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At least 1,621 records · Page 90Linked to original sources
PubMed1996

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster.

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2) d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.

Animals↗
PubMed2002

RT-PCR splicing analysis of the NF1 open reading frame.

Neurofibromatosis 1 (NF1) is an autosomal dominant condition whose molecular diagnosis is challenging because of the large size of the gene and the vast number of unique NF1 gene mutations. Some splicing and nonsense mutations have been shown to cause exon skipping. Recently, temperature-induced abnormal splicing has been found in NF1 in ex-vivo tissues. This prompted us to investigate the entire NF1 transcript for such aberrant splicing. We found several novel exon skips that appeared de novo or were present initially and increased in aged/cooled blood: exon 20, exons 20 and 21 combined, exon 33, exon 34, exon 37, exon 40, exon 45, exons 43 and 45 combined, part of exon 43, and the first codon of exon 12b. Some aberrant splice forms were undetectable when blood was drawn into Qiagen PAXgene tubes, rather than EDTA vacutainers, and we demonstrate how these aberrant splicing events are a potential pitfall for RNA-based NF1 mutation characterization. The same reverse transcription/polymerase chain reaction strategy was used to screen for novel NF1 alternative splicing in Schwann cells and seven other tissues. Even though no Schwann-specific alternative exons were identified, we found minor novel splicing isoforms differentially expressed such as skips of exon 37 and exon 40. Skipping of exon 43, part of exon 43, and the first codon of exon 12b were found in all tissues analyzed. These forms suggest greater tissue-based variability in the NF1 message than was previously thought and may indicate minor amounts of heterogeneity at the protein level.

Alternative Splicing↗
PubMed2004

Qualitative and quantitative analysis of the effect of splicing mutations in propionic acidemia underlying non-severe phenotypes.

In this work we analyze splicing mutations identified in propionic acidemia patients to clarify their functional effects and their involvement in the disease phenotype. Two mutations in the PCCA gene detected in homozygous patients and involving consensus splice sequences (IVS21+3del4 and IVS22-2A>G) were shown to produce some normal splicing in patients' cells, at very low levels, which were quantitated by real-time PCR methods, and which presumably are sufficient to moderate the phenotype. We have also analysed the effect of mutations c.653A>G and IVS10-11del6 in the PCCB gene present in heterozygous patients with mild phenotype. The c.653A>G mutation is located in the last codon of exon 6 and interferes with the correct spliceosomal assembly activating a cryptic splice site within exon 6, which leads to an in-frame six-nucleotide deletion (delV217-K218). Minigene analysis and sequence-specific hybridization probes using real-time PCR methods showed that no normally spliced transcript is detectable in the patients' fibroblasts. The IVS10-11del6 mutation shortens the polypyrimidine tract of the 3'-splice site of exon 11, resulting in exon skipping. Some normal transcript is detectable by allele-specific hybridization probes. These analyses suggest that, in some cases, the regulation of gene splicing can potentially play an important role in human disease influencing phenotypic parameters.

Amino Acid Metabolism, Inborn Errors↗
PubMed1989

Evidence that a nuclear matrix protein participates in premessenger RNA splicing.

The role of nuclear matrix proteins in premessenger RNA splicing has been investigated using antibodies raised against isolated rat liver nuclear matrix and cross-reactive with a 65-kDa HeLa cell nuclear matrix protein (IGA-65). IGA-65 is an internal nuclear matrix component which can be solubilized as a component of nuclear splicing extracts, by the action of endogenous ribonucleases, EDTA, and DTT during extract preparation. Preincubation of splicing extract with antibodies against IGA-65 (anti-IGA-65) inhibited in vitro splicing of exogenous adenovirus precursor RNA. Furthermore, assembly of precursor RNA into active spliceosome complexes was inhibited by pretreatment of extracts with anti-IGA-65, suggesting a role for IGA-65 during early spliceosome assembly. The IGA-65 present in splicing extracts was distinguishable from known U-snRNP and hnRNP proteins on protein gels. Furthermore, electrophoresis of splicing extract on native gels indicated that IGA-65 was present in protein complexes different from those containing U-snRNPs or hnRNP C protein. The data support identification of complexes containing IGA-65 as nuclear factors involved in pre-mRNA splicing and, by extension, suggest a role for the nuclear matrix during processing in vivo.

