells and somatic cells of the testis, and the expression of both RIalpha 1a and 1b mRNAs were stimulated by cAMP in Sertoli cells. In sperm, the RIalpha protein was expressed after meiosis, and targeted to various subcellular structures via anchoring proteins. The RIalpha 1c haploid-specific mRNA, therefore, may be important for the regulation of RIalpha expression in sperm.

5' Untranslated Regions↗
PubMed2001

Spatial and temporal dynamics of two alternatively spliced regulatory factors, lens epithelium-derived growth factor (ledgf/p75) and p52, in the nucleus.

Regulatory factors, lens epithelium-derived growth factor (LEDGF)/p75 and p52, are generated from a single LEDGF gene by alternative splicing. They have identical amino acid residues between positions 1-325, but 205 and 8 of the remaining residues are different in LEDGF and p52, respectively. LEDGF promotes growth and survival of many cell types. It has an antiapoptotic function and is a weak general transcriptional co-activator. p52 is a transcriptional activator and an essential splicing factor. We investigated the spatial and temporal dynamics of LEDGF/p75 and p52, each being tagged with a fluorescent protein, during the cell cycles of CHO-K1, MCDK, and NRK cells in culture. Both LEDGF/p75 and p52 were localized predominantly in the nucleus. LEDGF/p75 was distributed diffusely in the nucleoplasm in the G1-phase and attached to chromatin heterogeneously during the G2 and M-phases of cells. In contrast, p52 was localized in the nuclear periphery during the G1-phase and formed a speckle pattern at the S-phase. It formed a cylindrical pattern around the chromosomes during the M-phases of cells. LEDGF and p52 on sister chromatids migrated into daughter cells. Thus, LEDGF/p75 and p52 are localized in distinct nuclear compartments where they can activate transcription or splicing of pre-mRNAs.

Alternative Splicing↗
PubMed1999

Reverse transcription-polymerase chain reaction products of alternatively spliced mRNAs form DNA heteroduplexes and heteroduplex complexes.

Reverse transcription-polymerase chain reaction (RT-PCR) is frequently used to simultaneously detect mRNA isoforms, which are generated by alternative splicing. Here we characterize two previously unrecognized RT-PCR products of vascular endothelial growth factor (VEGF) RNA. DNA products with apparent sizes of 600 and 1200 base pairs (bp) were detected at high cycle numbers. Heat denaturation of the smaller product and subsequent reannealing revealed that it was a heteroduplex consisting of two different DNA strands. These were identified by DNA sequencing as the amplification products of two VEGF transcripts, i.e. VEGF121 and VEGF165, which differ by the presence of one exon. S1 nuclease analysis showed that this exon is bulged out as a single-stranded loop. Purified heteroduplexes in solution were found to form a 1200-bp DNA product which could be reconverted into 600-bp DNA heteroduplexes by mild denaturation at 70 degreesC. These findings suggest that this product is formed by base pairing of complementary heteroduplex loops and represents a novel four-stranded DNA structure.

Alternative Splicing↗
PubMed1999

Changes in the pattern of fibronectin mRNA alternative splicing in acute experimental mesangioproliferative nephritis.

Fibronectins (FN) regulate cell migration, proliferation, and matrix formation during tissue injury. In humans, up to 20 different FN isoforms are generated by alternative splicing in three regions called EIIIA, EIIIB, and V, which have been implicated in the process undergoing wound healing and embryonic development. Specifically, EIIIA- and EIIIB-containing isoforms have been implicated in the regulation of cell proliferation and migration, whereas FN isoforms containing the full-length V region (named V120) are ligands to the VLA-4 integrin. To study the changes in the expression of FN isoforms in the anti-Thy-1 nephritis, an acute and self-resolutive model of mesangioproliferative nephritis, we analyzed the FN splicing patterns by means of ribonuclease protection assays. At Day 7 after anti-Thy-1 monoclonal injection, time of the maximal matrix expansion and glomerular hypercellularity, EIIIA+, EIIIB-, and V120 FN mRNA isoforms were increased. In accordance with the mRNA studies, FN proteins, including the EIIIA and V120 regions, increased in the mesangium of nephritic rats, as assayed by immunohistochemistry. Coinciding with the EIIIA and V120 isoforms up-regulation, there was an increase in mesangial cell proliferation and in the number of VLA-4+ infiltrating cells. At Day 14, in parallel with a remission of the above-mentioned changes, there was a decline in the mRNA and protein FN isoforms increased in the previous phase. The marked and reversible changes in the pattern of FN isoforms and their temporal association with other indicators of glomerular injury suggest that certain FN isotypes are important and coordinated components of the mechanisms attempting to reverse glomerular damage.

