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PubMed1991

Relationship between alternatively spliced exons and functional domains in tropomyosin.

Smooth and striated muscle alpha-tropomyosins differ as a consequence of alternative splicing of exons 2 and 9 encoding amino acid residues 39-80 and 258-284, respectively [Ruiz-Opazo, N., Weinberger, J. & Nadal-Ginard, B. (1985) Nature (London) 315, 67-70]. To understand the relationship between alternatively spliced exons and functional domains in tropomyosin, recombinant unacetylated striated muscle, smooth muscle, and chimeric rat alpha-tropomyosins (+H3N-tropomyosins) expressed in and purified from Escherichia coli were analyzed. The functional differences between the isoforms can be primarily ascribed to exon 9. +H3N-Tropomyosins with the smooth muscle exon 9 bound to skeletal muscle filamentous actin with at least a 5-fold higher affinity than +H3N-tropomyosins with the striated muscle exon 9. On the other hand, in the presence of Ca2+, troponin increased the affinity of +H3N-tropomyosins with the striated muscle exon 9 at least 50-fold, whereas it had little effect on +H3N-tropomyosins with the smooth muscle exon 9. The unique striated muscle alpha-tropomyosin exon 9 seems to be specialized for Ca(2+)-insensitive interaction with troponin on the thin filament. The unique smooth muscle alpha-tropomyosin exon 2 was associated with a slightly lower actin affinity than the striated muscle exon 2. Although the regions encoded by exons 2 and 9 correspond to functional domains, they are not recognizable as independent units or structural domains in the extended coiled-coil structure of this fibrous actin binding protein.

Actins↗

PubMed2005

Induction of apoptosis by the overexpression of an alternative splicing variant of mitochondrial thioredoxin reductase.

Mammalian cells harbor two forms of thioredoxin reductase (TrxR), cytosolic TrxR1 and mitochondrial TrxR2, both of which are involved in the redox regulation of cell growth and apoptosis. Furthermore, several alternative splicing variants of TrxR1 and TrxR2 have been identified. However, little remains known with regard to their functions in cells. Here, we report an alternative splicing variant of TrxR2 (TrxR2A), which displays a 3-bp deletion in the coding region and an insertion of 1228 bp in the 3'-UTR, between the stop codon and the SECIS element, of the TrxR2 cDNA. In order to determine the cellular function of TrxR2A, we established TrxR2A-inducible HeLa cell lines in which TrxR2A transcription was regulated via a Tet-off expression system. We observed that the induction of TrxR2A resulted in increased apoptosis, due to the reduction of NADPH and alterations in cellular ROS levels. These results suggest that TrxR2A may play a vital role in the regulation of TrxR2 and may confer functional complexity onto the thioredoxin system.

Alternative Splicing↗

PubMed2005

Alternative splicing generates a smaller assortment of CaV2.1 transcripts in cerebellar Purkinje cells than in the cerebellum.

P/Q-type calcium channels control many calcium-driven functions in the brain. The CACNA1A gene encoding the pore-forming CaV2.1 (alpha1A) subunit of P/Q-type channels undergoes alternative splicing at multiple loci. This results in channel variants with different phenotypes. However, the combinatorial patterns of alternative splice events at two or more loci, and hence the diversity of CaV2.1 transcripts, are incompletely defined for specific brain regions and types of brain neurons. Using RT-PCR and splice variant-specific primers, we have identified multiple CaV2.1 transcript variants defined by different pairs of splice events in the cerebellum of adult rat. We have uncovered new splice variations between exons 28 and 34 (some of which predict a premature stop codon) and a new variation in exon 47 (which predicts a novel extended COOH-terminus). Single cell RT-PCR reveals that each individual cerebellar Purkinje neuron also expresses multiple alternative CaV2.1 transcripts, but the assortment is smaller than in the cerebellum. Two of these variants encode different extended COOH-termini which are not the same as those previously reported in Purkinje cells of the mouse. Our patch-clamp recordings show that calcium channel currents in the soma and dendrites of Purkinje cells are largely inhibited by a concentration of omega-agatoxin IVA selective for P-type over Q-type channels, suggesting that the different transcripts may form phenotypic variants of P-type calcium channels in Purkinje cells. These results expand the known diversity of CaV2.1 transcripts in cerebellar Purkinje cells, and propose the selective expression of distinct assortments of CaV2.1 transcripts in different brain neurons and species.

Alternative Splicing↗

PubMedDate not supplied

Alternative splicing of murine T-cell receptor beta-chain transcripts.

