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PubMed2006

Alternative splicing: new insights from global analyses.

Recent analyses of sequence and microarray data have suggested that alternative splicing plays a major role in the generation of proteomic and functional diversity in metazoan organisms. Efforts are now being directed at establishing the full repertoire of functionally relevant transcript variants generated by alternative splicing, the specific roles of such variants in normal and disease physiology, and how alternative splicing is coordinated on a global level to achieve cell- and tissue-specific functions. Recent progress in these areas is summarized in this review.

Alternative Splicing↗

PubMed1995

Alternative splicing results in two different transcripts for H-protein of the glycine cleavage system in the C4 species Flaveria trinervia.

Alternative splicing is a well-known post-transcriptional regulatory mechanism in eukaryotic organisms but there are only very few reports on alternative splicing in plants. The analysis of cDNAs encoding H-protein of the glycine decarboxylase multi-enzyme complex from the C4 species Flaveria trinervia revealed the presence of two transcript populations that differ in the length of their coding regions by six nucleotides. Otherwise, including their 3' nontranslated region, they are identical. From a genomic Southern analysis and from the sequencing of several independent cDNA clones it is evident that both types of transcript are derived from a single-copy gene. This gene, FTgdcsH, has been cloned and sequenced. It comprises four short exons. The two alternative splice sites are located at the end of intron 1. The shorter transcript closely corresponds to published H-protein mRNA sequences from other organisms. The longer transcript encodes two additional alanine residues very close to the N-terminus of the mature H-protein. A quantification of the relative amounts of both transcripts in different organs revealed that, with 80-90% of the total H-protein mRNA, the alternative mRNA dominates in leaves whereas roots contain more of the 'correctly' spliced transcript. It is concluded that, in F. trinervia and with a distinct organ preference, alternative splicing leads to the synthesis of two different H-proteins of the glycine cleavage system.

Alternative Splicing↗

PubMed2006

PLP1 alternative splicing in differentiating oligodendrocytes: characterization of an exonic splicing enhancer.

Proteolipid protein (PLP) and DM20 are generated by alternative splicing of exon 3B of PLP1 transcript in differentiating oligodendrocytes. We investigated the role of exonic splicing enhancers (ESE) in the selection of PLP 5' donor site, focusing on putative ASF/SF2, and SC35 binding motifs in exon 3B on the basis of mutations that cause disease in humans. Mutations in a putative ASF/SF2 binding motif (nucleotides 406-412) reduced PLP 5' donor site selection, whereas a mutation in a putative SC35 binding motif (nucleotides 382-389) had no effect. UV crosslinking and immunoprecipitation (IP) assays using an antibody to ASF/SF2 showed that the ASF/SF2 protein specifically binds to the ESE (nucleotides 406-412). The single nucleotide mutations that reduced PLP splice site selection greatly diminished ASF/SF2 protein binding to this motif. We next tested the effect of overexpressed ASF/SF2 on PLP 5'splice selection in differentiating oligodendrocytes. ASF/SF2 positively regulates PLP splice site selection in a concentration-dependent manner. Disruption of the putative ASF/SF2 binding site in exon 3B reduced the positive effect of ASF/SF2 on PLP splicing. We conclude that an ESE in exon3B regulates PLP 5' donor site selection and that ASF/SF2 protein participates in the regulation of PLP alternative splicing in oligodendrocytes.

Alternative Splicing↗

PubMed2005

The L-type calcium channel alpha 1C subunit gene undergoes extensive, uncoordinated alternative splicing.

The alpha1C subunit is the pore-forming protein for the L-type calcium channel. Previous studies indicate that there is possible tissue-specific alternative splicing of this gene. In this study we cloned the entire open reading frame of the alpha1C subunit cDNA from adult rat cardiac myocytes in a single piece (6.64 kb). Using 75 positive clones that were identified by restriction enzyme mapping, we tested the alternative splicing patterns of the Ca(v) 1.2 gene that encodes the alpha1C subunit protein and focused on five loci: IS6, post-IS6, IIIS2, IVS3, and the c-terminus. The results indicate that: (1) alternative splicing occurs in most of the loci, giving rise to two or three different isoforms at those sites; (2) there is a predominant form for each splicing site, (3) there does not appear to be consistent coordination of splicing at multiple loci of this gene. Alternative splicing is not tissue-specific in most regions.

