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PubMed1997

Alternative splicing and structure of the human and mouse SFRS5/HRS/SRp40 genes.

HRS/SRp40/SFRS5 (HRS) is an SR (serine-arginine-rich) protein which regulates both alternative splicing and basal splicing. HRS mRNA contains several transcripts, including HRS-SF and HRS-LF which have different temporal patterns of expression in proliferating liver. As previously reported, HRS-SF mRNA encodes the SR splicing factor. However, the identity of HRS-LF remained unknown. Here, we cloned and characterized the mouse HRS gene, partial human HRS gene, and several cDNAs derived from HRS-LF mRNA. The mouse HRS gene spans 5050 bp and contains eight exons and seven introns. HRS-LF mRNA contains a 1.2 kb insert within the SF mRNA with stop codons in all three reading frames. A comparison of HRS-LF and the HRS gene revealed that HRS-LF mRNA is an intron-retaining product which contains intron 5. At most, HRS-LF encodes a truncated HRS protein with one RNA binding domain. Interestingly, intron 5 demonstrates 90% identity between the mouse and human HRS genes, implying that intron 5 might play an important role in regulating HRS gene splicing or expression.

Alternative Splicing↗

PubMed2006

Characterization of porcine TAP genes: alternative splicing of TAP1.

The transporter associated with antigen processing (TAP) is a heterodimer composed of TAP1 and TAP2 subunits that belong to the ATP-binding cassette family of transporters. TAP translocates small peptides (usually 8- to 12-amino-acid-long) from the cytosol to the endoplasmic reticulum for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. The translocated peptides are required for the stable cell surface expression of MHC class I molecules. Virus-encoded proteins, which inhibit TAP activity, include ICP47 from herpes simplex virus and US6 from human cytomegalovirus. We have previously shown that ICP47 downregulated porcine MHC class I [swine leukocyte Ag class I (SLA I)] cell-surface expression in the pig epithelial cell line PK(15). Here we show that SLA I cell-surface expression in the pig epithelial cell line LLC-PK1 is relatively unaffected by expression of ICP47. Anticipating that this might be due to differences in the primary structure of TAP1 or TAP2 expressed by these two cell lines, cDNAs from PK(15) and LLC-PK1 encoding the complete open reading frames of porcine TAP1 and TAP2 were cloned and sequenced. Porcine TAP1 and TAP2 exhibited 80% amino acid identity with their human orthologs. Two splice variants of TAP1 were found. In LLC-PK1 cells, an alternatively spliced TAP1 transcript was detected, which was predicted to encode a protein with nine fewer amino acids. While the deleted amino acids may be in close proximity to the putative peptide/ICP47-binding site, we were unable to demonstrate that this imparted an apparent resistance to the effects of ICP47 on SLA I surface expression.

ATP-Binding Cassette Transporters↗

PubMed2000

Alternative splicing of the human PTEN/MMAC1/TEP1 gene.

The human tumour suppressor gene PTEN/MMAC1/TEP1 encodes a lipid and protein phosphatase. Using RT-PCR, alternatively spliced forms of PTEN mRNA, encoding full-length PTEN and two forms of the protein truncated at the C-terminal end, were detected in normal human tissue. Cultured tumour and non-tumour cell lines show similar splicing patterns.

Alternative Splicing↗

PubMed1991

Analysis of alternatively spliced human immunodeficiency virus type-1 mRNA species, one of which encodes a novel tat-env fusion protein.

A polymerase chain reaction-based analysis was used to define the structures of the mRNAs that encode human immunodeficiency virus type-1 (HIV-1) regulatory and structural proteins in infected H9 cells. Twenty alternatively spliced mRNAs encoding the vif, vpr, env, nef, tat, and rev proteins were characterized. An evaluation of the coding potentials of these transcripts recognized both leaky scanning and reinitiation at downstream initiation codons as mechanisms that may operate during translation of many of the polycistronic messages. Two new splice acceptor sites, one at nt 6018 defining a new mRNA coding for the env and vpu proteins and another at nt 8671 defining a novel tat-env fusion transcript, were characterized. The latter transcript expressed a novel protein p17tev that was immunoprecipitated by both polyclonal tat antibodies and monoclonals directed towards the C-terminal region of gp41. The p17tev protein was able to transactivate transcription from the HIV-1 LTR in transient transfection assays. The use of multiple alternative splice donor and acceptor sites and the generation of novel proteins may confer evolutionary advantages on the viral mutants encoding them and influence the course of clinical disease.