Variable processing of heteronuclear RNA into multiple, defined species of messenger RNA is a well-established phenomenon in a number of systems, including myelin basic protein, calcitonin, troponin T4 and immunoglobulins. Within a single B cell, immunoglobulin heavy-chain peptides often exist as two related but distinct species, a membrane-bound form and a secreted form, both of which originate from the same germline constant(C)-region gene via alternative RNA processing pathways. Furthermore, at certain stages of B-cell development, a single variable(V)-region element can be concurrently expressed in immunoglobulins of both the IgM and IgD isotypes, presumably resulting from the alternative splicing of one variable-region exon to different constant-region genes. We report here the existence of an alternative RNA splicing pathway available to murine T-cell receptor (TCR) beta-chain transcripts. Sequence analysis of beta-chain complementary DNA clones reveals a C beta 1 species containing a 72-base pair (bp) insertion between the joining (J beta) and C beta elements. This sequence is inserted via an alternative splicing pathway available to C beta 1 transcripts. The optional exon is located between the J beta 1 cluster and the first exon of C beta 1. Interestingly, this element can be spliced to C beta 2 in the New Zealand White mouse, in which the C beta 1 gene is deleted. Use of the alternative splicing pathway varies between 1% and 18% of total C beta clones, depending on the source of isolation.

Animals↗

PubMed1998

LAR tyrosine phosphatase receptor: proximal membrane alternative splicing is coordinated with regional expression and intraneuronal localization.

Examination of null-mutant Drosophila and Leukocyte Common Antigen-Related (LAR)-deficient transgenic mice has demonstrated that the LAR protein tyrosine phosphatase (PTP) receptor promotes neurite outgrowth. In the absence of known ligands, the mechanisms by which LAR-type PTP receptors are regulated are unknown. We hypothesized that an alternatively spliced eleven amino acid proximal membrane segment of LAR (LAR alternatively spliced element-a; LASE-a) contributes to regulation of LAR function. Human, rat and mouse LAR cDNA sequences demonstrated that the predicted eleven amino acid inserts in rat and mouse are identical and share nine of eleven residues with the human insert. LASE-a splicing led to the introduction of a Ser residue into LAR at a position analogous to Ser residues undergoing regulated phosphorylation in other PTPs. In-situ studies revealed increasingly region-specific expression of LASE-a containing LAR transcripts during postnatal development. RT-PCR analysis of cortical and hippocampal tissue confirmed that the proportion of LAR transcripts containing LASE-a decreases during development. Immunostaining of cultured PC12 cells, cerebellar granule neurons, dorsal root ganglia and sciatic nerve sections with antibody directed against the LASE-a insert demonstrated signal in cell bodies but little if any along neurites. In contrast, staining with antibody directed to a separate domain of LAR showed accumulation of LAR along neurites. The findings that LASE-a splicing is conserved across human, rat and mouse, that the LASE-a insert introduces a Ser at a site likely to be targeted for regulated phosphorylation and that developmentally regulated splicing is coordinated with specific regional and intraneuronal localization point to important novel potential mechanisms regulating LAR-type tyrosine phosphatase receptor function in the nervous system.

Alternative Splicing↗

PubMed2002

Alternative splicing of the GABA(A) receptor alpha 4 subunit creates a severely truncated mRNA.

GABA(A) receptors, important sites of drug action, are chloride channels composed of 5 subunits chosen from among 19 or more. Alternative splicing for alpha 5, alpha 6, and rho 1 subunits results in truncated proteins which appear to lack function. We report a similar, relatively common (about 20%) form of alternative splicing of the alpha 4 subunit mRNA in mice and humans which, remarkably, creates a severely truncated message containing only the first two and last coding exons, with a frameshift in between. The only apparent translation product includes a short piece (39 amino acids) of the N-terminus right after the signal peptide. The splicing was developmentally and regionally regulated; the highest proportions of truncated alpha 4 mRNA, about 40%, were observed in embryonic day 18 whole brain and adult cerebellum. The truncated mRNA, when coexpresssed in human embryonic kidney (HEK) 293 cells with the complete alpha 4 subunit and beta1 and gamma 2 S subunits, reduced observed GABA currents without kinetic alterations. No such effect of truncated alpha 4 was observed with alpha1 subunit-containing receptors. Thus, the truncated alpha 4 N-terminus may play a post-translational regulatory role in intracellular folding/glycosylation/assembly of the alpha 4 subunit.

Alternative Splicing↗

PubMed2004

Muscleblind proteins regulate alternative splicing.

Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF- and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets.

Alternative Splicing↗

PubMed2001

Alternative splicing of amino-terminal Tau mRNA in rat spinal cord during development and following axonal injury.

