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At least 1,603 records · Page 89Linked to original sources
PubMed1991

A novel splicing factor is an integral component of 200S large nuclear ribonucleoprotein (InRNP) particles.

In previous studies we have shown that nuclear transcripts of several pre-mRNAs can be released from nuclei of mammalian cells in the form of large nuclear ribonucleoprotein (InRNP) particles. By electron microscopy, these particles appeared as compact composite structures, 50 nm in diameter, which invariably sedimented at the 200S region in sucrose gradients. In order to identify putative protein splicing factors associated with the 200S InRNP particles, a panel of monoclonal antibodies directed against these particles were screened for their ability to inhibit splicing of pre-mRNA in vitro. In this study we have focused on a nuclear protein of 88 kd in molecular weight, which is an integral component of the InRNP complex and is recognized by monoclonal antibodies from a specific clone. This protein has been identified here as a novel splicing factor by, (i) antibody inhibition of splicing in vitro and (ii) depletion of splicing activity from HeLa cell nuclear extract after removing the 88 kd polypeptide by immunoadsorption, and complementation of the depleted activity with an affinity-purified 88 kd antigen. This splicing factor has further been shown to be required for the assembly of an active splicing complex.

Animals↗
PubMed1994

Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport.

The influenza virus RNA segment 8 encodes two proteins, NS1 and NS2, by differential splicing. The collinear transcript acts as mRNA for NS1 protein, while the spliced mRNA encodes NS2 protein. The splicing of NS1 mRNA was studied in cells transfected with a recombinant plasmid that has the cDNA of RNA segment 8 cloned under the SV40 late promoter and polyadenylation signals. As described for influenza virus-infected cells, NS1 mRNA was poorly spliced to yield NS2 mRNA. However, inactivation of the NS1 gene, but not the NS2 gene, led to a substantial increase in the splicing efficiency, as shown by the relative accumulations of NS1 and NS2 mRNAs. This effect was not specific for NS1 mRNA, since the splicing of the endogenous SV40 early transcript was altered in such a way that t-Ag mRNA was almost eliminated. These changes in the splicing pattern coincided with a strong inhibition of the mRNA nucleocytoplasmic transport. Both NS1 and NS2 mRNAs were retained in the nucleus of cells expressing NS1 protein, but no effect was observed when only NS2 protein was expressed. Furthermore, other mRNAs tested, such as T-Ag mRNA and the non-spliceable nucleoprotein transcript, were also retained in the nucleus upon expression of NS1 protein, suggesting that it induced a generalized block of mRNA export from the nucleus.

Amino Acid Sequence↗
PubMed2004

Splice variants: a homology modeling approach.

Splice variants play an important role within the cell in both increasing the proteome diversity and in cellular function. Splice variants are also associated with disease states and may play a role in their etiology. Information about splice variants has, until now, mostly been derived from the primary transcript or through cellular studies. In this study information from the transcript and other studies is combined with tertiary structure information derived from homology models. Through this method we have determined that it is possible to effectively model splice variants. Forty models of splice variants for fourteen proteins were produced. Analysis of the models shows that deletions produce superior model validation values. Additions to sequences where there is little homology become increasingly difficult to model with increasing sequence length. Many of the splicing events are associated with post-translational modification either in the N-terminal region by changing the signal peptide or by affecting the number or availability of glycosylation sites. Often the alternative exon combinations are associated with loss or gain of whole structural units, as opposed to just changing small loop regions. Losing part of the secondary structure may destabilize neighboring parts of the same secondary structure. Detailed analysis is given of four biomedically relevant proteins (Beta-site Amyloid Precursor Protein Cleaving enzyme (BACE), Interleukin-4, Frataxin and Hereditary hemochromatosis protein) and their associated splice variant models. The visualization of these possible structures provides new insights about their functionality and the possible etiology of associated diseases.

Alternative Splicing↗
PubMed1997

Novel splicing of an IGF2 polymorphic region in human adrenocortical carcinomas.

