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PubMed1997

Human tenascin-C: identification of a novel type III repeat in oral cancer and of novel splice variants in normal, malignant and reactive oral mucosae.

Tenascin-C is a mosaic, linear glycoprotein that is up-regulated during many normal and pathological processes involving either cell migration or tissue morphogenesis, such as invasion of malignant cells and wound healing. Human tenascin-C contains 8 consecutive type III fibronectin (TNCfn) domains that are involved in alternative splicing and potentially generate a large number of isoforms that code for tenascin-C proteins with subtly different functions. Human tenascin-C splice variants were investigated by RT-PCR in a range of normal and pathological oral mucosal tissues. A novel, 9th human TNCfn domain involved in alternative splicing was identified. It shares 70% nucleic acid and 55% protein sequence homology with chicken TNCfn-ad2. As in avians, this novel repeat was located between TNCfn-B and TNCfn-ad1 and accordingly was designated human TNCfn-ad2. Human TNCfn-ad2 was detected in only 2 of 10 oral cancers. However, TNCfn-ad2 was absent from 40 normal, reactive, pre-malignant and other oral mucosal specimens investigated. Previous studies have described 8 splice variant transcripts for human tenascin-C. By systematic investigation we identified further novel splice variants for human tenascin-C. Furthermore, our results indicate that many potential splice variants probably do not exist in the tissues investigated. Thus, we have demonstrated that human tenascin-C transcripts generate a complex but selected repertoire of different alternative splice products.

Alternative Splicing↗
PubMed1999

Expression of alternatively spliced mdm2 transcripts correlates with stabilized wild-type p53 protein in human glioblastoma cells.

A puzzling finding in various human tumors, including glioblastoma multiforme (GBM), is the stabilization of wild-type (wt) p53 protein. The biological significance of this phenomenon and the mechanism by which it occurs are unexplained. Recent reports have revealed that mdm2 exerts its negative regulation on the p53 signal by directly binding p53 protein and thereby instigating its proteasomal degradation. mdm2 has been shown to exist in alternatively spliced forms in human ovarian and bladder carcinomas, and recently in GBM, with loss or disruption of its p53 binding domain. Here we report that alternatively spliced transcripts of mdm2 are present in 7 of 16 human GBM primary cell cultures and in the established GBM cell lines LN 229 and LN 18. Sequencing demonstrated loss of the amino terminal p53 binding domain in these alternatively spliced mdm2 transcripts, and an out-of-frame splicing in the majority of cases. A significant correlation between the presence of mdm2 splice variants and increased expression of wt p53 protein was observed. Furthermore, in the presence of an mdm2 splice variant, wt p53 stabilization occurred despite coincident MDM2 amplification. Our findings suggest that wt p53 protein stabilization may arise as a consequence of alternative splicing of mdm2. Such a mechanism might account for wt p53 protein accumulation in GBM cells, even in the presence of MDM2 gene amplification.

Binding Sites↗
PubMed1999

Regulation of fibronectin alternative splicing during peripheral nerve repair.

Wallerian degeneration following peripheral nerve injury is associated with increased production of fibronectin and other extracellular matrix molecules that are thought to enhance repair. We have shown previously that alternative splicing of the mRNA for fibronectin also changes following sciatic nerve lesions so as to reexpress forms of mRNA seen during embryogenesis. In the present study, we have examined the role of the regenerating axons in the regulation of this splicing. We have compared the patterns of fibronectin mRNA splicing seen in sciatic nerve development with that seen in cut nerves (that do not regenerate), crushed nerves (that regenerate successfully), and Schwann cells cultured in forskolin so as to mimic axonal signals. By using a reverse transcriptase polymerase chain reaction assay to examine all three regions of fibronectin mRNA splicing in a quantitative manner, we found that embryonic patterns of fibronectin mRNA splicing appear rapidly following injury and are not then altered by reestablishment of axons in the nerve. In addition, we found that forskolin has no effect on fibronectin mRNA splicing in cultured cells. We conclude that axonal signals do not regulate the pattern of fibronectin alternative splicing in peripheral nerve repair.

Alternative Splicing↗
PubMed1996

Dynamic organization of pre-mRNA splicing factors.