Tau is a family of microtubule-associated phosphoproteins in which isoform variation is produced by alternative splicing of a single gene and posttranslational modifications. Tau isoforms that include exon 10 are overexpressed in frontotemporal dementia and progressive supranuclear palsy. Therefore, we examined the expression of tau mRNA splice variants during axonal regeneration and abortive regeneration. Previous work in our laboratory demonstrated that expression of exon 10 tau isoforms during regeneration and abortive regeneration was altered and partially recapitulated the developmental patterns of tau isoform expression. Using RT-PCR, we examined the alternative splicing of exons 2 and 3 in tau during early postnatal development and regeneration in the rat spinal cord. The levels of tau lacking exons 2 and 3 were high on the day of birth and rapidly declined. Conversely, tau isoforms containing exon 2 or exons 2 and 3 first appeared at low levels and steadily increased. During axonal regeneration, the levels of all three tau mRNA isoforms were significantly lower 7 days after injury. In a model of abortive regeneration, all of the tau isoforms were elevated 14 and 42 days postinjury. The relative levels of exon 2 and 3 tau splice variants were not altered during regeneration or abortive regeneration as occurred during development. These results suggest that tau isoform expression following neuronal injury does not recapitulate the developmental pattern and is not independently regulated as in development. Our previous results together with these data suggest that alterations in tau mRNA isoform expression that occur in neurodegeneration are not secondary to axonal injury but may be a more primary event underlying cytoskeletal derangement.

Aging↗

PubMed1993

Expression of alternatively spliced epidermal growth factor-like domains in aggrecans of different species. Evidence for a novel module.

The carboxyl-terminal globular domain of human aggrecan has been shown previously to contain an alternatively spliced epidermal growth factor (EGF)-like module. We have used reverse transcription/polymerase chain reactions on cartilage-derived RNAs to investigate the heterogeneity in the EGF-like domain content of aggrecans from five species (mouse, rat, dog, bovine, and human). A novel alternatively spliced EGF-like module was detected in human aggrecan, establishing the presence of two of these domains. Highly homologous domains are present in aggrecans of other species, and the expression of these modules is identical (4-8%). They share significant homology with EGF-like domains of differentiation proteins and coagulation factors and have a putative calcium binding site. In contrast to this novel domain (EGF2), the previously described EGF-like module (EGF1) is expressed at a high level exclusively in human aggrecan. The expression of the two EGF-like domains in human aggrecan appears to be independent. Although the function of these domains is not understood, the uniform expression of the EGF2 domain may indicate a general role of this aggrecan module, while the expression of the EGF1 domain may reflect species specificity.

Aggrecans↗

PubMed2001

Identification of an alternatively spliced variant of human CD86 mRNA.

CD86 is a costimulatory molecule constitutively expressed by human antigen presenting cells which interacts with CD28 and CTLA-4 expressed by T cells. We have recently reported the identification of an alternatively spliced CD86 mRNA variant (CD86deltaTM) characterized by the deletion of exon 6 which encodes for the transmembrane domain. We report here the identification of an alternatively spliced variant (called CD86deltaEC) expressed by nonstimulated human monocytes and characterized by the deletion of exons 4 and 5 which encode for the extracellular V-like and C-like domains, respectively. The activation of monocytes by IFNgamma (i) induces the preferential expression of the transcript encoding for the membrane form and (ii) increases the expression of CD86 and of the accessory molecules CD40, CD49d and CD54. These results suggest that resting human monocytes may constitutively express different forms of CD86 which can then influence the activation of T cells.

Alternative Splicing↗

PubMed2005

Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry.

The human proteome is a highly complex extension of the genome wherein a single gene often produces distinct protein forms due to alternative splicing, RNA editing, polymorphisms, and posttranslational modifications. Such biological variation compounded by the high sequence identity within gene families currently overwhelms the complete and routine characterization of mammalian proteins by MS. A new data base of human proteins (and their possible variants) was created and searched using tandem mass spectrometric data from intact proteins. This first application of top down MS/MS to wild-type human proteins demonstrates both gene-specific identification and the unambiguous characterization of multifaceted mass shifts (Deltam values). Such Deltam values found from the precise identification of 45 protein forms from HeLa cells reveal 34 coding single nucleotide polymorphisms, two protein forms from alternative splicing, and 12 diverse modifications (not including simple N-terminal processing), including a previously unknown phosphorylation at 10% occupancy. Automated protein identification was achieved with a median expectation value of 10(-13) and often occurred simultaneously with dissection of diverse sources of protein variability as they occur in combination. Top down MS therefore has a bright future for enabling precise annotation of gene products expressed from the human genome by non-mass spectrometrists.

