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PubMed2003

Impact of splice-site mutations of the human MDR1 cDNA on its stability and expression following retroviral gene transfer.

The multidrug resistance 1 (MDR1) gene transfer to hematopoietic cells for protection against cytotoxic drugs has received considerable attention in gene therapy. However, ectopic expression of MDR1 from retroviral vectors has been hampered by its genetic instability resulting from cryptic splice sites within the cDNA. We have evaluated the efficiency of retroviral MDR1 vectors with introduced mutations of the MDR1 cryptic splice donor (cSD) located at nucleotide +339 and of the cryptic splice acceptor (cSA) at nucleotide +2319 of the cDNA. Sequence alterations of the cSD reduced the expression of MDR1 P-glycoprotein (P-gp), even when generated as silent mutations. A silent mutation of the cSA reduced the splicing activity shifting the splice acceptor site one base downstream; however, it significantly improved the expression of P-gp. The incidence of wild-type MDR1 pregenome splicing was markedly reduced when vectors were produced in human 293 packaging cells as opposed to murine PG13 and GP+envAm12. We conclude that complete splice correction of MDR1 in retroviral vectors may only be achieved with extensive alterations of the cDNA or neighboring vector sequences and that the splicing is significantly influenced by the choice of the packaging cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

GTP cyclohydrolase I mRNA: novel splice variants in the slime mould Physarum polycephalum and in human monocytes (THP-1) indicate conservation of mRNA processing.

GTP cyclohydrolase I (EC 3.5.4.16) is the first enzyme in the biosynthesis of tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)-biopterin] in mammals and of folic acid in bacteria. Here we have characterized the GTP cyclohydrolase I gene structure and two mRNA species from Physarum polycephalum, an acellular slime mould that synthesizes H(4)-biopterin and metabolites of the folic acid biosynthetic pathway. Its GTP cyclohydrolase I gene consists of seven exons, and the two GTP cyclohydrolase I cDNA species isolated from Physarum encode for proteins with 228 (25.7 kDa) and 195 (22.1 kDa) amino acids. Furthermore, we identified two previously undescribed mRNA species in interferon-gamma-treated human myelomonocytoma cells (THP-1) in addition to the cDNA coding for the fully functional 250-residue (27.9 kDa) protein, which is identical with that in human phaeochromocytoma cells. One of the new splice variants codes for a 233-residue (25.7 kDa) protein, whereas the other codes for the full-length protein but is alternatively spliced within the 3'-untranslated region. In heterologous expression, the shorter proteins of Physarum as well as of THP-1 cells identified here are degraded by proteolysis. Accordingly, only the 27.9 kDa protein was detectable in Western blots from THP-1 cell extracts. Quantification of GTP cyclohydrolase I mRNA species in different human cell types with and without cytokine treatment showed that in addition to the correct mRNA the two splice variants isolated here, as well as the two splice variants known from human liver, are strongly induced by cytokines in cell types with inducible GTP cyclohydrolase I (THP-1, dermal fibroblasts), but not in cell types with constitutive GTP cyclohydrolase I expression (SK-N-SH, Hep-G2). As in human liver, splicing of the new mRNA variant found in THP-1 cells occurs at the boundary of exons 5 and 6. Strikingly, the 195-residue protein from Physarum is alternatively spliced at a homologous position, i.e. at the boundary of exons 6 and 7. Thus alternative splicing of GTP cyclohydrolase I at this position occurs in two species highly distant from each other in terms of evolution. It remains to be seen whether variant proteins encoded by alternatively spliced GTP cyclohydrolase I mRNA transcripts do occur in vivo and whether they participate in regulation of enzyme activity.

Alternative Splicing↗
PubMed2005

Deletions within a CA-repeat-rich region of intron 4 of the human SP-B gene affect mRNA splicing.

