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PubMed2004

Insulinomas and expression of an insulin splice variant.

BACKGROUND: Insulinomas are beta-cell tumours characterised by uncontrolled insulin secretion even in the presence of hypoglycaemia. However, the mechanisms allowing such excessive insulin secretion are not known. Insulin secretion can occur only when the beta-cell insulin stores have been replenished by insulin biosynthesis, which is mainly controlled by translation. Such specific translational regulation often involves the 5' untranslated region. We have identified an insulin splice variant in isolated human pancreatic islets of non-diabetic donors that retains 26 bp of intron 1 and thereby changes the 5' untranslated region, but leaves the coding region unchanged. This splice variant has increased translation efficiency in vitro and in vivo compared with native insulin mRNA. However, splice variant expression is less than 1% of native insulin mRNA in normal islets. METHODS: To test whether this splice variant is involved in insulin production by human insulinomas, we extracted RNA from nine laser-captured surgical insulinoma samples and from isolated islets of nine donors who did not have diabetes. We then determined the ratio of splice variant to native insulin mRNA by quantitative real-time RT-PCR. FINDINGS: The mean ratio of the splice variant to native insulin mRNA was increased more than 50-fold in insulinomas compared with normal islets, and this difference was present in all nine human insulinomas. Overexpression of the splice variant therefore seems to be a general characteristic of insulinomas and is estimated to contribute about 90% to insulin synthesis by these tumours. INTERPRETATION: Overexpression of the insulin splice variant with increased translation efficiency in insulinomas might explain how these tumours maintain high levels of insulin synthesis and secretion leading to hyperinsulinaemia-the hallmark of this disease.

Alternative Splicing↗
PubMed1997

Organization of the murine reduced folate carrier gene and identification of variant splice forms.

RT-PCR analysis of the reduced folate carrier (RFC) from L1210 and murine erythroleukemia cells led to the identification of three clones which appeared to result from the use of alternative splice sites. The nucleotide sequence of each splice form predicts a protein that contains at least the first 7 transmembrane domains of the parental RFC protein followed by a novel hydrophilic carboxyl terminus of 33, 72, or 105 amino acid residues. Sequence analysis of cDNA clones isolated from murine liver and the results of 5'-RACE from L1210 cells indicated that RFC also utilizes alternate 5'-terminal exons. To understand how the alternatively spliced RFC transcripts and multiple 5'-termini were generated, the genomic organization of RFC was determined. The gene is comprised of at least 8 exons, the first two of which encode the alternative 5' termini. Based on sequence identity with cDNAs encoding RFC from hamster and rat, however, it appears that additional 5' exons may be present. Two of the RFC splice variants result from the use of a cryptic splice donor site within exon 4 and the third results from the use of a cryptic splice acceptor site within exon 5. In addition, the splice variant form that encodes the largest protein also utilizes an alternative exon located between exons 5 and 6. The apparent use of alternative transcriptional start sites and the identification of several RFC splice forms raises the possibility that unique RFC molecules may be generated that exhibit tissue- or cell line-specific distribution.

Alternative Splicing↗
PubMed2001

Alternative RNA splicing in the nervous system.

Tissue-specific alternative splicing profoundly effects animal physiology, development and disease, and this is nowhere more evident than in the nervous system. Alternative splicing is a versatile form of genetic control whereby a common pre-mRNA is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. In the nervous system, thousands of alternatively spliced mRNAs are translated into their protein counterparts where specific isoforms play roles in learning and memory, neuronal cell recognition, neurotransmission, ion channel function, and receptor specificity. The essential nature of this process is underscored by the finding that its misregulation is a common characteristic of human disease. This review highlights the current views of the biological phenomenon of alternative splicing, and describes evidence for its intricate underlying biochemical mechanisms. The roles of RNA binding proteins and their tissue-specific properties are discussed. Why does alternative splicing occur in cosmic proportions in the nervous system? How does it affect integrated cellular functions? How are region-specific, cell-specific and developmental differences in splicing directed? How are the control mechanisms that operate in the nervous system distinct from those of other tissues? Although there are many unanswered questions, substantial progress has been made in showing that alternative splicing is of major importance in generating proteomic diversity, and in modulating protein activities in a temporal and spatial manner. The relevance of alternative splicing to diseases of the nervous system is also discussed.