The human IGF2 gene lies on chromosome 11p15.5 and encodes for a mitogenic peptide. IGF2 is often overexpressed in many tumours including adrenal carcinomas. In this study while screening 12 adrenocortical carcinomas for heterozygosity at the Apa I and (CA)n repeat polymorphisms we observed a novel splicing event in two samples which showed both an allelic expression imbalance and preferential splicing for one of the alleles. Further examination revealed that the splicing was not confined to one particular site. Three of such splice products were isolated and cloned. Using RNase protection analysis the presence of this splicing event was demonstrated for both adrenocortical carcinoma samples and also in a Hep3B cell line. This suggested that the event may be occurring in all the samples. The presence of this splicing was then confirmed in all 12 adrenocortical carcinoma samples by PCR. These data suggest that the splicing event may be a general feature for IGF2 transcripts.

Adrenal Cortex Neoplasms↗
PubMed2000

The role of extracellular and cytoplasmic splice domains of alpha7-integrin in cell adhesion and migration on laminins.

The major laminin-binding integrin of skeletal, smooth, and heart muscle is alpha7beta1-integrin, which is structurally related to alpha6beta1. It occurs in three cytoplasmic splice variants (alpha7A, -B, and -C) and two extracellular forms (X1 and X2) which are developmentally regulated and differentially expressed in skeletal muscle. Previously, we have shown that ectopic expression of the alpha7beta-integrin splice variant in nonmotile HEK293 cells specifically induced cell locomotion on laminin-1 but not on fibronectin. To investigate the specificity and the mechanism of the alpha7-mediated cell motility, we expressed the three alpha7-chain cytoplasmic splice variants, as well as alpha6A- and alpha6B-integrin subunits in HEK293 cells. Here we show that all three alpha7 splice variants (containing the X2 domain), as well as alpha6A and alpha6B, promote cell attachment and stimulate cell motility on laminin-1 and its E8 fragment. Deletion of the cytoplasmic domain (excluding the GFFKR consensus sequence) from alpha7B resulted in a loss of the motility-enhancing effect. On laminin-2/4 (merosin), the predominant isoform in mature skeletal muscle, only alpha7-expressing cells showed enhanced motility, whereas cells transfected with alpha6A and alpha6B neither attached nor migrated on laminin-2. Adhesion of alpha7-expressing cells to both laminin-1 and laminin-2 was specifically inhibited by a new monoclonal antibody (6A11) specific for alpha7. Expression of the two extracellular splice variants alpha7X1 and alpha7X2 in HEK293 cells conferred different motilities on laminin isoforms: Whereas alpha7X2B promoted cell migration on both laminin-1 and laminin-2, alpha7X1B supported motility only on laminin-2 and not on laminin-1, although both X1 and X2 splice variants revealed similar adhesion rates to laminin-1 and -2. Fluorescence-activated cell sorter analysis revealed a dramatic reduction of surface expression of alpha6-integrin subunits after alpha7A or -B transfection; also, surface expression of alpha1-, alpha3-, and alpha5-integrins was significantly reduced. These results demonstrate selective responses of alpha6- and alpha7-integrins and of the alpha7 splice variants to laminin-1 and -2 and indicate differential roles in laminin-controlled cell adhesion and migration.

Antigens, CD↗
PubMed2002

Alternative splicing of brain-specific PTB defines a tissue-specific isoform pattern that predicts distinct functional roles.

Splicing of neural-specific exons is differentially regulated in neuronal and non-neuronal cells. The polypyrimidine tract binding protein (PTB) has been implicated as a negative regulator for exon splicing, whereas the brain-specific homolog of PTB, termed nPTB, promotes exon splicing exclusively in neurons. We have now isolated a novel mRNA splice variant of nPTB from non-neuronal cells. In contrast to the neural nPTB transcript, the expression of this novel isoform was absent from brain tissue and was generated in non-neuronal cells by alternative splicing to include five additional amino acid residues encoded by exon 9. In addition, we identified a brain-specific transcript containing a novel, alternatively spliced, internal exon 10. The exclusion of this 34-nucleotide exon 10 in non-neuronal tissues generates a premature termination codon and results in the truncation of the open reading frame. Our findings suggest that alternative splicing of nPTB has an important role in regulation of tissue-specific gene expression and thus in the functional activity of nPTB in neuronal and non-neuronal cells.