Acute Disease↗
PubMed2004

Altered expression pattern of alternatively spliced estrogen receptor beta transcripts in breast carcinoma.

Dysregulation of total estrogen receptor beta (ERbeta) expression has been implicated in breast tumorigenesis. The ERbeta gene yields five exon 8 alternatively spliced transcripts (ERbeta1-5), which encode proteins with different C-terminal amino acids. Individual expression analysis of these transcripts may provide new insights into estrogen signaling in breast cancer. We measured mRNA levels of total ERbeta and its five isoforms in normal tissues, breast carcinomas from post-menopausal patients, and breast cancer cell lines by means of real-time reverse transcription-polymerase chain reaction and fluorescent fragment analysis. In various normal human tissues, ERbeta1-5 isoforms displayed different qualitative and quantitative expression patterns that were consistent with previous reports. Total ERbeta mRNA levels were significantly lower in breast tumors than in normal breast tissues (38-fold lower, P < 0.001), mainly due to lower expression of ERbeta1 and ERbeta2 (ERbeta5 expression was similar in the two tissue types). This altered expression pattern of ERbeta isoforms in breast cancer should be taken into account in future ERbeta-based clinical applications.

Aged↗
PubMed2003

APAF-1-ALT, a novel alternative splicing form of APAF-1, potentially causes impeded ability of undergoing DNA damage-induced apoptosis in the LNCaP human prostate cancer cell line.

We have found a novel alternative splicing product of the apoptotic protease activating factor 1 (APAF-1), termed APAF-1-ALT, in the LNCaP human prostate cancer cell line. APAF-1-ALT harbors the caspase recruitment domain and an incomplete CED-4 like/ATPase domain, but lacks the WD-40 repeat units. The LNCaP cell expressed the full-length APAF-1 weakly and APAF-1-ALT rather abundantly, especially after mycoplasma infection. LNCaP underwent a retarded DNA damage-induced apoptosis, which was independent of caspase 9 activity. APAF-1-ALT functioned less effectively in inducing apoptosis than did APAF-1-XL, the full-length APAF-1, in transient transfection. Moreover, APAF-1-ALT interfered with APAF-1-XL's ability to induce apoptosis in transient double transfection experiment. These results indicate that APAF-1-ALT is a molecule that is deficient and impeded for mediating apoptosis and that it may contribute to the resistance to DNA damage-induced treatment observed in LNCaP.

Actins↗
PubMed1993

Preferential expression of alternatively spliced mRNAs encoding type II procollagen with a cysteine-rich amino-propeptide in differentiating cartilage and nonchondrogenic tissues during early mouse development.

Type II procollagen mRNAs are alternatively spliced: type IIA mRNA contains an exon encoding a cysteine-rich domain in the amino-propeptide and type IIB mRNA lacks this exon. In mouse embryos between 9.5 and 13.5 days, type IIA mRNA was the major form of Col2a-1 transcript expressed in both prechondrogenic and nonchondrogenic tissues and type IIB mRNAs were present in small amounts. After 12.5 days, type IIB mRNA levels increased rapidly and finally exceeded type IIA mRNAs. Type IIB mRNAs became the major Col2a-1 transcript by 14.5 days, predominantly expressed in maturing chondrocytes. By 17.5 days type IIB mRNAs account for 80% of the Col2a-1 transcripts. Expression of type IIA mRNAs follows the change in the growth pattern of the cartilaginous model of the axial and appendicular skeleton and of the otic capsule and nasal septum. In nonchondrogenic tissues, type IIA mRNAs are more commonly expressed in epithelial structures of ectodermal and endodermal origin than in nonepithelial tissues. The switching of expression from type IIA to type IIB mRNA as major Col2a-1 transcript may be associated with the commitment of precursor cells to the chondrocyte lineage and sites of type IIA mRNA expression may mark regions of potential cartilage growth. The differential expression pattern of type IIA mRNAs therefore points to an association of type IIA procollagen with chondrocyte differentiation during cartilage growth and some function early in embryogenesis in the epithelial organization of nonchondrogenic tissues.

