[Topoisomerase I in alternative splicing].
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Alternative Splicing↗
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Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing.
Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.
Alternative Splicing↗
Analysis of the linkage between fibronectin alternative spliced sites during ageing in rat tissues.
The modulation of fibronectin (FN) functions in cell-cell and cell-substrate interactions in a variety of physiological situations is achieved by the selective expression of different isoforms, which in the rat are generated by alternatively splicing of at least three regions of the molecule: EIIIA, EIIIB and V. Extensive information has been collected on the regulation of the differential processing of the single alternatively spliced regions but up to now there was scant knowledge about a possible coordinated expression of the three regions in the same transcript. Using a long range RT-PCR system we have shown that most of the splicing regulation is autonomous for each individual region but we have also observed a preferential expression of the EIIIA+ form linked to the EIIIB- variant that is age independent. Furthermore the analysis of the single regions showed interesting variations occurring in brain during the ageing. There is a decrease in the V120 form between the 6- and the 24-month-old rat brain (from 57% to 39%), whereas despite a constant prevalence of the EIIIA- form in the young rats (65%) there is a random individual variation of this form in the old animals. Noteworthy no variations have been detected in the EIIIB region (90% EIIIB-). These data suggest a role for the V and EIIIA regions, but not for the EIIIB, during the ageing process at least in brain, since no variations were detected in kidney between the 6- and 24-month-old animals.
Aging↗
Primary sequence of an alternatively spliced form of CR1. Candidate for the 75,000 M(r) complement receptor expressed on chimpanzee erythrocytes.
Chimpanzee erythrocytes express a 75,000 M(r) complement receptor (E-CR) that binds C3b bearing immune complexes and is recognized by an anti-CR1 mAb (E11). Human erythrocytes express the type 1 CR (CR1), the most common form being 220,000 M(r) and consisting of 30 short consensus repeats (SCRs) for its entire extracellular region. The purpose of this investigation was to determine the structure of the 75,000 M(r) chimpanzee E-CR. A chimpanzee cell line was identified that expressed a 220,000 M(r) CR1, and a 75,000 M(r) molecule that was recognized by E11 and could bind human C3i. Utilizing this cell line, chimpanzee CR1 cDNA was amplified in overlapping segments by the PCR, using primer pairs specific for various regions of human CR1 cDNA. Direct sequencing of the PCR-amplified products revealed 6044 nucleotides encoding the entire 220,000 M(r) chimpanzee CR1. This nucleotide sequence was 98.8% homologous to that of the human 220,000 M(r) CR1. Amplification using a CR1 primer from the signal peptide and from the cytoplasmic region yielded a 1985-bp PCR product, termed CR1a. The CR1a sequence was identical with the sequence encoding SCRs 1 to 6, SCRs 28 to 30, and the transmembrane and cytoplasmic regions of chimpanzee CR1. This alternatively spliced product of chimpanzee CR1 would encode a protein of 71,000 peptide m.w. with six potential N-glycosylation sites. Amplification employing a CR1 primer from SCR 1 and from the 3' untranslated region yielded a second PCR product of 1731 bp. This sequence, termed CR1b, encoded eight SCRs, followed by a hydrophobic region that ended in a stop codon. The first six SCRs of CR1b were closer in homology to the first six SCRs of a human CR1-like genomic sequence (97.4%) than to those of the chimpanzee CR1 (94.8%). Taken together, these sequence data suggest that the 75,000 M(r) chimpanzee E-CR is encoded by CR1a, an alternative splice variant of chimpanzee CR1. The CR1b is presumably derived from an RNA species related to the CR1-like genomic sequence previously described only in humans.
Alternative Splicing↗
Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript.
Somatic sexual differentiation in Drosophila melanogaster is accomplished by a hierarchy of genes of which one, Sex-lethal (Sxl), is required for the functional female-specific splicing of the transcripts of the immediately downstream regulatory gene, transformer (tra). The first exon of the tra primary transcript is spliced to one of two acceptor sites. Splicing to the upstream site yields a messenger RNA which is neither sex-specific nor functional, but that produced after splicing to the downstream acceptor site yields a functional female-specific mRNA. Here we address the question of how the Sxl gene product determines the alternative splicing of tra primary transcripts. One suggestion is that non-sex-specific splicing to the upstream acceptor is blocked in female flies by sex-specific factors, but neither the identity of the female-specific factors nor the mechanism of the blockage has been specified. We have now performed co-transfection experiments in which Sxl complementary DNA and the tra gene are expressed in Drosophila Kc cells. Moreover, we find that female Sxl-encoded protein binds specifically to the tra transcript at or near the non-sex-specific acceptor site, implying that the female Sxl gene product is the trans-acting factor that regulates the alternative splicing.