Alternative Splicing↗
PubMed1993

Requirements of a group I intron for reactions at the 3' splice site.

Structural elements of the Tetrahymena group I intron have been implicated in directing the exon-ligation transesterification reaction (the second step of self-splicing) to the correct 3' splice site. The 3' splice site also serves as the reaction site in a distinct transesterification reaction known as G-exchange and in a specific hydrolysis reaction. The dependence of the reactions on the internal guide sequence (IGS), an exon-binding element, was examined. Hydrolysis did not require the IGS, while G-exchange did, suggesting that one of the guanosine-binding sites employed during G-exchange requires the IGS. For the exon ligation reaction, the specification of the 3' splice site is thought to be influenced by the 3' exon-IGS pairing P10 and three structural elements in the intron: the universally conserved guanosine residue preceding the 3' splice site, the long-range pairing P9.0, and the triple stem-loop region composed of P9, P9.1 and P9.2. A systematic mutational study of the three structural elements was performed in order to clarify the mechanism of the specification of the 3' splice site for the reactions. For both transesterification reactions, the elements are important, with some superfluity in function. The specific hydrolysis reaction, however, shows even more dependence on the individual elements, especially the conserved guanosine. The results are discussed in terms of models for the mechanism of self-splicing.

Animals↗
PubMed1999

The maturase encoded by a group I intron from Aspergillus nidulans stabilizes RNA tertiary structure and promotes rapid splicing.

The AnCOB group I intron from Aspergillus nidulans self-splices, providing the Mg2+ concentration is >/= 15 mM. The splicing reaction is greatly stimulated by a maturase protein encoded within the intron itself. An initial structural and biochemical analysis of the splicing reaction has now been performed. The maturase bound rapidly to the precursor RNA (kon approximately 3 x 10(9) M(-1) min(-1)) and remained tightly bound (koff </= 0.04 min(-1)). The catalytic step of 5' splice-site cleavage occurred at a rate of up to 11 min(-1) under single turnover conditions. The maturase-assisted reaction of heat-denatured RNA proceeded at a rate of about 1 min(-1), arguing that there are early steps of folding that cannot be readily facilitated by the protein. pH analysis revealed a biphasic profile with a pKa of 7.0. The rate of the maturase-assisted reaction was independent of the Mg2+ concentration down to 3 mM. Self-splicing in optimal Mg2+ (>/= 150 mM) was tenfold slower, in part because of the existence of an equilibrium between folded and partially folded RNA. In contrast, the maturase very effectively stabilized tertiary structure in 5 mM Mg2+, a noticeable example being an interaction between the P8 helix and a GNRA sequence that constitutes the L2 terminal loop of the P2 helix. Formation of the 5' splice-site recognition helix was assisted by either the maturase or high concentrations of Mg2+. The maturase was required during splicing so it is not a true chaperone. However, RNase protection assays and kinetic studies suggest that the maturase recognizes and facilitates folding of an intron with limited tertiary structure and even incomplete secondary structure.

Aspergillus nidulans↗
PubMed1996

Expression of human foamy virus reverse transcriptase involves a spliced pol mRNA.

In human foamy virus (HFV) the reverse transcriptase is expressed independently of the Gag protein as a 127-kDa Pol precursor molecule. Evaluating the mechanism of Pol expression we identified a spliced mRNA which uses the main 5' splice donor and a splice acceptor site located in the gag gene. The significance of this spliced transcript for HFV Pol expression was studied by constructing a virus with a mutated splice acceptor site. This virus was unable to express detectable Pol proteins after transient transfection. Replication of the mutant was studied by a sensitive assay based on HFV transactivator-stimulated expression of an integrated lacZ gene under control of the HFV long terminal repeat. Whereas in the first 2 weeks after transfection the mutant replicated 3 to 5 order of magnitude less well than wild-type virus, extracellular titers obtained thereafter were similar to those of wild-type virus. This increase in replication competence was accompanied by a reversion of the mutated splice acceptor site. The results underlined the importance of the spliced pol transcript for HFV replication and pointed to a second mechanism of Pol expression. Indicator gene assays suggest that this other mechanism is likely to be a transactivator-dependent cryptic promoter in the gag gene which gives rise to Pol-encoding transcripts.