Alternative Splicing↗

PubMed2004

Alternative splicing in the voltage-sensing region of N-Type CaV2.2 channels modulates channel kinetics.

The CaV2.2 gene encodes the functional core of the N-type calcium channel. This gene has the potential to generate thousands of CaV2.2 splice isoforms with different properties. However, the functional significance of most sites of alternative splicing is not established. The IVS3-IVS4 region contains an alternative splice site that is conserved evolutionarily among CaValpha1 genes from Drosophila to human. In CaV2.2, inclusion of exon 31a in the IVS3-IVS4 region is restricted to the peripheral nervous system, and its inclusion slows the speed of channel activation. To investigate the effects of exon 31a in more detail, we generated four tsA201 cell lines stably expressing CaV2.2 splice isoforms. Coexpression of auxiliary CaVbeta and CaValpha2delta subunits was required to reconstitute currents with the kinetics of N-type channels from neurons. Channels including exon 31a activated and deactivated more slowly at all voltages. Current densities were high enough in the stable cell lines co-expressing CaValpha2delta to resolve gating currents. The steady-state voltage dependence of charge movement was not consistently different between splice isoforms, but on gating currents from the exon 31a-containing CaV2.2 isoform decayed with a slower time course, corresponding to slower movement of the charge sensor. Exon 31a-containing CaV2.2 is restricted to peripheral ganglia; and the slower gating kinetics of CaV2.2 splice isoforms containing exon 31a correlated reasonably well with the properties of native N-type currents in sympathetic neurons. Our results suggest that alternative splicing in the S3-S4 linker influences the kinetics but not the voltage dependence of N-type channel gating.

Alternative Splicing↗

PubMed2004

Smooth muscle-selective alternatively spliced exon generates functional variation in Cav1.2 calcium channels.

Voltage-gated calcium channels play a major role in many important processes including muscle contraction, neurotransmission, excitation-transcription coupling, and hormone secretion. To date, 10 calcium channel alpha(1)-subunits have been reported, of which four code for L-type calcium channels. In our previous work, we uncovered by transcript-scanning the presence of 19 alternatively spliced exons in the L-type Ca(v)1.2 alpha(1)-subunit. Here, we report the smooth muscle-selective expression of alternatively spliced exon 9(*) in Ca(v)1.2 channels found on arterial smooth muscle. Specific polyclonal antibody against exon 9(*) localized the intense expression of 9(*)-containing Ca(v)1.2 channels on the smooth muscle wall of arteries, but the expression on cardiac muscle was low. Whole-cell patch clamp recordings of the 9(*)-containing Ca(v)1.2 channels in HEK293 cells demonstrated -9 and -11-mV hyperpolarized shift in voltage-dependent activation and current-voltage relationships, respectively. The steady-state inactivation property and sensitivity to blockade by nifedipine of the +/-exon 9(*) splice variants were, however, not significantly different. Such cell-selective expression of an alternatively spliced exon strongly indicates the customization and fine tuning of calcium channel functions through alternative splicing of the pore-forming alpha(1)-subunit. The generation of proteomic variations by alternative splicing of the calcium channel Ca(v)1.2 alpha(1)-subunit can potentially provide a flexible mechanism for muscle or neuronal cells to respond to various physiological signals or to diseases.

Alternative Splicing↗

PubMed2005

Phenotyping-genotyping of alternatively spliced genes in one step: study of CYP3A5*3 polymorphism.

Alternative splicing is required to increase the mRNA diversity of many genes, but can also be responsible for the abnormal expression of genes. For example, the CYP3A5*3 defective allele is caused by a single nucleotide polymorphism in intron 3. This mutation activates a cryptic acceptor splice site, which leads to the insertion of an intronic sequence containing premature termination codons in the mature mRNA, and hence the very low CYP3A5 protein expression in 75% of the Caucasian population. In the present study, we propose a novel strategy based on the quantitative real-time polymerase chain reaction with SYBR Green I chemistry, followed by melting curve analysis, to demonstrate and quantify the amount of splice variant mRNA. Using oligonucleotides flanking the insertion site, two products with different size can be obtained, which can be resolved by melting curve analysis. The relative ratio of differently spliced RNA can be estimated at the plateau phase by using the peak height ratio. For the CYP3A5 gene, the genotype, the level of expression and the proportion of alternatively spliced products were determined in a single reaction without DNA sequencing.