Alternative Splicing↗

PubMed1997

CASP, a novel, highly conserved alternative-splicing product of the CDP/cut/cux gene, lacks cut-repeat and homeo DNA-binding domains, and interacts with full-length CDP in vitro.

Human CDP/cut and its murine counterpart, cux1/CDP are homeodomain repressor proteins in the family of Drosophila Cut. Northern blot analysis reveals complex alternative splicing, including forms too small to encode the full 1505 amino acid protein. We have characterized a CDP/cut alternatively spliced cDNA (CASP) of 3.4 kb. Human CASP, a predicted 678 amino acid polypeptide, shares 400 amino acids with CDP, but has an alternate N terminal exon of 20 aa, and the C-terminal 258 amino acids diverge from CDP/cut entirely. As the unique C-terminus of CASP lacks the three 'cut-repeats' and homeodomain of CDP/cut, we predict it does not bind DNA. Murine CASP, 96% similar to human, shares these features. Database searches identify homologs in chicken (86% identical to human CASP) and yeast (29% identical to human). Murine CASP mRNA is ubiquitous in mouse tissues and in tissue-culture cell lines. We generated a specific antiserum against the unique C-terminus of CASP, and used this reagent to demonstrate that CASP protein is expressed as an approx. 80 kDa protein in human and murine cells. Co-translation of in vitro-translated CDP and CASP mRNA, followed by immunoprecipitation with specific anti-CASP IgG, shows that CASP polypeptide can from a complex with CDP. Studies of the intron/exon structure of the murine cux/CDP/mCASP locus (>> 100 kb) reveal that the unique 3' exons of CASP are interposed between cut-repeats 2 and 3 of the cux gene. We speculate that a primordial CASP-like gene captured a cut-repeat-homeobox gene to give rise to the eukaryotic Cut/CDP family of proteins.

Alternative Splicing↗

PubMed2000

The interaction of calmodulin with alternatively spliced isoforms of the type-I inositol trisphosphate receptor.

A 592-amino acid segment of the regulatory domain of the neuronal type-I inositol 1,4,5-trisphosphate receptor (IP(3)R) isoform (type-I long, amino acids1314-1905) and the corresponding 552-amino acid alternatively spliced form present in peripheral tissues (type-I short, amino acids 1693-1733 deleted) were expressed as glutathione S-transferase fusion proteins. These domains encompass a putative calmodulin (CaM) binding domain and two protein kinase A phosphorylation sites. Both long and short fusion proteins retained the ability to bind CaM in a Ca(2+)-dependent manner as measured by CaM-Sepharose chromatography or a dansyl-CaM fluorescence assay. Both assays indicated that the short fusion protein bound twice the amount of CaM than the long form at saturating concentrations of CaM. In addition, the binding of the short form to CaM-Sepharose was inhibited by phosphorylation with protein kinase A, whereas the binding of the long form was unaffected. Full-length cDNAs encoding type-I long, type-I short, and type-III IP(3)R isoforms were expressed in COS cells, and the Ca(2+) sensitivity of [(3)H]IP(3) binding to permeabilized cells was measured. The type-I long isoform was more sensitive to Ca(2+) inhibition (IC(50) = 0.55 microM) than the type-I short (IC(50) = 5.7 microM) or the type-III isoform (IC(50) = 3 microM). In agreement with studies on the fusion proteins, the full-length type-I short bound more CaM-Sepharose, and this binding was inhibited to a greater extent by protein kinase A phosphorylation than the type-I long IP(3)R. Although type-III IP(3)Rs did not bind directly to CaM-Sepharose, hetero-oligomers of type-I/III IP(3)Rs retained the ability to interact with CaM. We conclude that the deletion of the SII splice site in the type-I IP(3)R results in the differential regulation of the alternatively spliced isoforms by Ca(2+), CaM, and protein kinase A.

Alternative Splicing↗

PubMed1984

Alternative splicing of a Drosophila tropomyosin gene generates muscle tropomyosin isoforms with different carboxy-terminal ends.