Animals↗
PubMed1998

SR protein and snRNP requirements for assembly of the Rous sarcoma virus negative regulator of splicing complex in vitro.

Retroviruses use unspliced RNA as mRNA for expression of virion structural proteins and as genomic RNA; the full-length RNA often constitutes the majority of the viral RNA in an infected cell. Maintenance of this large pool of unspliced RNA is crucial since even a modest increase in splicing efficiency can lead to impaired replication. In Rous sarcoma virus, the negative regulator of splicing (NRS) was identified as a cis element that negatively impacts splicing of viral RNA. Components of the splicing apparatus appear to be involved in splicing inhibition since binding of a number of splicing factors (snRNPs and SR proteins) and assembly of a large complex (NRS-C) in nuclear extracts correlate with NRS-mediated splicing inhibition. In determining the requirements for NRS complex assembly, we show that NRS-C assembly can be reconstituted by addition of total SR proteins to an S100 extract that lacks these factors. Of the purified SR proteins tested, SF2/ASF was functional in NRS-C assembly, whereas SC35 and SRp40 were not. The participation of snRNPs in NRS-C assembly was addressed by selectively depleting individual snRNPs with oligonucleotides and RNase H or by sequestering critical snRNA domains with 2'-O-methyl RNA oligonucleotides. The results indicate that in addition to U11 snRNP, U1 snRNP and SR proteins, but not U2 snRNP, are involved in NRS-C assembly.

Animals↗
PubMed1999

Inclusion of the NS2-specific exon in minute virus of mice mRNA is facilitated by an intronic splicing enhancer that affects definition of the downstream small intron.

Alternative splicing of pre-mRNAs plays a critical role in maximizing the coding capacity of the small parvovirus genome. The small-intron region of minute virus of mice (MVM) pre-mRNAs undergoes an unusual pattern of overlapping alternative splicing, using two donors, D1 and D2, and two acceptors, A1 and A2, within a region of 120 nucleotides, that governs the steady-state ratios of the various viral mRNAs. In a previous report we demonstrated that a complex interaction between both donor and acceptor sequences, as well as the constraints of size, defines the small intron and governs its alternative splicing. We also identified a G-rich intronic splicing enhancer sequence (IES) that appeared to function as both an intron- and an exon-defining element. In this report we further examined the components that govern MVM small-intron splicing. In fully processed wild-type mRNAs, A1 is used preferentially over A2. In this report, we show that in the context of the wild-type small intron the position of the downstream acceptor A2 was preferred, and the primary sequence of A1 must be stronger for it to be utilized at wild-type efficiency. Use of A2 in generation of the minor spliced forms D2/A2 required the IES because of a weak A2 polypyrimidine tract and because of the relative strength of A1. The small size of the intron and the relative position of the IES were also shown to play a critical role in donor and acceptor site selection. Finally, we have further characterized how the IES functions as an intronic enhancer of upstream exon definition. When the small intron was expanded, upstream exon inclusion was dependent upon the position of the IES. Within the context of the small intron, alterations of the small intron that overcame the requirement for the IES for splicing to A2 also permitted wild-type levels of upstream exon inclusion in the absence of the IES, suggesting that, in its natural context, the IES facilitates upstream exon inclusion by affecting small-intron definition.

Alternative Splicing↗
PubMed1999

Novel, nonconsensus cellular splicing regulates expression of a gene encoding a chemokine-like protein that shows high variation and is specific for human herpesvirus 6.