Alternative Splicing↗

PubMed2006

Crystal structure of the second LNS/LG domain from neurexin 1alpha: Ca2+ binding and the effects of alternative splicing.

Neurexins mediate protein interactions at the synapse, playing an essential role in synaptic function. Extracellular domains of neurexins, and their fragments, bind a distinct profile of different proteins regulated by alternative splicing and Ca2+. The crystal structure of n1alpha_LNS#2 (the second LNS/LG domain of bovine neurexin 1alpha) reveals large structural differences compared with n1alpha_LNS#6 (or n1beta_LNS), the only other LNS/LG domain for which a structure has been determined. The differences overlap the so-called hyper-variable surface, the putative protein interaction surface that is reshaped as a result of alternative splicing. A Ca2+-binding site is revealed at the center of the hyper-variable surface next to splice insertion sites. Isothermal titration calorimetry indicates that the Ca2+-binding site in n1alpha_LNS#2 has low affinity (Kd approximately 400 microm). Ca2+ binding ceases to be measurable when an 8- or 15-residue splice insert is present at the splice site SS#2 indicating that alternative splicing can affect Ca2+-binding sites of neurexin LNS/LG domains. Our studies initiate a framework for the putative protein interaction sites of neurexin LNS/LG domains. This framework is essential to understand how incorporation of alternative splice inserts expands the information from a limited set of neurexin genes to produce a large array of synaptic adhesion molecules with potentially very different synaptic function.

Alternative Splicing↗

PubMed1996

Functional properties of p54, a novel SR protein active in constitutive and alternative splicing.

The p54 protein was previously identified by its reactivity with an autoantiserum. We report here that p54 is a new member of the SR family of splicing factors, as judged from its structural, antigenic, and functional characteristics. Consistent with its identification as an SR protein, p54 can function as a constitutive splicing factor in complementing splicing-deficient HeLa cell S100 extract. However, p54 also shows properties distinct from those of other SR family members, p54 can directly interact with the 65-kDa subunit of U2 auxiliary factor (U2AF65), a protein associated with the 3' splice site. In addition, p54 interacts with other SR proteins but does not interact with the U1 small nuclear ribonucleoprotein U1-70K or the 35-kDa subunit of U2 auxiliary factor (U2AF35). This protein-protein interaction profile is different from those of prototypical SR proteins SC35 and ASF/SF2, both of which interact with U1-70K and U2AF35 but not with U2AF65. p54 promotes the use of the distal 5' splice site in E1A pre-mRNA alternative splicing, while the same site is suppressed by ASF/SF2 and SC35. These findings and the differential tissue distribution of p54 suggest that this novel SR protein may participate in regulation of alternative splicing in a tissue- and substrate-dependent manner.

Alternative Splicing↗

PubMed2002

Alternatively spliced TCR mRNA induced by disruption of reading frame.

Nonsense codons that prematurely terminate translation generate potentially deleterious truncated proteins. Here, we show that the T cell receptor-beta (TCRbeta) gene, which acquires in-frame nonsense codons at high frequency during normal lymphocyte development, gives rise to an alternatively spliced transcript [alternative messenger RNA (alt-mRNA)] that skips the offending mutations that generate such nonsense codons. This alt-mRNA is up-regulated by a transfer RNA-dependent scanning mechanism that responds specifically to mutations that disrupt the reading frame. The finding that translation signals regulate the levels of alternatively spliced mRNAs generated in the nucleus may alter the current view of how gene expression is controlled in eukaryotic cells.

Alternative Splicing↗

PubMed1996

H-protein of the glycine cleavage system in Flaveria: alternative splicing of the pre-mRNA occurs exclusively in advanced C4 species of the genus.