Animals↗

PubMed1998

The use of antibodies to the polypyrimidine tract binding protein (PTB) to analyze the protein components that assemble on alternatively spliced pre-mRNAs that use distant branch points.

We are using the rat beta-tropomyosin (beta-TM) gene as a model system to study the mechanism of alternative splicing. Previous studies demonstrated that the use of the muscle-specific exon is associated with the use of distant branch points located 147-153 nt upstream of the 3' splice site. In addition, at least one protein, the polypyrimidine tract binding protein (PTB), specifically interacts with critical cis-acting sequences upstream of exon 7 that are involved in blocking the use of this alternative exon in nonmuscle cells. In order to further study the role of PTB, monoclonal antibodies to PTB were prepared. Anti-PTB antibodies did not inhibit the binding of PTB to RNA because they were able to supershift RNA-PTB complexes. To determine if additional proteins interact with sequences within the pre-mRNA, 35S-met-labeled nuclear extracts from HeLa cells were mixed with RNAs and the RNA-protein complexes were recovered by immunoprecipitation using antibodies to PTB. When RNAs containing intron 6 were added to an 35S-met-labeled nuclear extract, precipitation with PTB antibodies showed a novel set of proteins. By contrast, addition of RNAs containing introns 5 or 7 gave the same results as no RNA, indicating that these RNAs are unable to form stable complexes with PTB. These results are in agreement with our previous studies demonstrating that PTB interacts with sequences within intron 6, but not with sequences within introns 5 and 7. When 35S-met-labeled HeLa nuclear extracts were mixed with biotinylated RNA containing intron 6 and the RNA-protein complexes were recovered using streptavidin-agarose beads, an identical pattern of proteins was observed when compared with the immunoprecipitation assay. Analysis of the proteins that assembled on introns 5, 6, or 7 using biotinylated RNA revealed a unique set of proteins that interact with each of these sequences. The composition of proteins interacting with sequences associated with the use of the 3' splice site of intron 6 included proteins of 30, 40, 55, 60, 65, 70, 80, and 100 kDa. Microsequencing identified two of the proteins to be Sam68 and the Far Upstream Element Binding Protein (FBP) from the c-myc gene. In addition, a comparison of the proteins that assemble on introns from the alpha- and beta-TM genes that utilize distant branch points revealed common as well as unique proteins that assemble on these introns. These studies identify a set of proteins, in addition to PTB, that are likely involved in the use of distant branch sites associated with the use of alternatively spliced introns.

Alternative Splicing↗

PubMed1994

The mouse Rxrb gene encoding RXR beta: genomic organization and two mRNA isoforms generated by alternative splicing of transcripts initiated from CpG island promoters.

Two major isoforms of retinoid X receptor beta (RXR beta; H-2RIIBP), encoded by the Rxrb gene, have been identified in the mouse. Northern analysis of Rxrb mRNA showed two close bands of 2.8 and 2.6 kb in many tissues and cell lines. They are designated as mRxr beta 1 and mRxr beta 2, respectively. Some rapidly growing cell lines and spleen tissue had about twofold more Rxr beta 1 mRNA than Rxr beta 2 whereas most adult tissues had similar amounts of both beta 1 and beta 2. Amino acid (aa) sequences deduced from cDNAs show an extra N-terminal domain of 72 aa for RXR beta 1 that is well conserved between mouse and human, but not found in RXR beta 2. These isoforms are generated from separate exons transcribed from different CpG island promoters and spliced into the common acceptor site in the transactivation domain by an alternative splicing. The Rxrb gene contains an intron in the midst of the first zinc-finger coding region. This is different from the retinoic acid receptor (RAR) and other nuclear receptor superfamily genes that contain an intron between the first and the second zinc-finger coding regions. These results, together with their unique ability to form heterodimers with other members of the superfamily, suggest a distinct phylogenic position for the Rxr genes.

Alternative Splicing↗

PubMed1990

Differentiation of mouse erythroleukemia cells enhanced by alternatively spliced c-myb mRNA.

C-myb, the normal cellular homolog of the retroviral transforming gene v-myb, encodes a nuclear, transcriptional regulatory protein (p75c-myb). C-myb is involved in regulating normal human hematopoiesis, and inhibits dimethyl sulfoxide-induced differentiation of Friend murine erythroleukemia (F-MEL) cells. An alternately spliced c-myb mRNA encodes a truncated version of p75c-myb (mbm2) that includes the DNA binding region and nuclear localization signal present in the c-myb protein, but does not contain the transcriptional regulatory regions. Constitutive expression of mbm2, in contrast to c-myb, here resulted in enhanced differentiation of F-MEL cells. These data suggest that the c-myb protooncogene encodes alternately spliced mRNA species with opposing effects on differentiation.