Alternative Splicing↗
PubMed1993

Characterization of the cathepsin B gene and multiple mRNAs in human tissues: evidence for alternative splicing of cathepsin B pre-mRNA.

We have cloned and characterized multiple messages for cathepsin B that differ in their 5' and 3' untranslated regions (UTRs) from human kidney and the hepatoma cell line HepG2. A comparison of these messages with the cloned human cathepsin B gene reveals that they arise by alternative splicing of a single gene. Processing at a cryptic intron donor site in exon 11 and splicing to exon 12 produces a 4.0-kb message with an alternate 3' UTR in addition to the 2.3-kb message described previously by Chan et al. (1986). Variable removal of exon 2 produces cathepsin B mRNAs which differ by 88 nucleotides in their 5'-UTRs. The ratio of the 2.3-kb to 4.0-kb transcript is about 2:1 in most of the tissues examined, but the ratio of mRNAs with variant 5' UTRs differs widely. Cathepsin B mRNAs lacking exon 2 are predominant in human tumors. In addition, human breast and colon carcinomas and a human melanoma contain a cathepsin B transcript that is also missing exon 3 encoding the signal peptide and 7 residues of the activation propeptide. An in vitro transcription/translation assay was used to demonstrate that this message could be translated from an internal methionine codon (residue 52), producing a 32-kD product lacking the signal peptide and more than half the propeptide. The transcription/translation assay also demonstrated that the variant messages differ in their rates of translation. The relative rates are about 8:2:1 for mRNA lacking exons 2 and 3 compared to mRNA lacking exon 2 and mRNA containing the full-length 5' end, respectively. These results suggest that the expression of cathepsin B in human tissues may be regulated in part at the level of mRNA processing.

Alternative Splicing↗
PubMed2005

Sequence conservation, relative isoform frequencies, and nonsense-mediated decay in evolutionarily conserved alternative splicing.

Studies of expressed sequence tag data sets have revealed large numbers of splicing variants for human genes, but it remains challenging to distinguish functionally important variants from aberrant splicing, clarify the nature of the alternative functions, and understand the signals that regulate splicing choices. To help address these issues, we have constructed and analyzed a large data set of 1,478 exon-skipping alternative splicing (AS) variants evolutionarily conserved in human and mouse. In about one-fifth of cases, one isoform appears subject to nonsense-mediated mRNA decay (NMD), supporting the idea that a major role of AS is to regulate gene expression; one-quarter of these NMD-inducing cases involve a conserved exon whose apparent sole purpose is to mediate destruction of the message when included. We explore sequence conservation likely related to splicing regulation, using in part a measure of the overall amount of conserved information in a sequence, and find that the increased conservation that has been observed within AS exons primarily affects synonymous sites, suggesting that regulatory signals significantly constrain synonymous substitution rates. We show that a lower frequency of the inclusion isoform relative to the exclusion isoform tends to be associated with weaker splice site signals, smaller exon size, and higher intronic sequence conservation, and provide evidence that all of these factors are under selection to control relative isoform frequencies. Some conserved instances of AS appear to represent aberrant splicing events that by chance have occurred in both species, and we develop a nonparametric likelihood approach to identify these.

Alternative Splicing↗
PubMed2001

Cloning and characterization of an alternatively spliced form of SR protein kinase 1 that interacts specifically with scaffold attachment factor-B.