Studies from several laboratories during the past few years have increased our understanding towards the dynamic organization of pre-mRNA splicing factors in the mammalian cell nucleus. Many well characterized splicing factors have been localized in a speckled pattern in the cell nucleus. Upon the activation of RNA polymerase II transcription, splicing factors are recruited to the sites of transcription from sites of reassembly and/or storage. Nascent intron-containing RNA transcripts are spliced at the sites of transcription. The speckled distribution of splicing factors in the nucleus is altered when either transcription or pre-mRNA splicing activities are interrupted suggesting that the organization of the splicing machinery in the interphase nucleus is a direct reflection of the transcriptional activity of the cell.

Animals↗
PubMed1998

Coordinated expression of splice variants for luteinizing hormone receptor messenger RNA during the development of bovine corpora lutea.

We characterized splice variants for LH receptor mRNA in the bovine corpus luteum and examined the levels of expression of all the splice variants during the development of corpus luteum. Total RNA was extracted from bovine corpora lutea, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to amplify a part of the extracellular and transmembrane domains. As a result, four distinct bands were observed on analyses with polyacrylamide gel electrophoresis, and sequencing of the products revealed that the largest form (519 bp) was the full-length A form and that the three shorter forms (438, 253, and 172 bp) were its splicing variants. Splicing patterns of the 438-, 253-, and 172-bp forms are identical to the F, B, and G forms, respectively, found in the ovine ovary. In order to examine the level of expression of all these splice variants simultaneously, semiquantitative RT-PCR was set up and performed. The relative intensities for all four forms of LH receptor mRNA significantly increased (P < 0.05) from Stage I (Days 1-4; Day 1 = day of ovulation) to IIS (Days 5-10, CL weight < 4 g), and from Stage IIS to IIL (Days 5-10, CL weight > or = 4 g). The intensities of all forms for LH receptor mRNA decreased slightly, but not significantly, from Stage IIL to III (Days 11-17). The ratios of the levels for all the forms did not change significantly during the development of the corpus luteum. Determination of the steady-state levels of LH receptor mRNA by Northern blotting showed similar changes during the development of the corpus luteum to changes of the splice variants examined by the semiquantitative RT-PCR. These results suggest that at least three splicing variants of LH receptor mRNA (i.e., F, B, and G forms) exist in the bovine corpus luteum and that the levels of all the forms of LH receptor mRNA increased in a coordinated manner during the development of bovine corpus luteum.

Alternative Splicing↗
PubMed1992

Mutations causing defective splicing in the human hprt gene.

Ten intron mutations and one exon mutation giving rise to defective splicing in the human gene for hypoxanthine phosphoribosyl transferase (hprt) in T-lymphocytes have been characterized. The splicing mutants were detected by PCR amplification of hprt cDNA and direct sequencing. Nine of the mutants showed skipping of whole exons or parts of exons in the cDNA, one mutant had an inclusion of an intron sequence into the cDNA, and one mutant showed both inclusion of an intron sequence and skipping of exons as well as a normal cDNA. Genomic PCR and direct sequencing of the splice sites involved showed one deletion of three base pairs and 10 different single base alterations to be responsible for these splice alterations. One mutation in the last base pair of exon 6 causing skipping of the entire exon 6 was found, whereas an identical mutation in the last base pair of exon 2 caused no aberrant splicing. It was also found that a deletion mutation in the pyrimidine rich stretch of the acceptor site of intron 7 caused skipping of the entire exon 8, whereas a base substitution in the last base of intron 7 caused exclusion of only the first 21 base pairs of exon 8 as a result of the activation of a cryptic acceptor site in exon 8. The results show that many different types of mutations at several different sites can cause splicing errors in the hprt gene and that the sequence differences between the splice sites influence the possible spectrum of mutations in each site.

Base Sequence↗
PubMed2006

Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing.

A substantial proportion of MLH1 and MSH2 gene mutations in hereditary nonpolyposis colon cancer syndrome (HNPCC) families are characterized by nucleotide substitutions, either within the coding sequence (missense or silent mutations) or in introns. The question of whether these mutations affect the normal function of encoding mismatch DNA repair proteins and thus lead to the predisposition to cancer is determinant in genetic testing. Recent studies have suggested that some nucleotide substitutions can induce aberrant splicing by disrupting cis-transcription elements such as exonic enhancers (ESEs). ESE disruption has been proposed to be the mechanism that underlies the presumed pathological missense mutations identified in HNPCC families. To investigate the prevalence of aberrant splicing resulting from nucleotide substitutions, and its relevance to predicted ESEs, we conducted a systematic RNA screening of a series of 60 patients who carried unrelated exonic or intronic mutations in MLH1 or MSH2 genes. Aberrant splicing was found in 15 cases, five of which were associated with exonic mutations. We evaluated the link between those splicing mutations and predicted putative ESEs by using the computational tools ESEfinder and RESCUE-ESE. Our study shows that the algorithm-based ESE prediction cannot be definitely correlated to experimental observations from RNA screening. By using minigene constructs and in vitro transcription assay, we demonstrated that nucleotide substitutions are the direct cause of the splicing defect. This is the first systematic screening for the effect of missense and silent mutations on splicing in HNPCC patients. The pathogenic splicing mutations identified in this study will contribute to the assessment of "unclassified variants" in genetic counseling. Our results also suggest that one must use caution when determining the pathogenic effect of a missense or silent mutation using ESE prediction algorithms. Analysis at the RNA level is therefore necessary.