There are few genes that are specific and diagnostic for human herpesvirus-6. U83 and U22 are two of them. U22 is unique, whereas U83 encodes distant similarity with some cellular chemokines. Reverse transcription-polymerase chain reaction, cDNA cloning, and sequence analyses show polyadenylated RNA transcripts corresponding to minor full-length and abundant spliced forms of U83 in human herpesvirus 6-infected cells. The splice donor and acceptor sites do not fit consensus sequences for either major GT-AG or minor AT-AC introns. However, the spliced form can also be detected in a U83 transfected cell line; thus the novel sites are used by cellular mechanisms. This intron may represent a new minor CT-AC splicing class. The novel splicing regulates gene expression by introducing a central stop codon that abrogates production of the chemokine-like molecule, resulting in an encoded truncated peptide. The use of metabolic inhibitors and an infection time course showed expression of the two RNA transcripts with immediate early kinetics. However, the full-length product accumulated later, dependent on virus DNA replication, similar to U22. Sequence analyses of 16 strains showed high variation (13%) in U83, with conservation of the novel splice sites. Representative strain variants had similar kinetics of expression and spliced products.

3' Untranslated Regions↗
PubMed1999

Processing of carnitine octanoyltransferase pre-mRNAs by cis and trans-splicing.

Trans-splicing is a mechanism by which two pre-mRNAs are processed to produce a mature transcript that contains exons from both precursors. This process has been described mostly in trypanosoma, nematodes, plant/algal chloroplasts and plant mitochondria [Bonen et al. (1993) FASEB J. 7, 40-46]. Our studies clearly demonstrate that a trans-splicing reaction occurs in the processing of the carnitine octanoyltransferase (COT) gene in rat liver. Three different mature transcripts of COT have been found in vivo, the canonical cis-spliced mRNA and two trans-spliced transcripts, in which either exon 2 or exons 2 and 3 are repeated. Splicing experiments in vitro also indicate the capacity of exon 2 to act either as a donor or as an acceptor of splicing, allowing the trans-splicing reactions to occur.

Alternative Splicing↗
PubMed1996

Expression of the Volvox gene encoding nitrate reductase: mutation-dependent activation of cryptic splice sites and intron-enhanced gene expression from a cDNA.

Use of the nitrate reductase encoding gene (nitA) as selection marker has facilitated the successful nuclear transformation of Volvox carteri. The Volvox nitA gene contains 10 introns. A stable nitA mutation in the Volvox recipient strain 153-81 resides in a G-to-A transition of the first nucleotide in the 5' splice site of nitA intron 2. This mutation resulted in at least three non-functional splice variants, namely: (1) intron 2 was not spliced at all; (2) a cryptic 5' splice site 60 nt upstream or (3) a cryptic 5' splice site 16 nt downstream of the mutation were activated and used for splicing. When we used nitA cDNA (pVcNR13) for transformation of V. carteri 153-81, a low efficiency of about 5 x 10(-5) transformants per reproductive cell was observed. Re-integration of either intron 1 (pVcNR15) or introns 9 and 10 (pVcNR16) in the transforming cDNA increased transformation rates to 5 x 10(-4). In parallel, pVcNR15-transformed Volvox exhibited growth rates that were 100-fold increased over the pVcNR13-transformed alga. This intron-enhancement of nitA gene expression appears to be associated with post-transcriptional processing and 'channelling' of the message. These data suggest an important role of splicing for gene expression in V. carteri.

Base Sequence↗
PubMed1996

The chloroplast chlL gene of the green alga Chlorella vulgaris C-27 contains a self-splicing group I intron.

The chlL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome of Chlorella vulgaris strain C-27 contains the first example of a split chlL gene, which is interrupted by 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequence is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5' and 3' splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3' splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3' splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.

Amino Acid Sequence↗
PubMed1999

In vitro translation of a 2.3-kb splicing variant of the hamster pgp1 gene whose presence in transfectants is associated with decreased drug resistance.