A recent paper reports on the occurrence of alternative splicing of H-protein pre-mRNA in the C4 species, Flaveria trinervia, that is organ-specifically regulated. The analysis of 11 other species of the genus, F. cronquistii and F. pringlei (C3), F. anomala, F. chloraefolia, F. floridana, F. linearis and F. pubescens (C3-C4 intermediate), F. brownii (C4-like), F. palmeri, F. bidentis and F. australasica (C4), revealed that this post-transcriptional effect is not specific for F. trinervia. It occurs in all the examined C4 species of the genus Flaveria except the less advanced C4-like species, F. brownii. Both the position and the direct effect of alternative splicing, the addition of two alanine residues near to the N-terminus of the derived mature H-protein, are invariant. A quantification of the relative amounts of both transcripts revealed that, as in F. trinervia, the alternative mRNA strongly dominates in leaves. In sharp contrast, none of the C3, C3-C4 intermediate, or C4-like species showed alternative splicing. By Western analysis both H-isoproteins have been detected in F. trinervia and their ratio approximately corresponds to the measured transcript levels. It is concluded that alternative splicing leads to the synthesis of two different H-proteins of the glycine cleavage system in all advanced Flaveria C4 species.

Alternative Splicing↗

PubMed2002

Alternative splicing in the alpha-galactosidase A gene: increased exon inclusion results in the Fabry cardiac phenotype.

Fabry disease is an inborn error of glycosphingolipid catabolism, resulting from deficient activity of lysosomal alpha-galactosidase A (alpha-Gal A). A rare alternative splicing that introduces a 57-nucleotide (nt) intronic sequence to the alpha-Gal A transcript from intron 4 of the gene has been identified. In addition, a novel midintronic base substitution that results in substantially increased alternative splicing has been identified in a patient with Fabry disease who has the cardiac variant phenotype. The sequence of the patient's intron 4 contains a single G-->A transversion at genomic nt 9331 (IVS4+919 G-->A ), located at the minus sign4 position of the 3' end of the intronic insertion (nts 9278--9334 in the genomic sequence). Minigene constructs containing the entire intron 4 sequence with G, A, C, or T at nt 9331 within an alpha-Gal A complementary DNA expression vector were prepared and expressed in COS-1 cells. Whereas transfection of the G or T minigenes transcribed predominantly normal-sized transcripts, the transfection of the A or C minigenes produced a large amount of the alternatively spliced transcript. These results suggest that the G-->A mutation, within an A/C-rich domain, results in increased recognition of the alternative splicing by an A/C-rich enhancer-type exonic splicing enhancer. The intronic mutation was not observed in 100 unrelated unaffected men but was present in 6 unrelated patients with cardiac Fabry disease. Reverse-transcriptase polymerase chain reaction of total RNA of various normal human tissues revealed that the alternatively spliced transcript was present in all of the samples, and especially at a higher ratio in the lung and muscle. The normal transcript was present in the patients' lymphoblasts and resulted in approximately 10% residual enzyme activity, leading to a cardiac phenotype of Fabry disease.

Alternative Splicing↗

PubMed2004

Alternative splicing of human telomerase reverse transcriptase may not be involved in telomerase regulation during all-trans-retinoic acid-induced HL-60 cell differentiation.

Alternative splicing of the human telomerase reverse transcriptase subunit (hTERT) suppresses telomerase activity during the development of human fetal kidney cells into mature cells. Tumor cell differentiation is the process of turning abnormal tumor cells into 'normal' cells accompanied by down-regulation of telomerase activity. However, the precise mechanism of the regulation of telomerase activity in differentiated cells is not fully understood. In this study, we observed the role of alternative splicing of hTERT in the regulation of telomerase activity in all-trans-retinoic acid (ATRA)-induced, differentiated HL-60 cells. ATRA-induced down-regulation of telomerase activity in differentiated HL-60 cells was associated with a decrease in hTERT and an increase in human telomerase-associated protein-1 (hTP1) transcription. Expression of full length variant hTERT alpha+ beta+ mRNA decreased in a dose- and time-dependent manner. The drop of hTERT beta- mRNA was time-dependent. hTERT alpha- and hTERT alpha- beta- mRNA were reduced dramatically after ATRA treatment. In the dose-effect study, hTERT alpha+ beta+ and hTERT beta- maintained a relatively stable ratio when telomerase activity decreased largely from treatment with 1 to 5 microM ATRA. Although the splicing pattern of hTERT mRNA was altered in time-effect research, the change was not related to the ATRA-treated decline of telomerase activity. The expression of alternative splicing variants of hTERT also decreased at the protein level. All these results suggested that alternative splicing of hTERT mRNA may not contribute to the suppression of telomerase activity during ATRA-induced HL-60 leukemia cell differentiation.