Animals↗

PubMed2002

The Xenopus laevis beta TrCP gene: genomic organization, alternative splicing, 5' and 3' region characterization and comparison of its structure with that of human beta TrCP genes.

beta TrCP plays a relevant role in the control of stability of several key protein factors. In Xenopus, beta TrCP acts as an inhibitor of Wnt signaling and dorsal axis formation. We determined the primary structure of the frog beta TrCP gene, which consists of 14 exons and 13 introns, spanning over 34 kb. Isoforms of x-beta TrCP have been found, which show differences in the NH(2) and COOH regions. NH(2) isoforms differ for the presence or absence of a 30 aa sequence, coded by exon III. In COOH isoforms, 19 C-terminal amino acids are replaced by three different amino acids. Occurrence of two 5' splice donor sites for splicing of intron XIII provides an explanation for these isoforms, based on alternative splicing. The DNA region of the putative beta TrCP promoter contains several TATA elements, one GCCAAT box, and putative binding sites for Ets, Tcf/Lef and NF-kappa B transcription factors. Two transcription initiation sites have been mapped downstream of TATA boxes proximal to ATG for start of translation. Comparison of the Xenopus and human beta TrCP genes indicates high conservation of exon nucleotide and amino acid sequences, size and organization; differences are limited to exons coding for N- and C-terminal regions.

3' Flanking Region↗

PubMed1989

Structure of the elastin gene and alternative splicing of elastin mRNA: implications for human disease.

The protein elastin is largely responsible for the elastic properties of vertebrate lungs, large blood vessels, and skin. The structure of the human, bovine, and chick elastin gene and protein monomer, tropoelastin, has recently been elucidated by using techniques of molecular biology. Extensive homology of amino acid sequence exists among the mammalian species and there is in addition strong conservation of nucleotide sequences in the 3' untranslated region of the gene. The translated exons are small and embedded in large expanses of introns. Sequences coding for the hydrophobic regions, responsible for the elastic properties of the molecule, and the alanine-lysine rich regions, responsible for crosslink formation between molecules, reside in separate exons and alternate for the most part in the elastin gene. S1 analyses and sequence analysis of cDNA and genomic clones have indicated that there is substantial alternative splicing of the primary elastin transcript. Variations in the structure of mRNAs resulting from alternative splicing could explain the existence of the multiple forms of tropoelastin observed electrophoretically in several species. Different kinds of splicing patterns could occur in human populations and may contribute to aging and pathological situations in the cardiovascular and pulmonary systems.

Aging↗

PubMed2004

Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts.

There are three types of mouse Mutyh mRNAs (type a, b and c) generated by alternative splicing, and type b mRNA is a major form among the three in most of the tissues examined. The level of type c mRNA is relatively high in brain. Type a and b mRNAs were expected to encode 57.7 kDa protein (MUTYHalpha), while type c mRNA had a partly different open reading frame encoding a 50.2 kDa protein (MUTYHbeta). An in vitro translation of type b and c mRNAs produced a 50 kDa MUTYHalpha and 47 kDa MUTYHbeta, respectively. MUTYHalpha and MUTYHbeta were detected in wild-type embryonic stem (ES) cells or thymocytes prepared from wild-type mice, but neither MUTYH-null ES cells nor thymocytes prepared from MUTYH-null mice. Both MUTYHalpha and MUTYHbeta were mainly localized in the nuclei and some in mitochondria in wild-type ES cells. Recombinant MUTYHalpha and beta were expressed as fusion proteins with thioredoxin in Escherichia coli, but only MUTYHalpha was partly soluble and thus could be purified. Recombinant MUTYHalpha possessed DNA glycosylase activities to excise adenine opposite 8-oxoguanine and guanine but not AP lyase activity.

Adenine↗

PubMed1997

Recognition of follicle stimulating hormone (alpha-subunit) by a recombinant receptor protein domain coded by an alternately spliced mRNA and expressed in Escherichia coli.