PURPOSE: P-glycoprotein (P-gp), a product of the Chinese hamster pgpl gene, confers multidrug resistance to mammalian cells in which it is overexpressed either by transfection or as a result of drug selection. It is encoded by a 4.3-kb mRNA and in its unglycosylated form has a predicted molecular weight of approximately 141 kDa. When a cDNA containing this sequence is transfected into drug-sensitive Chinese hamster lung cells and is expressed under the control of the beta-actin promoter, both the full-length 4.3-kb mRNA and a 2.3-kb transcript are produced. The latter results from a splicing event that utilizes near consensus 5' and 3' splicing signals resident in the full-length mRNA, and it has also been found to be present in cell lines that express the native gene. Therefore, it is a splicing product of pgpl per se. This report is concerned with the biological relevance of this transcript. METHODS: In vitro transcription and translation experiments were used to show that the putative open reading frame of the 2.3-kb transcript encodes a novel 57-kDa protein (p57pgp1) that contains transmembrane domains 9-12 and the C-terminal ATP binding fold of P-gp. To elucidate the function of p57pgp1, expression vectors containing cDNAs representing (1) the 2.3-kb transcript, (2) the full-length 4.3-kb mRNA, and (3) a splice-disabled 4.3-kb transcript in which production of the 2.3-kb transcript is eliminated by an in-frame mutation at the 3' splice site, were constructed and transfected into DC-3F cells. Additional expression vectors in which p57pgp1 represented the N-terminus of a green fluorescent protein fusion construct were also prepared and used for transient expression studies. RESULTS: Overexpression of the 2.3-kb transcript alone did not confer multidrug resistance. Transfectants in which both the 4.3-kb transcript and the 2.3-kb transcript were present, compared with transfectants in which no 2.3-kb transcript was expressed, but in which the level of expression of the 4.3-kb mRNA alone was the same, showed little change in cross-resistance pattern. However, the overall level of resistance in the latter cells was increased by approximately twofold. Hence the presence of the 2.3-kb transcript was associated with a decrease in drug resistance. In vitro transcription and translation experiments and transient expression studies indicate that p57pgp1 can be expressed both in vitro and in vivo. CONCLUSION: These results demonstrate that a splicing variant of pgp1 contains an open reading frame capable of translation in vitro and in vivo and suggest that alterations in splicing may contribute both directly and indirectly to the overall mechanism of pgp1-mediated multidrug resistance in CHL cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Self-splicing activity of the mitochondrial group-I introns from Aspergillus nidulans and related introns from other species.

The mitochondrial genome of Aspergillus nidulans contains several group-I introns. Each one has been assayed for its ability to self-splice in vitro in the absence of proteins. The intron from the apocytochrome b gene is unusual among subgroup IB4 introns in being able to self-splice, unlike a similar intron from Saccharomyces cerevisiae. The first intron in the cytochrome oxidase subunit-1 gene self-splices but only correctly completes the first step of splicing; cryptic 3' splice-sites are recognized instead and these are also used at a low frequency in vivo. The highly homologous intron from Podospora anserina completes both steps in vitro. The remaining introns do not self-splice. The correlation between subgroup category, the likely presence of specific tertiary interactions, and self-splicing activity is discussed.

Apoproteins↗
PubMed2001

Expression of intron-containing GUS constructs is reduced due to activation of a cryptic 5' splice site.

An intron-containing beta-glucuronidase (GUS) gene has been used widely in promoter analyses and as a plant transformation marker. Maximal plant gene expression requires accurate and efficient removal of the intron from the expressed pre-mRNA transcripts by splicing. Detailed analysis of splicing of potato ST-LS1 and pea legumin introns from GUS constructs revealed the activation of a cryptic 5' splice site in the GUS coding sequence 4 nt upstream from the authentic intron 5' splice site. About 40% of transcripts utilised the cryptic 5' splice site in tobacco protoplasts, reducing the translational potential of expressed pre-mRNA. The same cryptic splicing event was evident in transgenic tobacco leaves but at reduced levels. Mutations that removed the cryptic 5' splice site are associated with a two-fold enhancement in GUS activity in tobacco protoplasts, highlighting the need for careful examination of introns and their sites of insertion into gene constructs to minimise variability in gene activity and maximise gene expression.

Glucuronidase↗