Length variants within a CA-repeat-rich region of intron 4 of the human SP-B (pulmonary surfactant protein-B) gene are associated with several lung diseases. The hypothesis that SP-B intron 4 affects mRNA splicing was studied. SP-B minigenes containing exons 1-6 with a normal-sized intron 4 (pBi4normal) or intron 4 containing deletions (pBi4del) of 193, 211, 264 or 340 bp were expressed in CHO (Chinese hamster ovary) cells by transient transfection. Two forms of SP-B transcripts, normal and incompletely spliced, were detected. With pBi4normal, normal-sized SP-B mRNA was the predominant form and a very low amount of incompletely spliced mRNA was present, whereas with the pBi4del variants the amount of normal SP-B mRNAs was lower and the amount of incompletely spliced mRNA was relatively high. Reverse transcription-PCR results and sequencing data indicated that the incompletely spliced SP-B RNA contained intron 4 sequence, and this incompletely spliced RNA was also observed in normal lung. Lung cancer tissues with intron 4 deletions exhibited a larger amount of abnormally spliced RNAs compared with normal lung tissue or cancerous tissue with normal-sized intron 4. The results indicate that intron 4 length variants affect SP-B mRNA splicing, and that this may contribute to lung disease.

Animals↗
PubMed1994

Regulation of splicing is responsible for the expression of the muscle-specific 2a isoform of the sarco/endoplasmic-reticulum Ca(2+)-ATPase.

Tissue-specific alternative processing of sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) transcripts generates functionally different Ca2+ pump isoforms in muscle compared with non-muscle tissues. In non-muscle cells, the SERCA2 pre-mRNA can be polyadenylated at a site located between the donor and acceptor splice site of an intron which is only removed in muscle tissues. To define the cis-active elements involved in differential processing, we constructed a minigene (pCM beta SERCA2) containing the 3' end of the SERCA2 gene. When stably transfected into a myogenic cell line, minigene transcripts were differentially processed depending on the differentiation state of the cells. This proves that the essential elements required for regulated processing are present in the construct. Furthermore, co-transfection of the pCM beta SERCA2 minigene and a myogenin expression vector in a fibroblast cell line induced muscle-specific splicing of transcripts from pCM beta SERCA2. This shows that trans-acting factor(s) responsible for muscle-specific processing can be induced by one of the important regulatory genes of muscle differentiation. Inactivation of the non-muscle poly(A) site did not induce splicing in non-muscle cells. This excludes a simple competition model between splicing and polyadenylation, but it is consistent with splicing being very inefficient in non-muscle cells. Moreover, splicing could be induced in non-muscle cells by optimizing the muscle-specific donor splice site and/or by shortening the intron length. We therefore propose that expression of the muscle-specific SERCA2a isoform is the result of activation of an otherwise inefficient splicing process.

Animals↗
PubMed1997

Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript.

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) requires activation of an inefficient optional splice process at the 3' end of the primary gene transcript. The sequence elements required for this regulated splice event were studied by modifying a minigene containing the 3' end of the SERCA2 gene. An important requirement appears to be a strong muscle-specific acceptor site, as replacing it by a weak one prevented the induction of muscle-type splicing during myogenic differentiation. The induction of muscle-type splicing did not depend on positive cis-active sequences in the muscle-specific exon. On the other hand, replacement of a broad region around the acceptor site dramatically deregulated the expression pattern, as this modification strongly induced muscle-type splicing in undifferentiated muscle cells and in fibroblasts. This cis-active region is also involved in the suppression of the neuronal type of splicing. Furthermore selective replacement of the acceptor site as well as deletions or replacements in the muscle-specific exon induced muscle-type splicing to various extents in undifferentiated myogenic cells. Therefore sequence elements in the distal part of the optional intron and in the muscle-specific exon contribute to the suppression of muscle-specific SERCA2 splicing.

Alternative Splicing↗
PubMed1999

Cloning and characterization of two neural-salient serine/arginine-rich (NSSR) proteins involved in the regulation of alternative splicing in neurones.