Animals↗
Characterization and phosphorylation of PDE10A2, a novel alternative splice variant of human phosphodiesterase that hydrolyzes cAMP and cGMP.
We have isolated a novel alternative splice variant of human cAMP- and cGMP-hydrolyzing phosphodiesterase (PDE10A2) from human fetal lung. The N-terminal sequence of human PDE10A2 differed from that of human PDE10A1 reported previously. PDE10A1 and PDE10A2 expressed in COS-7 cells have cGMP K(m) values of 14 and 13 microM, low cAMP K(m) values of 0.28 and 0.22 microM, and high cAMP K(m) values of 0.96 and 1.1 microM, respectively, at high concentrations of cGMP and cAMP. PCR analysis demonstrated that both PDE10A1 and PDE10A2 transcripts are present in various human tissues and that PDE10A2 transcripts are a major form in some human tissues. The unique N-terminus of PDE10A2 has a putative phosphorylation site by cAMP-dependent protein kinase (cAK), but PDE10A1 does not. The recombinant PDE10A2 protein is preferentially phosphorylated by cAK, although the recombinant PDE10A1 protein is not phosphorylated by cAK.
Alternative Splicing↗
Evolution of the neuron-specific alternative splicing product of the c-src proto-oncogene.
The observation of a slower migrating form of pp60c-src in neural tissue of chicken and mouse has recently been shown to be due to an alternative transcript form of the c-src gene (Martinez et al.: Science 237:411-415, 1987; Levy et al.: Mol Cell Biol 7:4142-4145, 1987). An insertion of 18 basepairs between exons 3 and 4, presumed to be due to alternative splicing of a mini-exon, gives rise to six amino acid residues not found in the non-neuronal (termed fibroblastic) form of pp60c-src. We have addressed the question of the evolutionary origin of the c-src neuronal insert and its functional significance regarding neural-specific expression of the c-src gene. To this end we have investigated whether the c-src gene of a lower vertebrate (the teleost fish Xiphophorus) gives rise to a neural-specific transcript in an analogous manner. We could show that the fish c-src gene does encode for a "fibroblastic" and a "neuronal" form of transcript and that the neuronal transcript does indeed arise by way of alternative splicing of a mini-exon. The mini-exon is also 18 basepairs long and we could demonstrate directly that this exon lies within the intron separating exons 3 and 4. For comparative purposes we have examined whether the fish c-yes gene, the member of the src gene family most closely related to c-src, also encodes a neural tissue-specific transcript. No evidence for a second transcript form in brain was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Human and murine glycerol kinase: influence of exon 18 alternative splicing on function.
Glycerol kinase (GK) is a key enzyme in glycerol metabolism with two alternatively spliced forms-one with an 87bp insertion corresponding to exon 18 (GK+EX18), and one lacking exon 18 (GK-EX18). We report the expression of GK+/-EX18 in various tissues and cell lines, as well as their enzymatic characteristics and subcellular localization. RT-PCR revealed differential expression in tissues and cell lines. Northern blot analysis revealed that both forms of the murine ortholog, Gyk, were highly expressed in murine heart and increased during embryonic development. K(m) values for glycerol for GK+/-EX18 were not significantly different, although GK-EX18 had a higher V(max) for glycerol. GK-EX18 had a lower K(m) and V(max) for ATP than GK+EX18. Immunofluorescence experiments showed that GK+EX18 co-localized to the mitochondria and the perinuclear region while GK-EX18 had a diffuse expression pattern. These data suggest specific and divergent roles for GK+EX18 and GK-EX18 in cellular metabolism and development.
Alternative Splicing↗
Molecular mechanisms associated with the regulation of apoptosis by the two alternatively spliced products of c-Myb.