The muscle tropomyosin I (mTm I) gene from Drosophila melanogaster has been analyzed and shown to express a complex transcription unit consisting of two sets of tissue-specific mRNAs. A 1.3- and 1.6-kilobase set of mRNAs is expressed during myogenesis in embryos, and in myogenic cell cultures. The mRNAs encode a 34,000-dalton muscle tropomyosin isoform. The same mTm I gene expresses a different set of 1.7- and 1.9-kilobase mRNAs in thoracic flight muscle tissue of the adult. The thorax RNAs encode a new tropomyosin isoform resolved on two-dimensional gels. The structure of the gene has been determined, and we show that the embryonic and thoracic mRNAs are generated by alternative splicing. The alternate exon splicing patterns determine a different 27 amino acids at the carboxy-terminal end of the two tropomyosin isoforms. These results show that the carboxy-terminal domain of tropomyosin is highly regulated in determining tropomyosin function. The results also show that contractile protein isoforms can be generated by single as well as multiple genes.

Amino Acid Sequence↗

PubMed2003

Complex alternative splicing of the hKLK3 gene coding for the tumor marker PSA (prostate-specific-antigen).

PSA (prostate-specific antigen), the most useful serum marker for prostate cancer, is encoded by the hKLK3 gene and is present in the serum as a mixture of several molecular species. This work was performed to identify the hKLK3 transcripts in order to determine how many proteins resembling PSA are synthesized from the hKLK3 gene and secreted in blood. Combined Northern blotting, molecular cloning and database searching showed that the hKLK3 gene produces at least 15 transcripts ranging in size from 0.7 to 6.1 kb. Polysomal distribution analysis revealed that the transcripts shorter than 3.1 kb are efficiently translated in prostate cell line. A total of 12 hKLK3 transcripts have been completely or partially cloned. They result from alternative splicing or/and alternative polyadenylation involving complex regulation. They code for eight proteins: PSA, a truncated form of PSA (PSA-Tr), five PSA variants (PSA-RPs) and one protein (PSA-LM) unrelated to PSA. Using a specific antibody, we detected the PSA-RP2 variant in prostate tissue. All the variants share the same signal peptide and could contribute to the diversity of hKLK3 proteins in prostate fluid and blood.

Alternative Splicing↗

PubMed1997

Functional association between promoter structure and transcript alternative splicing.

It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription.

Alternative Splicing↗

PubMed1993

The human mRNA encoding the Goodpasture antigen is alternatively spliced.

The noncollagenous (NC1) domain of the human collagen alpha 3(IV)-chain is the primary target of autoantibodies produced in Goodpasture syndrome and, therefore, has been designated as the Goodpasture antigen. In this report, we show that Goodpasture antigen mRNA undergoes processing to at least two alternatively spliced forms in a variety of human tissues, resulting in the exclusion of sequence encoded by either one or two exons. Interestingly, no alternatively spliced forms were observed in bovine or rat tissues. The derived amino acid sequences of the two variant mRNA forms are identical and significantly shorter than that arising from the complete Goodpasture antigen mRNA. They lack the carboxyl-terminal region contributing to the formation of the Goodpasture epitope and all but one of the cysteines found in the complete form. These sequence characteristics suggest that, if translated, the variant Goodpasture antigen is likely to be defective in triple helix formation and no longer reactive with Goodpasture autoantibodies. Although each tissue expressing Goodpasture antigen displayed a specific mRNA pattern, the complete form was always the most abundant and was present at levels apparently unrelated to whether or not the organ of origin is a potential target in Goodpasture syndrome. Furthermore, the antigen sequence was identical in the kidneys of normal and Goodpasture-affected individuals, and no major differences in the expression of the complete and spliced forms were observed.

Alternative Splicing↗

PubMed2004

Cumulative inactivation of N-type CaV2.2 calcium channels modified by alternative splicing.

The Ca(V)2 family of voltage-gated calcium channels, present in presynaptic nerve terminals, regulates exocytosis and synaptic transmission. Cumulative inactivation of these channels occurs during trains of action potentials, and this may control short-term dynamics at the synapse. Inactivation during brief, repetitive stimulation is primarily attributed to closed-state inactivation, and several factors modulate the susceptibility of voltage-gated calcium channels to this form of inactivation. We show that alternative splicing of an exon in a cytoplasmic region of the Ca(V)2.2 channel modulates its sensitivity to inactivation during trains of action potential waveforms. The presence of this exon, exon 18a, protects the Ca(V)2.2 channel from entry into closed-state inactivation specifically during short (10 ms to 3 s) and small depolarizations of the membrane potential (-60 mV to -50 mV). The reduced sensitivity to closed-state inactivation within this dynamic range likely underlies the differential responsiveness of Ca(V)2.2 splice isoforms to trains of action potential waveforms. Regulated alternative splicing of Ca(V)2.2 represents a possible mechanism for modulating short-term dynamics of synaptic efficacy in different regions of the nervous system.

Action Potentials↗