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At least 1,783 records · Page 99Linked to original sources
PubMed1996

In vitro reconstitution of mammalian U1 snRNPs active in splicing: the U1-C protein enhances the formation of early (E) spliceosomal complexes.

We have established an in vitro reconstitution/splicing complementation system which has allowed the investigation of the role of mammalian U1 snRNP components both in splicing and at the early stages of spliceosome formation. U1 snRNPs reconstituted from purified, native snRNP proteins and either authentic or in vitro transcribed U1 snRNA restored both early (E) splicing complex formation and splicing-activity to U1-depleted extracts. In vitro reconstituted U1 snRNPs possessing an m3G or ApppG cap were equally active in splicing, demonstrating that a physiological cap structure is not absolutely required for U1 function. However, the presence of an m7GpppG or GpppG cap was deleterious to splicing, most likely due to competition for the m7G cap binding proteins. No significant reduction in splicing or E complex formation was detected with U1 snRNPs reconstituted from U1 snRNA lacking the RNA binding sites of the U1-70K or U1-A protein (i.e., stem-loop I and II, respectively). Complementation studies with purified HeLa U1 snRNPs lacking subsets of the U1-specific proteins demonstrated a role for the U1-C, but not U1-A, protein in the formation and/or stabilization of early splicing complexes. Studies with recombinant U1-C protein mutants indicated that the N-terminal domain of U1-C is necessary and sufficient for the stimulation of E complex formation.

Animals↗
PubMed1996

The 5' and 3' splice sites come together via a three dimensional diffusion mechanism.

We present evidence that the splice sites in mammalian pre-mRNAs are brought together via a three dimensional diffusion mechanism. We tested two mechanisms for splice site pairing: a lateral diffusion ('scanning') model and the currently favored three dimensional diffusion ('jumping') model. Two lines of evidence that distinguish between these two models are presented. The first utilized bipartite splicing substrates tethered by double-stranded RNA stems predicted to provide either a moderate or severe block to splice site pairing via a scanning mechanism. Splice site pairing via a jumping mechanism was expected to be unaffected or affected minimally. The second approach utilized a flexible poly(ethylene glycol) moiety within the intron. This insertion was predicted to reduce scanning efficiency but not the efficiency of a three dimensional diffusion mechanism. The best explanation for the data with the bipartite RNAs is that splice site pairing occurs through three dimensional diffusion. Kinetic analysis of the poly(ethylene glycol) containing substrate showed that neither the lag phase nor the initial rates of mRNA production and spliceosome assembly were affected by this insertion. Therefore, both experimental approaches supported the three dimensional diffusion model of splice site pairing.

Base Sequence↗
PubMed1998

Prediction of locally optimal splice sites in plant pre-mRNA with applications to gene identification in Arabidopsis thaliana genomic DNA.

Prediction of splice site selection and efficiency from sequence inspection is of fundamental interest (testing the current knowledge of requisite sequence features) and practical importance (genome annotation, design of mutant or transgenic organisms). In plants, the dominant variables affecting splice site selection and efficiency include the degree of matching to the extended splice site consensus and the local gradient of U- and G+C-composition (introns being U-rich and exons G+C-rich). We present a novel method for splice site prediction, which was particularly trained for maize and Arabidopsis thaliana. The method extends our previous algorithm based on logitlinear models by considering three variables simultaneously: intrinsic splice site strength, local optimality and fit with respect to the overall splice pattern prediction. We show that the method considerably improves prediction specificity without compromising the high degree of sensitivity required in gene prediction algorithms. Applications to gene identification are illustrated for Arabidopsis and suggest that successful methods must combine scoring for splice sites, coding potential and similarity with potential homologs in non-trivial ways. A WWW version of the SplicePredictor program is available at http:/gnomic.stanford.edu/volker/SplicePredi ctor.html/

Algorithms↗
PubMed1999

Accurate selection of a 5' splice site requires sequences within fibronectin alternative exon B.

Inclusion of fibronectin alternative exon B in mRNA is developmentally regulated. Here we demonstrate that exon B contains two unique purine-rich sequence tracts, PRE1 and PRE2, that are important for proper 5' splice site selection both in vivo and in vitro. Targeted mutations of both PREs decreased the inclusion of exon B in the mRNA by 50% in vivo. Deletion or mutation of the PREs reduced removal of the downstream intron, but not the upstream intron, and induced the activation of cryptic 5' splice sites in vitro. PRE-mediated 5' splice selection activity appears sensitive to position and sequence context. A well characterized exon sequence enhancer that normally acts on the upstream 3' splice site can partially rescue proper exon B 5' splice site selection. In addition, we found that PRE 5' splice selection activity was preserved when exon B was inserted into a heterologous pre-mRNA substrate. Possible roles of these unique activities in modulating exon B splicing are considered.