Antibodies↗
PubMed1986

Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem.

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.

Anticodon↗
PubMed1990

Alternative splicing of hepatitis B virus RNAs in HepG2 cells transfected with the viral DNA.

We identified a novel spliced RNA of 2.6 kb from a human hepatoma cell line HepG2 transfected with the hepatitis B virus (HBV) genome. The splicing acceptor site of the novel 2.6-kb RNA (position 489) was shown to be common to that of the previously described 2.1-kb spliced RNA which codes for an altered core antigen lacking the carboxy-terminal amino acid, cysteine. However, the donor site of the 2.6-kb RNA is different from any of the spliced RNA reported and located at 538 nucleotides (nt) downstream of the donor site of the 2.1-kb RNA. Introduction of single-base change mutations in the consensus sequence of the donor site of the 2.1-kb RNA maintained the splicing by using the cryptic donor site. The amount of the 2.6-kb spliced RNA was unchanged by these mutations. These results suggest independent regulations for the synthesis of the 2.1- and 2.6-kb spliced RNAs.

Amino Acid Sequence↗
PubMed1991

Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing.

Retroviruses splice only a fraction of their primary RNA transcripts to subgenomic mRNA. The unspliced RNA is transported to the cytoplasm, where it serves as genomic RNA as well as mRNA for the gag and pol genes. Deletion of sequences from the Rous sarcoma virus gag gene, which is part of the intron of the subgenomic mRNAs, was previously observed to result in an increase in the ratio of spliced to unspliced RNA. These sequences, which we termed a negative regulator of splicing (NRS), can be moved to the intron of a heterologous gene resulting in an accumulation of unspliced RNA in the nucleus. We have used such constructs, assayed by transient expression in chicken embryo fibroblasts, to define the minimal sequences necessary to inhibit splicing. Maximal NRS activity was observed with a 300-nt fragment containing RSV nts 707-1006; two noncontiguous domains within this fragment, one of which contains a polypyrimidine tract, were both found to be essential. The NRS element was active exclusively in the sense orientation in two heterologous introns tested and in both avian and mammalian cells. Position dependence was also observed, with highest activity when the NRS was inserted in the intron near the 5' splice site. The NRS element was also active at an exon position 136 nts upstream of the 5' splice site but not at sites further upstream. In addition, it did not affect the splicing of a downstream intron.

Animals↗
PubMed1992

Viral spliced RNA are produced, encapsidated and reverse transcribed during in vivo woodchuck hepatitis virus infection.

By the use of reverse transcription followed by polymerase chain reaction (RT-PCR), we have identified one shorter than full-length, pregenomic viral RNA species in liver samples of woodchucks chronically infected with the woodchuck hepatitis virus (WHV). The spliced WHV RNA of about 2.4 kb in length was cloned and partially sequenced. The splicing donor and acceptor sites of this novel RNA are located, respectively, 130 nucleotides downstream of the ATG initiation codon of the core gene and 21 nucleotides upstream of the initiation codon of the pre-S2 surface gene. The splicing event generates a new core-polymerase fusion protein and removes the terminal protein domain and the spacer region of the polymerase gene. A nucleotide probe specific for the splice junction was used following RT-PCR, to further confirm the existence of this spliced RNA in the liver of seven WHV-infected woodchucks. Deleted viral DNA molecules corresponding to the 2.4 kb spliced RNA were also detected in the liver and, to a lesser extent, in the serum of infected woodchucks, suggesting that this spliced RNA can be encapsidated and reverse-transcribed during the course of natural WHV infection.

Amino Acid Sequence↗
PubMed1981

Beta + thalassemia: aberrant splicing results from a single point mutation in an intron.