The c-myb proto-oncogene encodes two alternatively spliced mRNAs, which in turn code for proteins of 75 kDa and 89 kDa. It is at present unclear whether the two isoforms of c-Myb perform identical functions or whether they mediate different biological effects. To assess their role in apoptotic death of hematopoietic cells, we expressed the two isoforms of c-Myb in the murine myeloid cell lines 32Dcl3 and FDCP1. Our results show that while ectopic overexpression of p75 c-Myb results in the acceleration of cell death, similar overexpression of p89 c-Myb results in the protection of cells from apoptotic death. An analysis of gene expression changes with mouse cDNA expression arrays revealed that while p75 c-Myb blocked the expression of glutathione S-transferase micro mRNA, p89 c-Myb greatly enhanced the expression of this gene. These results were further confirmed by Northern blot analysis. Ectopic overexpression of the glutathione S-transferase micro gene in 32Dcl3 cells resulted in protection of cells from interleukin-3 withdrawal-induced cell death similar to that seen with the ectopic overexpression of p89 c-Myb. These results suggest that the two isoforms of c-Myb differentially regulate apoptotic death of myeloid cells through differential regulation of glutathione S-transferase micro gene expression.
Alternative Splicing↗
DNA binding and 3'-5' exonuclease activity in the murine alternatively-spliced p53 protein.
In this study we show that the naturally occurring C-terminally alternative spliced p53 (referred to as AS-p53) is active as a sequence-specific DNA binding protein as well as a 3'-5'-exonuclease in the presence of Mg2+ ions. The two activities are positively correlated as the sequence-specific DNA target is more efficiently degraded than a non-specific target. In contrast, a mutated AS-p53 protein that is deficient in DNA binding lacks exonuclease activity. The use of modified p53 binding sites, where the 3'-phosphate is replaced by a phosphorothioate group, enabled the inhibition of DNA degradation under the binding conditions. We demonstrate that AS-p53 interacts with its specific DNA target by two distinct binding modes: a high-affinity mode characterized by a low-mobility protein-DNA complex at the nanomolar range, and a low-affinity mode shown by a high-mobility complex at the micromolar range. Comparison of the data on the natural and the modified p53 binding sites suggests that the high-affinity mode is related to AS-p53 function as a transcription factor and that the low-affinity mode is associated with its exonuclease activity. The implications of these findings to a specific cellular role of AS-p53 are discussed.
Alternative Splicing↗
Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript.
The technique of whole-genome polymerase chain reaction was used to study the DNA binding properties of the product of the wt1 gene. The zinc finger region of this gene is alternatively spliced such that the major transcript encodes a protein with three extra amino acids between the third and fourth fingers. The minor form of the protein binds specifically to DNA. It is now shown that the major form of wt1 messenger RNA encodes a protein that binds to DNA with a specificity that differs from that of the minor form. Therefore, alternative splicing within the DNA binding domain of a transcription factor can generate proteins with distinct DNA binding specificities and probably different physiological targets.
Amino Acid Sequence↗
Alternative splicing of pre-mRNAs encoding the nonstructural proteins of minute virus of mice is facilitated by sequences within the downstream intron.
mRNAs R1 and R2 of the parvovirus minute virus of mice encode the two essential viral regulatory proteins NS1 and NS2. Both RNAs are spliced between map units 44 and 46 (nucleotides 2280 and 2399); R2 RNAs are additionally spliced upstream between map units 10 and 39 (nucleotides 514 and 1989), using a nonconsensus donor and poor 3' splice site. The relative accumulation of R1 and R2 is determined by alternative splicing: there is twice the steady-state accumulation of R2 relative to that of R1 throughout viral infection, though they are generated from the same promoter and have indistinguishable stabilities. Here we demonstrate that efficient excision of the large intron to generate R2 is dependent on at least the initial presence, in P4-generated pre-mRNAs, of sequences within the downstream small intron. This effect is orientation dependent and related to the size of the intervening exon. Prior splicing of the small intron is unnecessary. Excision of the large intron is enhanced by changing its donor site to consensus, but only in the presence of the small intron sequences. Excision of the large intron is also enhanced by improving the polypyrimidine tract within its 3' splice site; however, in contrast, this change renders excision of the large intron independent of the downstream small intron. We suggest that sequences within the small intron play a primary role in efficient excision of the upstream large intron, perhaps as the initial entry site(s) for an element(s) of the splicesome, which stabilizes the binding of required factors to the polypyrimidine tract within the 3' splice site of the large intron.
Alternative Splicing↗
Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA.
The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 on the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effects. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron, Thus UK-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.
Alternative Splicing↗
Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties.