BACKGROUND: In neurones, alternative splicing regulates the functions of many gene products. However, the molecular basis of neural-specific splicing, and how splicing regulation is modulated in different neurones remains to be determined. RESULTS: We cloned two new SR proteins, Neural-salient SR proteins (NSSR) 1 and 2, which are present at higher levels in brain and testis. During the differentiation, NSSR 1 is detected only in the neuronal stage. Both the purified recombinant NSSR 1 and 2 proteins enhance the in vitro splicing activity of nuclear extract. Moreover, recombinant NSSR 1 protein enhances the assembly of ribonucleoprotein complexes with S100 fraction. Over-expression of NSSR 2 prevents the inclusion of either the Flip or Flop exons in the splicing of the GluR-B gene, resulting in an increase in the abnormal exon-skipping product. In contrast, transient transfection with NSSR 1 promotes the inclusion of the Flip exon so that the abnormal product is spliced to the mature spliced form. This suppression of exon skipping by NSSR 1 is observed even with co-transfection of NSSR 2. CONCLUSIONS: NSSR 1 and 2 were cloned from mouse cDNA libraries. Results indicate that NSSR 1 may play a crucial role in the regulation of alternative splicing in neurones.

Alternative Splicing↗
PubMed2000

Differential mRNA expression of the two mineralocorticoid receptor splice variants within the human brain: structure analysis of their different DNA binding domains.

In human brain tissue, cortisol action, at basal concentrations, is mediated by the mineralocorticoid receptor (MR). An in-frame insertion of 12 bp in the MR-DNA-binding domain due to alternative splice site usage between exons 3 and 4 results in an MR mRNA splice variant (MR+4) encoding a receptor protein with four additional amino acids compared to the wild-type MR protein. To elucidate the questions of sex, age, and/or tissue dependent differences of the relative amount of the two mRNA subtypes, we examined 131 fresh human brain tissue samples from temporal and frontal lobe or hippocampus. One hundred and twenty samples were obtained from patients with epilepsy and 11 samples from patients with brain tumours. A small but significant difference of the MR+4 mRNA splice variant proportions in cortex (9.5 +/- 0.8%) and subcortical white matter (6.6 +/- 0.7%) of the temporal lobe could be detected, indicating differential MR splice variant expression within these brain areas. Moreover, the splice variant ratios in samples of the temporal lobe cortex collected from patients with epilepsy differed from samples of patients with brain tumours. These data point to an altered expression of the MR splice variants in epilepsy, and strengthen the supposition of a tissue specific alternative splicing of the MR mRNA. The frequent occurrence of the MR+4 transcript raises the question of its functional significance. For this reason, an MR+4 DNA-binding-domain structure model was generated by computer-based homology modelling based on the known glucocorticoid receptor structure. The data obtained revealed no distorting effect of the inserted four amino acids on the adjacent secondary structures, thereby suggesting that both zinc fingers retain their function. The resulting structure of the MR+4 model leads to the supposition that the receptor retains its function. Moreover, databank analysis with respect to this kind of steroid receptor variation and our own sequence data of the closely related progesterone receptor sustained the hypothesis that only corticosteroid receptors were affected by this alternative splicing event.

Adult↗
PubMed1998

Aberrant splicing of intron 1 leads to the heterogeneous 5' UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content.