To assess the functional significance of putative proteins encoded by alternately spliced mRNA of the sheep testicular FSH receptor, a short form cDNA comprising of the first four exons (117 residues mature protein) was engineered for expression in Escherichia coli. The expressed protein of molecular mass 15 kDa was purified to homogeneity and verified by reaction with an antibody against a synthetic peptide sequence unique to the amino (N)-terminal region FSH receptor. The purified FSH receptor domain protein bound 125I-labeled hFSH in a ligand blot on polyvinylidine difluoride membranes. Further analyses by slot blot revealed high affinity of the immobilized protein with significant reaction at 10 pmol. As the immobilized receptor protein also reacted with structurally related hormones (125I-labeled LH/125I-labeled human chorionic gonadotropin), we confirmed that interaction most probably occurred via the common alpha-subunit of these glycoprotein hormones. Our results reveal that this N-terminal portion of the FSH receptor contain(s) major site(s) for hormone recognition that could be mediated via the alpha-subunit. A rabbit antibody to the receptor inhibited FSH action in receptor bearing cells, revealing the utility of such recombinant FSH receptor protein(s) for modulation of hormone action.

Alternative Splicing↗

PubMed1994

Functional analysis of an alternatively spliced estrogen receptor lacking exon 4 isolated from MCF-7 breast cancer cells and meningioma tissue.

An alternatively spliced mRNA coding for a variant estrogen receptor (ER) missing exon 4 (ERdelta4) was detected in the breast tumor cell line MCF7 and meningioma tissue by using the reversed transcriptase PCR technique. The trans-activational properties of this mutant ER were assessed in embryo carcinoma P19EC and human choriocarcinoma JEG3 cells by co-transfection of the ERdelta4 expression vector with an oxytocin promoter construct containing an estrogen-responsive element. ERdelta4 did not trans-activate the oxytocin promoter in either a hormone-dependent or -independent manner. Co-transfection of ERdelta4 together with the wtER did not show any interference of ERdelta4 on the stimulation of the oxytocin promoter by the wtER. ERdelta4 was translated in vitro. Its capacity to bind estradiol, and the binding of the variant to a synthetic estrogen-responsive element were compared to those of the wild-type receptor. ERdelta4 did not bind to a synthetic estrogen-responsive element, nor did it bind estradiol. Hence, ERdelta4 appears to be a silent variant and we speculate that it is without any role in tumor progression.

Alternative Splicing↗

PubMed1996

Species-specific alternative splicing of the epidermal growth factor-like domain 1 of cartilage aggrecan.

Aggrecan transcripts contain two alternatively spliced exons that code for two epidermal growth factor-like domains (EGF1 and EGF2). Whereas the EGF2 sequence is expressed at a uniform level among different species, the EGF1 sequence has been detected only in human aggrecan transcripts. In this study we have used the nested primer reverse transcription-PCR (RT-PCR) method to compare the expression of the EGF1 exon in human, bovine and dog aggrecan transcripts. Our results indicate that this exon is expressed in a species-specific manner. In addition to its significant expression level in human transcripts, the EGF1 sequence can be detected in a small portion of bovine aggrecan transcripts as shown with nested primer RT-PCR. In contrast, the same module is not detectable in dog aggrecan transcripts, although an EGF1 sequence is present in the dog aggrecan gene. The expression level of the EGF1 exon in the aggrecan transcripts correlates with the strength of the polypyrimidine tract upstream of the exon. The EGF1 sequence also shows much less conservation between the species than the EGF2 sequence. The species-specific expression and high sequence variation of the EGF1 exon imply that this sequence is likely to code for an aggrecan domain having no cartilage-specific function.

Aggrecans↗

PubMed2003

Regulation of gene expression in response to brain injury: enhanced expression and alternative splicing of rat prosaposin (SGP-1) mRNA in injured brain.

Prosaposin, the precursor of saposins or saps, is an injury-repair protein that acts on both neurons and glia. Previous studies identified the prosaposin gene as one of differentially expressed genes following nerve injury. In the present study, we investigated expression of prosaposin mRNA in injured brain utilizing rat models of focal cerebral ischemia and cortical stab wound in order to explore the significance of prosaposin in nerve injury. In ischemic brain, the level of prosaposin mRNA was elevated greater than 400% over controls within 5 days after ischemic insults. Importantly, this induction was accompanied by a 9-base splicing consistent with the alternative Exon-8 splicing of human prosaposin mRNA. In normal brain, two prosaposin mRNA species with and without the 9-base insertion were expressed at a ratio of 85:15; however, this equilibrium reverted to 5:95 following ischemic injury. Similar inductions were observed in stab wound brains. Immunohistochemical staining and in situ hybridization demonstrated an enhanced signal distribution of prosaposin mRNA and injury-induced prosaposin protein around the lesion. The data suggest the expression and processing of prosaposin mRNA may be crucially regulated not only for cerebral homeostasis but also during nerve regenerative and degenerative processes.