Alternative Splicing↗
PubMed2001

Analysis of exonic splicing enhancers in the mouse gonadotropin-releasing hormone (GnRH) gene.

We have previously reported that efficient removal of the first intron (intron A) of gonadotropin-releasing hormone (GnRH) pre-mRNA is the prerequisite event for post-transcriptional regulation. In the present study, using an in vitro HeLa splicing system, we examined the enhancing activities of exonic elements for GnRH pre-mRNA splicing. While not excised by exon 2 alone, intron A was efficiently excised when exon 3 and/or exon 4 was combined with exon 2, suggesting the presence of exonic splicing enhancers (ESEs) in exons 3 and 4. Purine-rich sequences located in the border of exons 2 and 3 (denoted ESE3) and in exon 4 (ESE4) revealed strong splicing enhancing activities. Mutation in ESE3 decreased pre-mRNA splicing, while mutation in purine-rich sequences in exon 2 did not. We further analyzed the functional activity of ESE4 by mutations or deletions of the ESE4 sequence that consists of three purine-repeats separated by two spacers and a putative hairpin constructing sequence. An UV cross-linking assay using the RNA sequence of ESE4 examined the presence of ESE4-specific binding proteins in the nuclear extracts from GT1 hypothalamic GnRH neurons. Collectively, this study indicates that a sequence context of ESE4 and its binding proteins may be crucially involved in enhanced GnRH pre-mRNA splicing. However, it should be further clarified as to which splicing factor(s) is responsible for ESE4-dependent GnRH pre-mRNA splicing.

Animals↗
PubMed2000

Functional recognition of 5' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly.

A sensitive assay based on competition between cis-and trans-splicing suggested that factors in addition to U1 snRNP were important for early 5' splice site recognition. Cross-linking and physical protection experiments revealed a functionally important interaction between U4/U6.U5 tri-snRNP and the 5' splice site, which unexpectedly was not dependent upon prior binding of U2 snRNP to the branch point. The early 5' splice site/tri-snRNP interaction requires ATP, occurs in both nematode and HeLa cell extracts, and involves sequence-specific interactions between the highly conserved splicing factor Prp8 and the 5' splice site. We propose that U1 and U5 snRNPs functionally collaborate to recognize and define the 5' splice site prior to establishment of communication with the 3' splice site.

Adenosine Triphosphate↗
PubMed2000

Splicing enhancement in the yeast rp51b intron.

Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1 snRNP, and to communication between splice sites across introns or exons mediated by protein-protein interactions. It has been previously shown that, in yeast, communication mediated by RNA-RNA interactions between the two ends of introns is a basis for splicing enhancement. We designed experiments of randomization-selection to isolate splicing enhancers that would work independently from RNA secondary structures. Surprisingly, one of the two families of sequences selected was essentially composed of 5' splice site variants. We show that this sequence enhances splicing independently of secondary structure, is exportable to heterologous contexts, and works in multiple copies with additive effects. The data argue in favor of an early role for splicing enhancement, possibly coincident with commitment complex formation. Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP binding to noncanonical locations is required for splicing enhancement.

5' Untranslated Regions↗
PubMed2001

The 3'-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo.