We have previously reported that three distinct patterns of waxy (Wx) gene transcript accumulation were present in 31 rice cultivars. The cultivars with high amylose content (group I) contain a 2.3 kb mature Wx mRNA, cultivars with intermediate amylose content (group II) produce both a 3.3 kb Wx pre-mRNA, which contains intron 1, and the 2.3 kb Wx mature mRNA, and cultivars with no amylose (group III) accumulate only the 3.3 kb Wx pre-mRNA. Analyses of the cDNAs reveals that four splice donor sites and three splice acceptor sites in intron 1 give rise to six splicing patterns in 2.3 kb Wx mRNA of group II cultivars. In addition, aberrant intron 1 excision causes either deletion of 4 or 5 nucleotides, or addition of 7 and 13 nucleotides at the junction of exon 1 and exon 2 of the 2.3 kb mRNA. In contrast, only one normal splicing pattern (one splice donor site and one splice acceptor site) was found in the 2.3 kb mRNA of group I cultivars. Nucleotide sequences of the Wx intron 1 in group I and group II cultivars differ by 16 individual bases. We suggest that these deletions or additions contribute to inefficient splicing of intron 1 from the 3.3 kb Wx pre-mRNA, as well as an aberrant splicing of the Wx intron 1 to produce the 2.3 kb mRNA with a heterogeneous 5' untranslated region (5'-UTR). As a consequence, the total amount of translatable Wx mRNA, and therefore the Wx protein and amylose content, are reduced in the group II cultivars compared with the group I cultivars.

Alternative Splicing↗
PubMed2005

[Alternative mRNA splicing, pathology and molecular therapeutics].

Pre-mRNA splicing operates towards at least 95 % of the transcript pool. It is subjected to a large number of variations, collectively regrouped under the term of alternative mRNA splicing, which occurs, on average, 6 to 8 times per pre-mRNA molecule. Consequently, many more proteins may be encoded from a single gene, which may satisfy a physiological need, or mark a pathological adaptation. The identification of mutations in sequences required for splicing, both constitutive and alternative, or for their control, has permitted to determine the causes of qualitative or quantitative variations in transcript levels associated with inherited diseases or cancer development. A number of molecular approaches have been undertaken to try to compensate for the effect of deleterious splicing mutations and to restore, at least in part, sufficient amounts of either the normal or a surrogate transcript. These include overexpression of splicing proteins, improvement of their activity by post-translational modification, splice-site increased or decreased usage, and RNA-mediated trans-splicing. Using such approaches, phenotypic improvements have been obtained in animal models, carrying new hopes for the development of therapeutic strategies aimed at correcting both inherited and acquired diseases that involve pre-mRNA splicing defects.

Alternative Splicing↗
PubMed2004

Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes.

The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones.

Alternative Splicing↗
PubMed2005

Evidence of functional selection pressure for alternative splicing events that accelerate evolution of protein subsequences.

Recently, it was proposed that alternative splicing may act as a mechanism for opening accelerated paths of evolution, by reducing negative selection pressure, but there has been little evidence so far that this mechanism could produce adaptive benefit. Here, we use metrics of very different types of selection pressures [e.g., against amino acid mutations (Ka/Ks), against mutations at synonymous sites (Ks), and for protein reading-frame preservation] to address this question by genomewide analyses of human, chimpanzee, mouse, and rat. These data show that alternative splicing relaxes Ka/Ks selection pressure up to 7-fold, but intriguingly this effect is accompanied by a strong increase in selection pressure against synonymous mutations, which propagates into the adjacent intron, and correlates strongly with the alternative splicing level observed for each exon. These effects are highly local to the alternatively spliced exon. Comparisons of these four genomes consistently show an increase in the density of amino acid mutations (Ka) in alternatively spliced exons and a decrease in the density of synonymous mutations (Ks). This selection pressure against synonymous mutations in alternatively spliced exons was accompanied in all four genomes by a striking increase in selection pressure for protein reading-frame preservation, and both increased markedly with increasing evolutionary age. Restricting our analysis to a subset of exons with strong evidence for biologically functional alternative splicing produced identical results. Thus alternative splicing apparently can create evolutionary "hotspots" within a protein sequence, and these events have evidently been selected for during mammalian evolution.

Alternative Splicing↗
PubMed1986

Alternative branch points are selected during splicing of a yeast pre-mRNA in mammalian and yeast extracts.