Serine/arginine protein kinases have been conserved throughout evolution and are thought to play important roles in the regulation of mRNA processing, nuclear import, germline development, polyamine transport, and ion homeostasis. Human SRPK1, which was first identified as a kinase specific for the SR family of splicing factors, is located on chromosome 6p21.2-p21.3. We report here the cloning and characterization of SRPK1a, which is encoded by an alternatively processed transcript derived from the SRPK1 gene. SRPK1a contains an insertion of 171 amino acids at its NH(2)-terminal domain and is similar to SRPK1 in substrate specificity and subcellular localization. Moreover, both isoforms can induce alternative splicing of human tau exon 10 in transfected cells. Using the yeast two-hybrid assay, we found that the extended NH(2)-terminal domain of SRPK1a interacts with Scaffold Attachment Factor-B, a nuclear scaffold-associated protein. Confirmation of this interaction was provided by in vitro binding assays, as well as by co-immunoprecipitation from 293T cells doubly transfected with SRPK1a and SAF-B. Our studies suggest that different SRPK family members are uniquely regulated and targeted and thus the multiple SRPK kinases present in higher eukaryotes may perform specialized and differentiable functions.

Alternative Splicing↗
PubMed2002

Identification and functional characterization of the novel isoforms of bovine norepinephrine transporter produced by alternative splicing.

Analyzing variation of bovine norepinephrine transporter (NET) at the 3'-region by RT-PCR in the adrenal glands and the brain revealed four isoforms of NET produced by alternative splicing of four cassettes (C0, C1, C2 and C3) encoded by exons 12-15, designated bNET1a (C0-C1-C2, formerly designated bNET1), bNET1b (C0-C2), bNET2a (C0-C1-C3) and bNET2b (C0-C3, formerly designated bNET2), respectively. Expression of these isoforms in COS-7 cells revealed that the isoforms that contain the C1 cassette encoded by exon 13 (bNET1a and bNET2a) showed a significant increase in [(3)H]norepinephrine uptake and [(3)H]nisoxetine binding, whereas the isoforms which lack the C1 cassette (bNET1b and bNET2b) failed to display those activities despite the selection of either exon 14 or exon 15. These results suggest that the region encoded by exon 13 is indispensable for NET functional expression.

Alternative Splicing↗
PubMed1997

Overlapping expression and redundant activation of mesenchymal fibroblast growth factor (FGF) receptors by alternatively spliced FGF-8 ligands.

FGF-8 is a member of the family of fibroblast growth factors and is expressed during vertebrate embryo development. Eight potential FGF-8 isoforms are generated by alternative splicing in mice, several of which are expressed during embryogenesis in epithelial locations. The significance of the multiple isoforms is currently unknown. In this report, we investigate the expression patterns and the specificity of the FGF-8 isoforms for known fibroblast growth factor (FGF) receptors. RNAs for seven of the eight potential isoforms are present at multiple sites of embryonic Fgf8 expression. None of the FGF-8 isoforms exhibited activity when assayed with BaF3 cells expressing the "b" splice forms of FGF receptors 1-3, which are mostly expressed in epithelial tissues. Mesenchymally expressed "c" splice forms of FGF receptors 2 and 3 and FGF receptor 4 were activated by several FGF-8 isoforms. These findings are consistent with the hypothesis that the multiple FGF-8 isoforms are functionally redundant and function to signal in paracrine (epithelial to mesenchymal) contexts.

Alternative Splicing↗
PubMed2000

Identification of an alternatively spliced seprase mRNA that encodes a novel intracellular isoform.

Seprase is a homodimeric 170-kDa integral membrane gelatinase that is related to the ectoenzyme dipeptidyl peptidase IV. We have identified an alternatively spliced seprase messenger from the human melanoma cell line LOX that encodes a novel truncated isoform, seprase-s. The splice variant mRNA is generated by an out-of-frame deletion of a 1223-base pair exonic region that encodes part of the cytoplasmic tail, transmembrane, and the membrane proximal-central regions of the extracellular domain (Val(5) through Ser(412)) of the seprase 97-kDa subunit (seprase-l). The seprase-s mRNA has an elongated 5' leader (548 nucleotides) that harbors at least two upstream open reading frames that inhibit seprase-s expression from a downstream major open reading frame. Deletion mutagenesis of the wild type splice variant cDNA confirms that initiation of the seprase-s coding sequence begins with an ATG codon that corresponds to Met(522) of seprase-l. The seprase-s open reading frame encodes a 239-amino acid polypeptide with an M(r) approximately 27,000 that precisely overlaps the carboxyl-terminal catalytic region of seprase-l.

Alternative Splicing↗