We have analyzed the molecular basis of beta + thalassemia by studying the expression of a cloned beta-globin gene in HeLa cells. This beta-globin gene was isolated from a beta + thalassemic patient and differs from the normal beta-globin genes by only a single point mutation within the first intron. The beta + thalassemic and the normal beta-globin genes were cloned into an SV40-pBR328 vector and introduced into HeLa cells by calcium phosphate coprecipitation. We assayed the RNA from these transfected HeLa cells by S1 nuclease mapping and cDNA sequencing to detect the nature of the defect in beta-globin gene expression. While the transcripts of the normal beta-globin gene are processed correctly, the first intron of the beta + thalassemic beta-globin gene is incorrectly spliced in about 90% of the mRNA because of an additional 3; splice site that has been created by the point mutation. This incorrectly spliced mRNA is effectively exported to the cytoplasm, where it would conceivably be translated to give a truncated globin chain of 35 amino acids. The remaining 10% of the mRNA transcribed from the beta+ thalassemic globin gene is correctly spliced and can therefore be translated to give normal beta-globin. In addition to the incorrect splicing of the first intron, the splicing of both introns is retarded, which results in the accumulation of unspliced pre-mRNA. This suggests that removal of the first intron might facilitate splicing of the second intron.

Base Sequence↗
PubMed1984

Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate.

Four RNA products have been characterized from a soluble system that accurately splices purified substrate RNA. The labeled substrate RNA contained the first and second exons of the major late transcription unit of adenovirus 2 and a shortened form of the first intervening sequence. One of these RNA products was the excised intervening sequence which accumulated quantitatively with the level of splicing. This RNA species has an unusual structure and is modified at internal sites. A potential intermediate in the splicing pathway which is cleaved at the 5' splice site of the first exon has been isolated. This RNA species is also modified at sites within the first intervening sequence. These results suggest that the splicing of mRNA precursors may involve sites in the intervening sequence, cleavage at the 5' splice site, cleavage at the 3' splice site, and ligation of the two exons.

Adenoviruses, Human↗
PubMed1985

Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing.

Biochemical components (splicing factors) interact with specific intron regions during pre-mRNA splicing in vitro. The pre-mRNA specifically associates with factors at both the branch point and the 5' splice site and these RNA-factor interactions are maintained in the intron-containing RNA processing products. The first detectable event, the ATP-dependent association of a factor (or factors) with the branch point, is mediated by at least one factor containing an essential nucleic acid component. Mutant RNA substrates that lack either the 5' splice site or the vast majority of exon sequences can still associate with the branch point binding factor(s). However, this branch point-factor interaction does not occur with a mutant RNA substrate that contains the branch point but that lacks the 3' splice site consensus sequence. These results suggest that selection of the 3' splice site accompanied by the association of a factor with the branch point may be the initial step in mammalian pre-mRNA splicing.

Adenosine Triphosphate↗
PubMed1986

Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor.

Oligonucleotide-directed mutagenesis has been used to alter highly conserved sequences within the intervening sequence (IVS) of the Tetrahymena large ribosomal RNA precursor. Mutations within either sequence element 9L or element 2 eliminate splicing activity under standard in vitro splicing conditions. A double mutant with compensatory base changes in elements 9L and 2 has accurate splicing activity restored. Thus, the targeted nucleotides of elements 9L and 2 base-pair with one another in the IVS RNA, and pairing is important for self-splicing. Mutant splicing activities are restored by increased magnesium ion concentrations, supporting the conclusion that the role of the targeted bases in splicing is primarily structural. Based on the temperature dependence, we propose that a conformational switch involving pairing and unpairing of elements 9L and 2 is required for splicing.

Animals↗
PubMed1988

The C. elegans trans-spliced leader RNA is bound to Sm and has a trimethylguanosine cap.

mRNA splicing in C. elegans is unusual: most introns are very short (approximately 50 bases), and many mRNAs receive a leader by trans-splicing. The donor in trans-splicing is a 94 nucleotide molecule, termed the leader RNA, that contributes its 5' 22 nucleotides to a variety of mRNAs. We show here that C. elegans has the usual snRNAs, which presumably catalyze the splicing reactions. As expected, they are bound to the Sm antigen and have 2,2,7-methylguanosine caps. Remarkably, the trans-spliced leader RNA is also Sm-associated and has this special cap. Hence, a molecule discovered as a substate of splicing has properties of molecules heretofore known only to facilitate splicing of other RNAs. Mature mRNAs that have received the leader evidently lack 2,2,7-methylguanosine caps, suggesting that these caps are removed or altered during processing.