We describe a new approach to elucidate the role of 3'-end processing in pre-mRNA splicing in vivo using the influenza virus NS1A protein. The effector domain of the NS1A protein, which inhibits the function of the CPSF and PABII factors of the cellular 3'-end-processing machinery, is sufficient for the inhibition of not only 3'-end formation but also the splicing of single-intron pre-mRNAs in vivo. We demonstrate that inhibition of the splicing of single-intron pre-mRNAs results from inhibition of 3'-end processing, thereby establishing that 3'-end processing is required for the splicing of a 3' terminal intron in vivo. Because the NS1A protein causes a global suppression of 3'-end processing in trans, we avoid the ambiguities caused by the activation of cryptic poly(A) sites that occurs when mutations are introduced into the AAUAAA sequence in the pre-mRNA. In addition, this strategy enabled us to establish that the function of a particular 3'-end-processing factor, namely CPSF, is required for the splicing of single-intron pre-mRNAs in vivo: splicing is inhibited only when the effector domain of the NS1A protein binds and inhibits the function of the 30-kDa CPSF protein in 3'-end formation. In contrast, the 3'-end processing factor PABII is not required for splicing. We discuss the implications of these results for cellular and influenza viral mRNA splicing.

Amino Acid Sequence↗
PubMed2001

ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.

Adenosine Triphosphate↗
PubMed2002

In vivo selection of better self-splicing introns in Escherichia coli: the role of the P1 extension helix of the Tetrahymena intron.

In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons. The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5' and 3' splice sites maintained. The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro. Random mutation libraries were constructed to identify active intron variants in E. coli. All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5' end of the intron. Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E. coli. Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo. In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 microM GTP concentration. The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration. Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed. This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo.

Animals↗
PubMed1999

Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron.

Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes. Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site. To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E. coli. Amplification of intron-rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA. Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected. The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA. The intron is spliced 10-fold less efficiently in E. coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site.

Animals↗
PubMed2003

An in vitro screening system for protein splicing inhibitors based on green fluorescent protein as an indicator.

This paper describes an in vitro fluorometric assay system for protein splicing based on the RecA intein of Mycobacterium tuberculosis and a modified green fluorescent protein (GFP). The assay takes advantage of the fact that polypeptides inserted adjacent to residue 129 of GFP cause the protein to form inclusion bodies when expressed in Escherichia coli and to be incapable of fluorophore formation. However, when the inserted polypeptide is an intein, the renatured fusion protein can undergo protein splicing and chromophore formation. Comparison of chromophore formation by renatured GFP-intein fusion and renatured GFP showed that under optimal conditions (pH 6.5 and 20 degrees C) protein splicing is significantly slower than GFP chromophore formation. Taking advantage of the reversible inhibition of protein splicing by zinc ion, a fluorometric protein splicing assay was developed in which the denatured fusion protein of GFP and the RecA intein was purified on a metal ion affinity column and renatured in the presence of 2 mM ZnCl2. When diluted into appropriate buffers, protein splicing could be initiated by the addition of a molar excess of EDTA and followed fluorometrically. This assay should be valuable as a high-throughput screening system for protein splicing inhibitors as potential antimycobacterial agents and as tools for studying the mechanism of protein splicing.

Bacterial Proteins↗
PubMed1991

Alternative secondary structures in the 5' exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA.

The natural splice junction of the Tetrahymena large ribosomal RNA is flanked by hairpins that are phylogenetically conserved. The stem immediately preceding the splice junction involves nucleotides that also base pair with the internal guide sequence of the intervening sequence during splicing. Thus, precursors which contain wild-type exons can form two alternative helices. We have constructed a series of RNAs where the stem-loop in the 5' exon is more or less stable than in the wild-type precursor, and tested them in both forward and reverse self-splicing reactions. The presence of a stable hairpin in ligated exon substrates interferes with the ability of the intervening sequence to integrate at the splice junction. Similarly, the presence of the wild-type hairpin in the 5' exon reduces the rate of splicing 20-fold in short precursors. The data are consistent with a competition between unproductive formation of a hairpin in the 5' exon and productive pairing of the 5' exon with the internal guide sequence. The reduction of splicing by a hairpin that is a normal feature of rRNA structure is surprising; we propose that this attenuation is relieved in the natural splicing environment.

Animals↗
PubMed1998

A novel Arg362Ser mutation in the sterol 27-hydroxylase gene (CYP27): its effects on pre-mRNA splicing and enzyme activity.