5' Untranslated Regions↗
PubMed2001

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms.

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.

Alternative Splicing↗
PubMed2003

Dependence of pre-mRNA introns on PRP17, a non-essential splicing factor: implications for efficient progression through cell cycle transitions.

Saccharomyces cerevisiae PRP17 (CDC40) encodes a second-step pre-mRNA splicing factor with a role in cell division. The functions of Prp17 in specific cell cycle transitions were examined using temperature-sensitive alleles in arrest/release experiments. We find that G(1)/S and G(2)/M transitions depend on Prp17. G(1)-synchronized prp17::LEU2 cells arrest at non-permissive temperatures as unbudded haploid cells with low levels of CLN1, CLB5 and RNR1 transcripts. This indicates a Prp17 execution point at or prior to Start. Reduced levels of alpha-tubulin protein, a mitotic spindle component, underlie the benomyl sensitivity of prp17 mutants and possibly their G(2)/M arrest. Splicing of TUB1 and TUB3 transcripts, which encode alpha-tubulin, was analyzed in prp17 and other second-step factor mutants. TUB1 splicing is inefficient in prp17, prp16 and prp22, and marginally affected in prp18, slu7-1 and psf1-1. TUB3 splicing is similarly affected. In vitro splicing with TUB3 pre-mRNA demonstrates a compromised second step in prp17::LEU2 extracts, implicating a direct role for Prp17 in its efficient splicing. Genomic replacement of an intronless TUB1 gene relieves the benomyl sensitivity of prp17 mutants; however, they remain temperature sensitive, implying multiple limiting factors for mitosis. The data suggest that integration of splicing with the cell cycle is important for G(1)/S and G(2)/M transitions.

Cell Cycle↗
PubMed2003

Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1.

CDC5L and PLRG1 are both spliceosomal proteins that are highly conserved across species. They have both been shown to be part of sub- spliceosomal protein complexes that are essential for pre-mRNA splicing in yeast and humans. CDC5L and PLRG1 interact directly in vitro. This interaction is mediated by WD40 regions in PLRG1 and the C-terminal domain of CDC5L. In order to determine whether this interaction is important for the splicing mechanism, we have designed peptides corresponding to highly conserved sequences in the interaction domains of both proteins. These peptides were used in in vitro splicing experiments as competitors to the cognate sequences in the endogenous proteins. Certain peptides derived from the binding domains of both proteins were found to inhibit in vitro splicing. This splicing inhibition could be prevented by preincubating the peptides with the corresponding partner protein that had been expressed in Escherichia coli. The results from this study indicate that the interaction between CDC5L and PLRG1 is essential for pre-mRNA splicing and further demonstrate that small peptides can be used as effective splicing inhibitors.

Amino Acid Sequence↗
PubMed2004

Telomerase downregulation induced by the G-quadruplex ligand 12459 in A549 cells is mediated by hTERT RNA alternative splicing.

Ligand 12459, a potent G-quadruplex-interacting agent that belongs to the triazine series, was previously shown to downregulate telomerase activity in the human A549 lung carcinoma cell line. We show here that the downregulation of telomerase activity is caused by an alteration of the hTERT splicing pattern induced by 12459, i.e. an almost complete disappearance of the active (+alpha,+beta) transcript and an over-expression of the inactive -beta transcript. Spliced intron 6 forming the -beta hTERT transcript contained several tracks of G-rich sequences able to form G-quadruplexes. By using a specific PCR-stop assay, we show that 12459 is able to stabilize the formation of these G-quadruplex structures. A549 cell line clones selected for resistance to 12459 have been analyzed for their hTERT splicing pattern. Resistant clones are able to maintain the active hTERT transcript under 12459 treatment, suggesting the appearance of mechanisms able to bypass the 12459-induced splicing alterations. In contrast to 12459, telomestatin and BRACO19, two other G-quadruplex-interacting agents, have no effect on the hTERT splicing pattern in A549 cells, are cytotoxic against the A549-resistant clones and display a lower efficiency to stabilize hTERT G-quadruplexes. These results lead us to propose that 12459 impairs the splicing machinery of hTERT through stabilization of quadruplexes located in the hTERT intron 6. Differences of selectivity between 12459, BRACO19 and telomestatin for these hTERT quadruplexes may be important to explain their respective activity and inactivity against hTERT splicing.

Acridines↗
PubMed2004

Silencer elements as possible inhibitors of pseudoexon splicing.