Pre-mRNA splicing in yeast and higher eukaryotes proceeds by similar pathways, in which a probable splicing intermediate and the excised intron are in a lariat configuration. To compare the pre-mRNA splicing mechanisms in yeast and higher eukaryotes, we have analyzed the RNA products resulting from in vitro processing of a yeast intron-containing pre-mRNA in HeLa cell and yeast extracts. In yeast, the RNA branch (2'-5' phosphodiester bond) of the RNA lariat forms at the third adenosine of the TACTAAC box in vivo and in vitro. In contrast, in the HeLa cell extract, the yeast pre-mRNA is accurately spliced, but the RNA lariats contain RNA branches located significantly closer to the 3' splice site than the TACTAAC box. In yeast, mutant pre-mRNAs that lack the TACTAAC box are not spliced in vivo or in vitro. However, these same mutant pre-mRNAs are accurately spliced in the HeLa cell extract. Therefore, although pre-mRNA splicing in yeast and higher eukaryotes proceeds by the same basic pathway, there are substantial differences in the specificity of the biochemical components that mediate the formation of the RNA processing products.

Base Sequence↗
PubMed1986

Fractionation and characterization of a yeast mRNA splicing extract.

We have fractionated a yeast whole cell extract that can accurately splice synthetic actin and CYH2 pre-mRNAs. Three fractions, designated I, II, and III, have been separated by use of ammonium sulfate fractionation and chromatography on heparin agarose. Each fraction alone has no splicing activity. Fractions I and II allow the first step of the splicing reaction to proceed, giving rise to the splicing intermediates, free exon 1, and intron-exon 2. Addition of fraction III completes the reaction. Micrococcal nuclease treatment of the whole cell extract or of either fraction I or II abolished splicing activity, indicating that fractions I and II have RNA moieties that are required in the splicing reaction. The nature of the RNAs was examined using antibodies directed against the trimethylated cap structure unique to small nuclear RNAs. Preincubation of the whole cell extract with protein A-Sepharose coupled to trimethylated cap antibody abolished splicing activity. This indicates that at least one essential RNA component contains a trimethyl cap. Thus, in yeast as in mammalian systems, small nuclear RNAs are involved in mRNA splicing.

Cell Fractionation↗
PubMed1987

Accurate 5' splice-site selection in mouse kappa immunoglobulin light chain premessenger RNAs is not cell-type-specific.

In mature mouse B lymphocytes, immunoglobulin kappa light chain transcripts contain an intervening sequence separating the recombined variable (V) plus joining (J) exon from the distant constant (C) exon. After V-J recombination, this intervening sequence can include as many as three unused but very similar J-region 5' splice sites. Each of these sites is potentially functional if the gene is appropriately recombined. It is unclear how the splicing machinery distinguishes among these 5' splice sites, always choosing the most upstream site. We used synthetic transcripts of kappa gene sequences containing J3 and J4 in both the germ-line and the recombined configurations to study the pattern of 5' splice-site selection in vitro. We find that both HeLa cell and lymphocyte nuclear extracts fail to discriminate between the J3- and J4-region 5' splice sites. In contrast, after transfection into HeLa cells, similar kappa light chain transcripts are spliced correctly at the most upstream 5' splice site--that which is used in kappa-producing cells. We conclude that accurate 5' splice-site selection in the mouse kappa light chain is neither cell-type- nor species-specific. Potential mechanisms for this controlling step in gene expression are discussed.

Animals↗
PubMed1988

Possible evolution of splice-junction signals in eukaryotic genes from stop codons.