A novel C to A mutation in the sterol 27-hydroxylase gene (CYP27) was identified by sequencing amplified CYP27 gene products from a patient with cerebrotendinous xanthomatosis (CTX). The mutation changed the adrenodoxin cofactor binding residue 362Arg to 362Ser (CGT 362Arg to AGT 362Ser), and was responsible for deficiency in the sterol 27-hydroxylase activity, as confirmed by expression of mutant cDNA into COS-1 cells. Quantitative analysis showed that the expression of CYP27 gene mRNA in the patient represented 52.5% of the normal level. As the mutation occurred at the penultimate nucleotide of exon 6 (-2 position of exon 6-intron 6 splice site) of the gene, we hypothesized that the mutation may partially affect the normal splicing efficiency in exon 6 and cause alternative splicing elsewhere, which resulted in decreased transcript in the patient. Transfection of constructed minigenes, with or without the mutation, into COS-1 cells confirmed that the mutant minigene was responsible for a mRNA species alternatively spliced at an activated cryptic 5' splice site 88 bp upstream from the 3' end of exon 6. Our data suggest that the C to A mutation at the penultimate nucleotide of exon 6 of the CYP27 gene not only causes the deficiency in the sterol 27-hydroxylase activity, but also partially leads to alternative pre-mRNA splicing of the gene. To our knowledge, this is the first report regarding effects on pre-mRNA splicing of a mutation at the -2 position of a 5' splice site.

Alternative Splicing↗
PubMed1998

Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties.

The rice Waxy gene encodes a granule-bound starch synthase (GBSS) necessary for the synthesis of amylose in endosperm tissue. We have previously shown that a CT microsatellite near the transcriptional start site of the GBSS gene can distinguish 7 alleles that accounted for more than 80% of the variation in apparent amylose content in an extended pedigree of 89 US rice cultivars (Oryza sativa L.). Furthermore, all the cultivars with 18% or less amylose were shown to have the sequence AGTTATA at the putative leader intron 5' splice site, while all cultivars with a higher proportion of amylose had AGGTATA. Here we demonstrate that this single-base mutation reduces the efficiency of GBSS pre-mRNA processing and results in alternate splicing at three cryptic sites. The predominant 5' splice site in CT18 low-amylose varieties is 93 bp upstream of the splice site used in intermediate and high amylose varieties and is immediately 5' to the CT microsatellite that we previously demonstrated to be tightly correlated with amylose content. Use of the leader intron 5' splice site at either -93 or -1 in conjunction with the predominant 3' splice site results in formation of a small open reading frame 38 bp upstream of the normal ATG and out of frame with it. This open reading frame is not produced when any of the 5' leader intron splice sites are used in conjunction with an alternate 3' splice site five bases further downstream which was observed in all rice varieties tested.

Alternative Splicing↗
PubMed2003

Analysis of splice-site mutations of the alpha-galactosidase A gene in Fabry disease.

Fabry disease is an X-linked disease caused by a defective lysosomal enzyme, alpha-galactosidase A, and characterized by skin lesions and multiorgan involvement, including kidney, heart, and the central nervous system. Currently more than 200 genotypes have been identified, including several aberrant splicing. However, most of the mutation analyses were performed using genomic sequencing only, and therefore some of the splicing mutations were misclassified as missense mutations. In order to predict the splicing event caused by each mutation, we conducted a literature search for all published mutations located near the splice sites, including exonic point mutations, and performed a splice-site score (SSS) analysis. The literature search identified 13 donor-site mutations, including four exonic mutations (S65T, D183S, K213N, and M267I), located at the end of exons 1, 3, 4, and 5, respectively, six acceptor-site mutations, and one new exon creation. All mutated splice sites, except for the one associated with the new exon creation, had a lower SSS than their respective natural sites. Cryptic or newly created sites were identified with SSS from 0.09 to 1.0. The predictions, based on SSS analysis, are in agreement with all six mutations with known cDNA sequence from the literature, including five mutations with exon skipping and one mutation with creation of a new acceptor site. For the S65T genotype, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis using RNA isolated from the whole-blood sample. We verified that a weak cryptic site (SSS = 0.09) 14 nucleotides downstream was activated and resulted in an insertion of 14 bp and a frameshift stop at codon 106. This change is more consistent with the clinical presentation of the patient, the classical Fabry disease, than the amino acid substitution (S65T), which does not affect the enzyme function. In conclusion, the SSS analysis is very useful for predicting splicing events and genotype/phenotype correlation in Fabry disease. As different mechanisms may be involved in pre-mRNA splicing, it is important to obtain cDNA sequencing for molecular diagnosis.