Adaptor Proteins, Signal Transducing↗
PubMed1983

Evidence against a scanning model of RNA splicing.

A cloned rabbit beta-globin gene was modified by introducing a DNA fragment containing the 5' splice region of the large intron upstream or downstream of its natural counterpart. Analogous constructions were carried out with the 3' splice region. The genes were linked to SV40 DNA, transiently expressed in HeLa cells and the transcripts analyzed by S1 mapping. In all cases, the splice site further removed from the intron was utilized to the complete exclusion of its counterpart. This finding argues persuasively against a simple scanning model of RNA splicing, in which the splicing enzyme(s) attaches at the 'donor' spliced region and moves along the intron until it encounters the closest 'acceptor' splice region. A model compatible with the currently known facts is presented.

Base Sequence↗
PubMed1983

Aberrant splicing of Drosophila alcohol dehydrogenase transcripts in Saccharomyces cerevisiae.

We have investigated the ability of transcripts of the Drosophila melanogaster alcohol dehydrogenase gene to be spliced in Saccharomyces cerevisiae. The alcohol dehydrogenase gene was cloned in S. cerevisiae on a 2 micron DNA-based vector and a hybrid yeast actin-Drosophila alcohol dehydrogenase gene was constructed to demonstrate that transcripts encoded on a 2 micron plasmid could be accurately and efficiently spliced. Transcription of the Drosophila gene occurred in yeast with and without a yeast promoter. The transcripts were polyadenylated and terminated approximately 600 nucleotides distal to the polyadenylation site used in Drosophila. In yeast no splicing of the two introns within the alcohol dehydrogenase coding sequence was detected. However, the leader sequence was apparently spliced using the same 3' splice site as is used in adult flies, but a different 5' splice site. This result may be partly explained by the existence in the Drosophila gene of a sequence which is believed to be required for splicing in S. cerevisiae.

Alcohol Dehydrogenase↗
PubMed1987

Differential splicing yields novel adenovirus 5 E1A mRNAs that encode 30 kd and 35 kd proteins.

In addition to the protein products of the adenovirus E1A 13S and 12S mRNAs, monoclonal antibodies specific for the E1A proteins immunoprecipitate polypeptides with relative mol. wt of 30,000 (30 kd) and 35,000 (35 kd) from extracts of infected cells. The 30 kd and 35 kd proteins are encoded by novel mRNAs referred to as the 10S and 11S mRNAs, respectively. These two mRNAs arise from differential splicing of the E1A precursor RNA. For the 10S mRNA, the precursor is spliced twice, once removing the region between nucleotides 637 and 854 and once between 974 and 1229. The splice between nucleotides 974 and 1229 is identical to the one used for the processing of the 12S mRNA. Synthesis of the 11S mRNA also utilizes two splicing events. One of these is identical to the 637/854 splice of the 10S mRNA, and the other removes the region between nucleotides 1112 and 1229, a splice junction also found in the 13S mRNA. All four mRNAs used the same reading frame and, therefore, code for related proteins. The products of the 10S and 11S mRNAs are identical to the products of the 12S and 13S mRNAs, respectively, except for an internal stretch of 27 amino acids removed by the 637/854 splice. Within this segment is a group of amino acid residues that is highly conserved between different adenovirus serotypes. Mutant adenoviruses in which the wild-type E1A sequences have been replaced with cDNA copies of the 10S or 11S mRNAs are defective for growth on HeLa cells suggesting that this region is important for viral growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus Early Proteins↗
PubMed1987

Self-splicing of a group II intron in yeast mitochondria: dependence on 5' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.

Base Sequence↗
PubMed1988

Splice site selection dominates over poly(A) site choice in RNA production from complex adenovirus transcription units.