Alternative Splicing↗

PubMed2006

ASPIC: a web resource for alternative splicing prediction and transcript isoforms characterization.

Alternative splicing (AS) is now emerging as a major mechanism contributing to the expansion of the transcriptome and proteome complexity of multicellular organisms. The fact that a single gene locus may give rise to multiple mRNAs and protein isoforms, showing both major and subtle structural variations, is an exceptionally versatile tool in the optimization of the coding capacity of the eukaryotic genome. The huge and continuously increasing number of genome and transcript sequences provides an essential information source for the computational detection of genes AS pattern. However, much of this information is not optimally or comprehensively used in gene annotation by current genome annotation pipelines. We present here a web resource implementing the ASPIC algorithm which we developed previously for the investigation of AS of user submitted genes, based on comparative analysis of available transcript and genome data from a variety of species. The ASPIC web resource provides graphical and tabular views of the splicing patterns of all full-length mRNA isoforms compatible with the detected splice sites of genes under investigation as well as relevant structural and functional annotation. The ASPIC web resource-available at http://www.caspur.it/ASPIC/--is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility.

Algorithms↗

PubMed1998

Alternative splicing of LTBP-3.

LTBPs bind the 100-kDa latent TGF-beta complex and thereby regulate TGF-beta assembly, tissue localization, and function. However, the 100-kDa complex is not always associated with LTBP, and, conversely, evidence suggests that LTBP has a distinct role in the extracellular matrix. As yet, there are no data to explain how the binding interaction between LTBP and the 100-kDa complex is regulated. This report provides the first direct evidence of alternative splicing of an LTBP gene. Two alternative splice sites in the mouse LTBP-3 gene have been identified based on in vivo and in vitro studies. Alternative splicing at one site in particular was found to disrupt a structural motif involved in the binding interaction with the 100-kDa latent TGF-beta complex. Therefore, alternative splicing may represent a molecular mechanism by which the uncomplexed form of LTBP-3 is produced, and, as a corollary, by which the 100-kDa latent TGF-beta 1 complex is produced.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Manipulation of alternative splicing by a newly developed inhibitor of Clks.

The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes. The importance of alternative splicing is further illustrated by the increasing number of human diseases that have been attributed to mis-splicing events. Appropriate spatial and temporal generation of splicing variants demands that alternative splicing be subjected to extensive regulation, similar to transcriptional control. The Clk (Cdc2-like kinase) family has been implicated in splicing control and consists of at least four members. Through extensive screening of a chemical library, we found that a benzothiazole compound, TG003, had a potent inhibitory effect on the activity of Clk1/Sty. TG003 inhibited SF2/ASF-dependent splicing of beta-globin pre-mRNA in vitro by suppression of Clk-mediated phosphorylation. This drug also suppressed serine/arginine-rich protein phosphorylation, dissociation of nuclear speckles, and Clk1/Sty-dependent alternative splicing in mammalian cells. Consistently, administration of TG003 rescued the embryonic defects induced by excessive Clk activity in Xenopus. Thus, TG003, a novel inhibitor of Clk family will be a valuable tool to dissect the regulatory mechanisms involving serine/arginine-rich protein phosphorylation signaling pathways in vivo, and may be applicable for the therapeutic manipulation of abnormal splicing.

Alternative Splicing↗

PubMed2002

Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.

Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

5' Untranslated Regions↗

PubMed2001

Alternative splicing of KCNQ2 potassium channel transcripts contributes to the functional diversity of M-currents.

The region of alternative splicing in the KCNQ2 potassium channel gene was determined by RNase protection analysis of KCNQ2 mRNA transcripts. Systematic analysis of KCNQ2 alternative splice variant expression in rat superior cervical ganglia revealed multiple variant isoforms. One class of KCNQ2 splice variants, those that contained exon 15a, was found to have significantly different kinetics to those of the other isoforms. These transcripts encoded channel subunits that, when co-expressed with the KCNQ3 subunit, activated and deactivated approximately 2.5 times more slowly than other isoforms. Deletion of exon 15a in these isoforms produced a reversion to the faster kinetics. Comparison of the kinetic properties of the cloned channel splice variants with those of the native M-current suggests that alternative splicing of the KCNQ2 gene may contribute to the variation in M-current kinetics seen in vivo.

Alternative Splicing↗