Animals↗
PubMed1988

A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly.

Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Here we show that unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. We identify a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, we demonstrate that U2AF binds to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing.

Binding Sites↗
PubMed1990

Destruction of U2, U4, or U6 small nuclear RNA blocks trans splicing in trypanosome cells.

We have used permeable cells of Trypanosoma brucei to analyze the role of snRNAs in the trans splicing process. Degradation of U2, U4, or U6 snRNA by site-directed cleavage with complementary deoxyoligonucleotides and RNAase H inhibits trans splicing of the spliced leader (SL) RNA and newly synthesized alpha-tubulin pre-mRNAs. Cleavage of U snRNAs abolishes the appearance of putative trans splicing reaction intermediates and products, namely, linear branched molecules consisting of the SL intron joined to high molecular weight RNA (Y structures) and free SL intron. This indicates that U snRNAs are required for an early step in trans splicing. alpha-tubulin transcripts synthesized in the absence of trans splicing are unstable, suggesting that the addition of the SL sequence stabilizes pre-mRNAs against degradation. Our results provide direct evidence for the participation of U2 and U4/U6 snRNPs in trans splicing.

Animals↗
PubMed1991

A U-rich tract enhances usage of an alternative 3' splice site in yeast.

There has been a long-standing belief that the mechanisms of mammalian and yeast splicing differ fundamentally in their requirement for a pyrimidine-rich motif preceding the 3' splice site. Using an in vivo assay, we have tested the influence of uridine content on competition between alternative 3' splice sites in yeast. We find that a uridine-rich tract preceding a PyAG greatly enhances its ability to compete as a splice acceptor. Moreover, a proximal PyAG is often overlooked if a more distal PyAG occurs in a superior sequence context; this observation cannot be accounted for by simple scanning models. Finally, we show that a distal (greater than 30 nucleotide) 3' splice site that is not preceded by uridines is a poor substrate for the second step of splicing; this argues that recognition of a uridine-rich motif is required for effective identification and utilization of distant splice sites.

Actins↗
PubMed1992

Mutations at the 3' splice site can be suppressed by compensatory base changes in U1 snRNA in fission yeast.

U1 snRNA is an essential splicing factor known to base pair with 5' splice sites of premessenger RNAs. We demonstrate that pairing between the universally conserved CU just downstream from the 5' junction interaction region and the 3' splice site AG contributes to efficient splicing of Schizosaccharomyces pombe introns that typify the AG-dependent class described in mammals. Strains carrying mutations in the 3' AG of an artificial intron accumulate linear precursor, indicative of a first step block. Lariat formation is partially restored in these mutants by compensatory changes in nucleotides C7 and U8 of U1 snRNA. Consistent with a general role in fission yeast splicing, mutations at C7 are lethal, while U8 mutants are growth impaired and accumulate linear, unspliced precursor to U6 snRNA. U1 RNA-mediated recognition of the 3' splice site may have origins in analogous intramolecular interactions in an ancestral self-splicing RNA.

Base Composition↗
PubMed1993

3' splice site recognition in S. cerevisiae does not require base pairing with U1 snRNA.

The conserved nucleotides 9 and 10 of U1 small nuclear RNA (snRNA) have been proposed to base pair with either 5' exon or 3' splice site sequences. In S. pombe, U1 snRNA pairing with the conserved 3' splice site is required for the first step of splicing and viability. In contrast, we show that S. cerevisiae U1 mutants at positions 9 and 10 are fully functional. Splicing of several genes is normal in these strains, ruling out an essential base pairing between U1 snRNA and 3' splice sites. U1 snRNA positions 9 and 10 are shown to be involved in 5' splice site selection through their interaction with exon sequences. Our results demonstrate that some snRNA-pre-mRNA interactions are not evolutionarily conserved and that 3' splice site recognition occurs by different mechanisms in various organisms.

Base Sequence↗