DNA Mutational Analysis↗
PubMedDate not supplied

Cofactor requirements of splicing of purified messenger RNA precursors.

The origin and functions of introns in protein coding genes is one of the enigmas of molecular biology. Splicing processes that remove intervening sequences from precursor RNAs must have either predated or co-evolved with introns. Inferences about the origin of introns and the possible modes of regulation of splicing should emerge from an understanding of the biochemical mechanisms of splicing. The biochemistry of splicing of tRNA and rRNA precursors has rapidly advanced with the development of in vitro reactions containing soluble components that duplicate in vivo reactions. We have recently shown that accurate splicing of an adenovirus mRNA precursor occurs during a coupled transcription/splicing reaction in a soluble whole cell extract. We now report that an exogenous RNA substrate containing the first and second leaders of adenovirus 2 is accurately spliced when added to an extract of HeLa cells. ATP and Mg2+ are essential cofactors for the reaction. The time course of splicing is unusual; a lag of 45 min is observed before the appearance of splicing product.

Adenosine Triphosphate↗
PubMedDate not supplied

A role for branchpoints in splicing in vivo.

The nucleotides immediately surrounding intron/exon junctions of genes transcribed by RNA polymerase B can be derived from 'consensus' sequences for donor and acceptor splice sites by only a few base changes. Studies in vivo have underlined the importance of these junction nucleotides for splicing. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing. However, the recent discovery that, in vitro, introns are excised in a lariat form where the 5' end of the intron is joined via a 2'-5'-phosphodiester linkage to an A residue (branchpoint acceptor) close to the 3' end of the intron, suggests that internal intron sequences may nonetheless be important for splicing. Indeed, in yeast nuclear genes, the internal sequence 5'-TACTAAC-3' (or close homologue) is essential for splicing in vivo. A proposed consensus sequence for branchpoints in mammalian introns is 5'-CT(A/G)A(C/T)-3'. This sequence resembles the essential yeast internal sequence. Are branchpoints involved in the splicing of introns of higher eukaryotes in vivo? We show here that a branchpoint sequence from a human globin gene (5'-CTGACTCTCTCTG-3') greatly enhances the efficiency of splicing of a 'synthetic' intron in HeLa cells. A mutated branchpoint sequence, 5'-CTCCTCTCTCTG-3', in which the branchpoint acceptor nucleotide A has been deleted and the neighbouring purine G mutated to a C, does not exhibit this enhancing capability. We conclude that branchpoints have an important function in the splicing process in vivo.

Base Sequence↗
PubMed2002

A genomic view of alternative splicing.

Recent genome-wide analyses of alternative splicing indicate that 40-60% of human genes have alternative splice forms, suggesting that alternative splicing is one of the most significant components of the functional complexity of the human genome. Here we review these recent results from bioinformatics studies, assess their reliability and consider the impact of alternative splicing on biological functions. Although the 'big picture' of alternative splicing that is emerging from genomics is exciting, there are many challenges. High-throughput experimental verification of alternative splice forms, functional characterization, and regulation of alternative splicing are key directions for research. We recommend a community-based effort to discover and characterize alternative splice forms comprehensively throughout the human genome.

Alternative Splicing↗