Splice-junction sequence signals are strongly conserved structural components of eukaryotic genes. These sequences border exon/intron junctions and aid in the process of removing introns by the RNA splicing machinery. Although substantial research has been undertaken to understand the mechanism of splicing, little is known about the origin and evolution of these splice signal sequences. Based on the previously published theory that the primitive genes evolved in pieces from primordial genetic sequences to avoid the interfering stop codons, a "stop-codon walk" mechanism is proposed in this paper to have assisted in the evolution of coding genes. This mechanism predicts the presence of stop codons in splice-junction signals inside the introns. Evidence of the consistent presence of stop codons in the splice-junction signals, in a position where they are expected, is shown by the analysis of codon statistics in these signal sequences in the GenBank databank. The results suggest that the splice-junction signals may have evolved from stop codons as a consequence of a selective pressure to avoid stop codons during the original evolution of coding genes. They also suggest that other splice signals within the introns, such as the branch-point sequence, may have evolved from stop codons for similar reasons.

Biological Evolution↗
PubMed1991

Reconstitution of a group I intron self-splicing reaction with an activator RNA.

The self-splicing rRNA intron of Tetrahymena thermophila belongs to a subgroup of group I introns that contain a conserved extra stem-loop structure termed P5abc. A Tetrahymena mutant precursor RNA lacking this P5abc is splicing-defective under standard conditions (5 mM MgCl2/200 mM NH4Cl, pH 7.5) in vitro. However, the mutant precursor RNA by itself is capable of performing the self-splicing reaction without P5abc under different conditions (15 mM MgCl2/2 mM spermidine, pH 7.5). We have investigated the functional role of the P5abc in the mechanism of the self-splicing reaction. When an RNA consisting of the P5abc but lacking the rest of the Tetrahymena intron is incubated with the mutant precursor, the self-splicing reaction proceeds highly efficiently under standard conditions (5 mM MgCl2/200 mM NH4Cl, pH 7.5). Two steps of the bimolecular self-splicing reaction can be performed accurately by a shortened precursor RNA containing all essential components required in the self-splicing reaction and an activator RNA consisting of the P5abc. Gel-mobility-shift assays suggest that two molecules associate by a direct RNA-RNA interaction during the splicing reaction. The results imply that there might exist other small RNAs whose role is to activate ribozymes.

Animals↗
PubMed1991

Alternative splicing and genomic structure of the Wilms tumor gene WT1.

The chromosome 11p13 Wilms tumor susceptibility gene WT1 appears to play a crucial role in regulating the proliferation and differentiation of nephroblasts and gonadal tissue. The WT1 gene consists of 10 exons, encoding a complex pattern of mRNA species: four distinct transcripts are expressed, reflecting the presence or absence of two alternative splices. Splice I consists of a separate exon, encoding 17 amino acids, which is inserted between the proline-rich amino terminus and the zinc finger domains. Splice II arises from the use of an alternative 5' splice junction and results in the insertion of 3 amino acids between zinc fingers 3 and 4. RNase protection analysis demonstrates that the most prevalent splice variant in both human and mouse is that which contains both alternative splices, whereas the least common is the transcript missing both splices. The relative distribution of splice variants is highly conserved between normal fetal kidney tissue and Wilms tumors that have intact WT1 transcripts. The ratio of these different WT1 mRNA species is also maintained as a function of development in the mouse kidney and in various mouse tissues expressing WT1. The conservation in structure and relative levels of each of the four WT1 mRNA species suggests that each encoded polypeptide makes a significant contribution to normal gene function. The control of cellular proliferation and differentiation exerted by the WT1 gene products may involve interactions between four polypeptides with distinct targets and functions.

Amino Acid Sequence↗
PubMed1993

Inverse splicing of a group II intron.

I describe the self-splicing of an RNA that consists of exon sequences flanked by group II intron sequences. I find that this RNA undergoes accurate splicing in vitro, yielding an excised exon circle. This splicing reaction involves the joining of the 5' splice site at the end of an exon to the 3' splice site at the beginning of the same exon; thus, I term it inverse splicing. Inverse splicing provides a potential mechanism for exon scrambling, for exon deletion in alternative splicing pathways, and for exon shuffling in gene evolution.

Alternative Splicing↗