The adenovirus late genes are organized in a complex transcription unit in which the production of multiple RNAs is controlled by specific RNA processing events. Adding to the complexity is the fact that the early E3 transcription unit is wholly contained within the late transcription unit. Within this overlapping region there are splicing events and poly(A) site choices specific to each transcription unit. Early in infection, there is near exclusive use of the E3 splice sites with little L4 poly(A) site use whereas the reverse is true late in infection. Using a plasmid containing an intact E3 transcription unit, we demonstrate that the L4 poly(A) site, located in the first E3 intron, is not used either in a mock-infected cell or a late infected cell; nor is it used if the same transcription unit is driven by the major late promoter. In addition, the nature of the poly(A) site appeared to be unimportant since the E3 poly(A) site was also not used in the intron if inserted in place of the L4 poly(A) site. Thus, splice site selection appears to dominate over poly(A) site choice. This conclusion was confirmed by the observation that deletion of either the E3 splice donor (5'ss) or the E3 splice acceptor (3'ss) allowed the use of the L4 poly(A) site. Finally, the L4 poly(A) site within the E3 intron was also used when additional sequence, including splicing signals from the L4 region and the tripartite leader, was inserted between the promoter and the E3 processing sites. It appears that splicing is the dominant event in governing production of E3/L4 RNAs.

Adenoviruses, Human↗
PubMed1989

Pre-mRNA splicing mutants of Schizosaccharomyces pombe.

A collection of temperature sensitive (ts-) mutants was prepared by chemical mutagenesis of a wild type Schizosaccharomyces pombe strain. To screen the ts- mutants for pre-mRNA splicing defects, an oligodeoxynucleotide that recognizes one of the introns of the beta-tubulin pre-mRNA was used as a probe in a Northern blot assay to detect accumulation of intron sequences. This screening procedure identified three pre-mRNA splicing mutants from 100 ts- strains. The three mutants are defective in an early step of the pre-mRNA splicing reaction; none accumulate intermediates. The precursors that accumulate at 37 degrees C are polyadenylated. Analysis of the splicing of another pre-mRNA showed that the mutations are not specific for beta-tubulin. The total RNA pattern in the three splicing mutants appears to be normal. In addition, the amounts of the spliceosomal snRNAs are not drastically changed compared to the wild type and splicing of pre-tRNAs is not blocked. Genetic analyses demonstrate that all three splicing mutations are tightly linked to the ts- growth defects and are recessive. Crosses among the mutants place them in three complementation groups. The mutants have been named prp1, prp2 and prp3.

Crosses, Genetic↗
PubMed1989

Cell type specific trans-acting factors are involved in alternative splicing of human fibronectin pre-mRNA.

ED-A and ED-B are facultative type III homologies of fibronectin, encoded by alternatively spliced exons, described in man and in rat. A hybrid alpha-globin-fibronectin minigene containing the ED-B region from the human gene has been transfected in human cell lines derived from various tissues, in order to study the processing of the generated precursor RNA in the different cell environments. In most tested lines the pre-RNA is alternatively spliced and produces two mature RNAs, with and without the ED-B exon, in different ratios that closely resemble the corresponding endogenous fibronectin RNAs. In a hepatoma cell line, Hep 3B, only one RNA is produced, in which the ED-B exon is absent; the same pattern of splicing is observed in liver. The data show that all the information required to produce accurate and regulated alternative splicing of the ED-B exon is contained in the fragment used and cell specific factors are necessary for the pre-RNA to be differentially spliced in the various cell lines. In contrast, expression in Hep 3B of a similar gene containing the ED-A area failed to reproduce the liver specific splicing pattern. Therefore regulation of ED-A processing is likely to involve different mechanisms to those responsible for control of ED-B splicing.

Base Sequence↗
PubMed1989

Evidence from complementation assays in vitro that U5 snRNP is required for both steps of mRNA splicing.

We have established an in vitro complementation system that has allowed us to investigate the role of individual purified snRNPs in the splicing of pre-mRNA molecules. For the preparation of snRNP-depleted nuclear extracts we have first removed the majority of endogenous snRNPs from the nuclear extracts by one passage over an anti-m3G column and then degraded the remaining snRNPs with micrococcal nuclease. The mixture of snRNPs U1, U2, U4/U6 and U5, obtained by anti-m3G immuno-affinity chromatography, was functionally active and able to restore the splicing of snRNP-depleted nuclear extracts. Mono-Q chromatography was used for further fractionation of the snRNPs U1-U6. This produced three fractions that were highly enriched in snRNPs U1 and U2, U5 and U4/U6 respectively. Conditions were found where addition of the [U1, U2] and the U4/U6 snRNP fractions to the snRNP-depleted nuclear extracts gave rise to the formation of splice intermediates in the absence of any 3' cleavage/exon 1-exon 2 product formation. Only when purified 20S U5 snRNPs were added did both steps of the splicing reaction occur efficiently. Our data suggest that U5 snRNP is absolutely required for the second step of splicing and is needed further for efficient initiation of the splicing reaction. The requirement for U5 snRNPs for splicing was corroborated by glycerol gradient sedimentation analysis of the respective reconstituted pre-mRNP complexes. Stable and efficient formation of 50-60S spliceosomes was observed only in the presence of all snRNPs.