Alternative Splicing↗

PubMed2006

Identification and evolutionary analysis of novel exons and alternative splicing events using cross-species EST-to-genome comparisons in human, mouse and rat.

BACKGROUND: Alternative splicing (AS) is important for evolution and major biological functions in complex organisms. However, the extent of AS in mammals other than human and mouse is largely unknown, making it difficult to study AS evolution in mammals and its biomedical implications. RESULTS: Here we describe a cross-species EST-to-genome comparison algorithm (ENACE) that can identify novel exons for EST-scanty species and distinguish conserved and lineage-specific exons. The identified exons represent not only novel exons but also evolutionarily meaningful AS events that are not previously annotated. A genome-wide AS analysis in human, mouse and rat using ENACE reveals a total of 758 novel cassette-on exons and 167 novel retained introns that have no EST evidence from the same species. RT-PCR-sequencing experiments validated approximately 50 approximately 80% of the tested exons, indicating high presence of exons predicted by ENACE. ENACE is particularly powerful when applied to closely related species. In addition, our analysis shows that the ENACE-identified AS exons tend not to pass the nonsynonymous-to-synonymous substitution ratio test and not to contain protein domain, implying that such exons may be under positive selection or relaxed negative selection. These AS exons may contribute to considerable inter-species functional divergence. Our analysis further indicates that a large number of exons may have been gained or lost during mammalian evolution. Moreover, a functional analysis shows that inter-species divergence of AS events may be substantial in protein carriers and receptor proteins in mammals. These exons may be of interest to studies of AS evolution. The ENACE programs and sequences of the ENACE-identified AS events are available for download. CONCLUSION: ENACE can identify potential novel cassette exons and retained introns between closely related species using a comparative approach. It can also provide information regarding lineage- or species-specificity in transcript isoforms, which are important for evolutionary and functional studies.

Algorithms↗

PubMed2004

Revealing global regulatory features of mammalian alternative splicing using a quantitative microarray platform.

We describe the application of a microarray platform, which combines information from exon body and splice-junction probes, to perform a quantitative analysis of tissue-specific alternative splicing (AS) for thousands of exons in mammalian cells. Through this system, we have analyzed global features of AS in major mouse tissues. The results provide numerous inferences for the functions of tissue-specific AS, insights into how the evolutionary history of exons can impact on their inclusion levels, and also information on how global regulatory properties of AS define tissue type. Like global transcription profiles, global AS profiles reflect tissue identity. Interestingly, we find that transcription and AS act independently on different sets of genes in order to define tissue-specific expression profiles. These results demonstrate the utility of our quantitative microarray platform and data for revealing important global regulatory features of AS.

Alternative Splicing↗

PubMed1998

CD44 isoform expression follows two alternative splicing pathways in breast tissue.

The repertoire of distinct CD44 protein isoforms is generated by means of alternative pre-mRNA splicing of 10 variable exons located in the central region of the CD44 gene. We have used human breast ductal carcinoma as a model to identify two alternative splicing pathways of the CD44 pre-mRNA variable region that account for the generation of all of the CD44 isoforms described in breast tissue. An alternative splicing pathway that reflects inclusion of variable exons in a gradual 3'-to-5' fashion is evidenced in breast ductal carcinoma and its lymph node metastases. This pathway is compatible with a mechanism that generates the standard form of CD44 (devoid of variable exons) and is distinguishable from an alternative splicing pathway that involves exclusively variant exon 3 and is observable in both normal and carcinoma breast tissue. We show that both pathways are detectable in the same cell type in the breast and provide a speculative model by which these splicing routes could take place.

Alternative Splicing↗

PubMed2002

Profiling alternative splicing on fiber-optic arrays.

The human transcriptome is marked by extensive alternative mRNA splicing and the expression of many closely related genes, which may be difficult to distinguish using standard microarray techniques. Here we describe a sensitive and specific assay for parallel analysis of mRNA isoforms on a fiber-optic microarray platform. The method permits analysis of mRNA transcripts without prior RNA purification or cDNA synthesis. Using an endogenously expressed viral transcript as a model, we demonstrated that the assay readily detects mRNA isoforms from as little as 10-100 pg of total cellular RNA or directly from a few cells. Multiplexed analysis of human cancer cell lines revealed differences in mRNA splicing and suggested a potential autocrine mechanism in the development of choriocarcinomas. Our approach may be useful in the large-scale analysis of the role of alternative splicing in development and disease.

Alternative Splicing↗