The rice Waxy gene encodes a granule-bound starch synthase (GBSS) necessary for the synthesis of amylose in endosperm tissue. We have previously shown that a CT microsatellite near the transcriptional start site of the GBSS gene can distinguish 7 alleles that accounted for more than 80% of the variation in apparent amylose content in an extended pedigree of 89 US rice cultivars (Oryza sativa L.). Furthermore, all the cultivars with 18% or less amylose were shown to have the sequence AGTTATA at the putative leader intron 5' splice site, while all cultivars with a higher proportion of amylose had AGGTATA. Here we demonstrate that this single-base mutation reduces the efficiency of GBSS pre-mRNA processing and results in alternate splicing at three cryptic sites. The predominant 5' splice site in CT18 low-amylose varieties is 93 bp upstream of the splice site used in intermediate and high amylose varieties and is immediately 5' to the CT microsatellite that we previously demonstrated to be tightly correlated with amylose content. Use of the leader intron 5' splice site at either -93 or -1 in conjunction with the predominant 3' splice site results in formation of a small open reading frame 38 bp upstream of the normal ATG and out of frame with it. This open reading frame is not produced when any of the 5' leader intron splice sites are used in conjunction with an alternate 3' splice site five bases further downstream which was observed in all rice varieties tested.
Alternative Splicing↗
No alteration in tau exon 10 alternative splicing in tangle-bearing neurons of the Alzheimer's disease brain.
Defective splicing of tau mRNA, promoting a shift between tau isoforms with (4R tau) and without (3R tau) exon 10, is believed to be a pathological consequence of certain tau mutations causing frontotemporal dementia. By assessing protein and mRNA levels of 4R tau and 3R tau in 27 AD and 20 control temporal cortex, we investigated whether altered tau splicing is a feature also in Alzheimer's disease (AD). However, apart from an expected increase of sarcosyl-insoluble tau in AD, there were no significant differences between the groups. Next, by laser-capture microscopy and quantitative PCR, we separately analyzed CA1 hippocampal neurons with and without neurofibrillary pathology from six of the AD and seven of the control brains. No statistically significant differences in 4R tau/3R tau mRNA were found between the different subgroups. Moreover, we confirmed the absence of significant ratio differences in a second data set with laser-captured entorhinal cortex neurons from four AD and four control brains. Finally, the 4R tau/3R tau ratio in CA1 neurons was roughly half of the ratio in temporal cortex, indicating region-specific differences in tau mRNA splicing. In conclusion, this study indicated region-specific and possibly cell-type-specific tau splicing but did not lend any support to overt changes in alternative splicing of tau exon 10 being an underlying factor in AD pathogenesis.
Aged↗
Alternative splicing gives rise to two forms of the p68 Ca2(+)-binding protein.
The p68 Ca2+ and phospholipid binding protein of the lipocortin/calpactin family appears to exist as two forms. These may be resolved into a closely-spaced polypeptide doublet by SDS-PAGE. The cloning and sequencing of p68 revealed an apparent 18 nucleotide alternative splice sequence, which could account for this observation. We show here that an antiserum directed against a synthetic peptide corresponding to the region containing the splice sequence, recognises only the upper band of the p68 doublet by both immunoprecipitation and Western blotting. These results are consistent with alternative splicing being responsible for the generation of the two forms of p68.
Amino Acid Sequence↗
ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform.
BACKGROUND: Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system. RESULTS: In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian. CONCLUSIONS: Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.
Alternative Splicing↗
Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development.
We report that the alternatively spliced isoforms of nonmuscle myosin heavy chain II-B (NHMC II-B) play distinct roles during mouse brain development. The B1-inserted isoform of NMHC II-B, which contains an insert of 10 amino acids near the ATP-binding region (loop 1) of the myosin heavy chain, is involved in normal migration of facial neurons. In contrast, the B2-inserted isoform, which contains an insert of 21 amino acids near the actin-binding region (loop 2), is important for postnatal development of cerebellar Purkinje cells. Deletion of the B1 alternative exon, together with reduced expression of myosin II-B, results in abnormal migration and consequent protrusion of facial neurons into the fourth ventricle. This protrusion is associated with the development of hydrocephalus. Restoring the amount of myosin II-B expression to wild-type levels prevents these defects, showing the importance of total myosin activity in facial neuron migration. In contrast, deletion of the B2 alternative exon results in abnormal development of cerebellar Purkinje cells. Cells lacking the B2-inserted isoform show reduced numbers of dendritic spines and branches. Some of the B2-ablated Purkinje cells are misplaced in the cerebellar molecular layer. All of the B2-ablated mice demonstrated impaired motor coordination.
Alternative Splicing↗