Centrifugation, Density Gradient↗
PubMed1989

A conserved base pair within helix P4 of the Tetrahymena ribozyme helps to form the tertiary structure required for self-splicing.

Site-specific mutagenesis of the self-splicing Tetrahymena intron has been used to investigate the function of C109-G212, a conserved base pair in the P4 stem of group I introns. Mutation of C109 to G affects splicing only slightly, whereas mutation of G212 to A or C reduces the rate of splicing substantially (500-fold reduction in kcat/Km under standard in vitro splicing conditions for the G212C mutant). Splicing activity of the compensatory double mutant (C109G:G212C) is intermediate between those of the two single mutants. Thus, the stability of the P4 stem as well as the identity of the base at position 212 are important for self-splicing. Single and double mutants containing the G212C substitution have a decreased temperature optimum for self-splicing and are partially Mg2+ suppressible, both indicative of structural destabilization. Chemical structure mapping indicates that the mutations do not redirect the global folding of the RNA, but affect the structure locally and at one other site (A183) that is distant in the secondary structure. We propose that, in addition to its pairing in P4, G212 is involved in a base triplet or an alternate base pair that contributes to the catalytically active tertiary structure of the ribozyme.

Animals↗
PubMed1990

Self-splicing of the mobile group II intron of the filamentous fungus Podospora anserina (COI I1) in vitro.

The first intron of the mitochondrial gene coding for cytochrome oxidase subunit I (COI I1) of Podospora anserina can undergo self-splicing in vitro at high concentrations of NH4Cl or KCl. Under these conditions cleavage at the 5' splice junction takes place without branch formation probably via hydrolysis by water or OH- and the intron is released in a linear form. In vitro transcripts that contain mutated introns with large deletions in nonconserved domain IV comprising greater than 50% of the intronic sequence display a more efficient splicing reaction and, surprisingly, 5' cleavage via transesterification and lariat formation is re-established to a low degree under NH4Cl. In contrast to the self-splicing group II introns aI5 gamma and bI1 from yeast mitochondria cleavage at the 3' splice site of the Podospora intron is reduced and cleavage by hydrolysis in trans (i.e. exon reopening) is almost completely suppressed. Both observations could be interpreted as a result of unfavourable spatial conformations of the intron that (i) lead to a steric hindrance of the 5' exon to attack the 3' splice site in cis and (ii) block intron-dependent cleavage reaction of the ligated exons in trans. Alternatively, the possibility that a weak overall interaction of the postulated exon- with the corresponding intron-binding sites (EBS-IBS pairings) is responsible for the remarkable differences to the self-splicing reaction of other group II introns is discussed.

Ascomycota↗
PubMed1990

In vitro splicing of mutually exclusive exons from the chicken beta-tropomyosin gene: role of the branch point location and very long pyrimidine stretch.

The chicken beta-tropomyosin gene contains 11 exons, two of which are spliced into mRNA only in skeletal muscle. One pair of alternative exons, 6A and 6B, is found in the middle of the gene; they are spliced in a mutually exclusive manner. The non-muscle splice 6A-7 is by far the predominant in vitro reaction in a HeLa cell nuclear extract. A minor product is the 6A-6B splice, which is excluded in all tissues. This minor product results from the use of a branch point located 105 nt upstream of the 3' end of the intron separating exons 6A and 6B. The region between the branch point sequence and the final AG contains a stretch of approximately 80 pyrimidines. We have examined the role of the distance of the branchpoint to the 3' splice site and of the sequences between these two elements. Our results suggest that at least two cis-acting elements contribute to the mutual exclusivity of exons 6A and 6B. The intron between exons 6A and 6B is intrinsically poorly 'spliceable' both because the branch point is too far upstream of the 3' end of the intron to give efficient splicing and because of the particular sequence lying between this branch point and the 3' splice site.

Animals↗