Human pre-mRNAs contain a definite number of exons and several pseudoexons which are located within intronic regions. We applied a computational approach to address the question of how pseudoexons are neglected in favor of exons and to possibly identify sequence elements preventing pseudoexon splicing. A search for possible splicing silencers was carried out on a pseudoexon selection that resembled exons in terms of splice site strength and exon splicing enhancer (ESE) representation; three motifs were retrieved through hexamer composition comparisons. One of these functions as a powerful silencer in transfection-based splicing assays and matches a previously identified silencer sequence with hnRNP H binding ability. The other two motifs are novel and failed to induce skipping of a constitutive exon, indicating that they might act as weak repressors or in synergy with other unidentified elements. All three motifs are enriched in pseudoexons compared with intronic regions and display higher frequencies in intronless gene-coding sequences compared with exons. We consider that a subpopulation of pseudoexons might rely on negative regulators for splicing repression; this hypothesis, if experimentally verified, might improve our understanding of exonic splicing regulatory sequences and provide the identification of a novel mutation target for human genetic diseases.

Animals↗
PubMed2005

SpliceInfo: an information repository for mRNA alternative splicing in human genome.

We have developed an information repository named SpliceInfo to collect the occurrences of the four major alternative-splicing (AS) modes in human genome; these include exon skipping, 5'-alternative splicing, 3'-alternative splicing and intron retention. The dataset is derived by comparing the nucleotide and protein sequences available for a given gene for evidence of AS. Additional features such as the tissue specificity of the mRNA, the protein domain contained by exons, the GC-ratio of exons, the repeats contained within the exons, and the Gene Ontology are annotated computationally for each exonic region that is alternatively spliced. Motivated by a previous investigation of AS-related motifs such as exonic splicing enhancer and exonic splicing silencer, this resource also provides a means of identifying motifs candidates and this should help to identify potential regulatory mechanisms within a particular exonic sequence set and its two flanking intronic sequence sets. This is carried out using motif discovery tools to identify motif candidates related to alternative splicing regulation and together with a secondary structure prediction tool, will help in the identification of the structural properties of such regulatory motifs. The integrated resource is now available on http://SpliceInfo.mbc.NCTU.edu.tw/.

Alternative Splicing↗
PubMed2006

EASI--enrichment of alternatively spliced isoforms.

Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts.

Alternative Splicing↗
PubMed2005

High-throughput alternative splicing quantification by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Alternative splicing is a significant contributor to transcriptome diversity, and a high-throughput experimental method to quantitatively assess predictions from expressed sequence tag and microarray analyses may help to answer questions about the extent and functional significance of these variants. Here, we describe a method for high-throughput analysis of known or suspected alternative splicing variants (ASVs) using PCR, primer extension and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Reverse-transcribed mRNA is PCR amplified with primers surrounding the site of alternative splicing, followed by a primer extension reaction designed to target sequence disparities between two or more variants. These primer extension products are assayed on a MALDI-TOF mass spectrometer and analyzed automatically. This method is high-throughput, highly accurate and reproducible, allowing for the verification of the existence of splicing variants in a variety of samples. An example given also demonstrates how this method can eliminate potential pitfalls from ordinary gel electrophoretic analysis of splicing variants where heteroduplexes formed from different variants can produce erroneous results. The new method can be used to create alternative variant profiles for cancer markers, to study complex splicing regulation, or to screen potential splicing therapies.

Actinin↗
PubMed1990

Surveying cis-acting sequences of pre-mRNA by adding antisense 2'-O-methyl oligoribonucleotides to a splicing reaction.

We chemically synthesized antisense 12 mer 2'-O-methylribonucleotides and surveyed a scanning (signal-tracking) process as well as sequences within a beta-globin transcript acting in the splicing reaction in vitro. The pre-mRNA transcript contained the sequences of the first exon, first intron, and a major part of the second exon of the human beta-globin gene. We found that the antisense 2'-O-methylribonucleotides could anneal effectively to the target site in the pre-mRNA during the splicing reaction. A 2'-O-methylribonucleotide complementary to the donor (5') splice site completely inhibited authentic splicing and activated an upstream cryptic donor site. A 2'-O-methylribonucleotide complementary to the branch site inhibited normal branch formation and greatly reduced subsequent generation of the spliced product. Six other 2'-O-methylribonucleotides complementary to loci in the exons or the intronic region between the donor and branch sites had no significant effect on the splicing reaction. These observations suggest that an extensive scanning of the present pre-mRNA across the six regions tested is not essential for the splicing reaction. We propose that a short antisense 2'-O-methylribonucleotide provides a practical and convenient method to examine cis-acting sequences of RNA. The advantages of this method in comparison with site-directed mutagenesis or deletion are discussed.

Base Sequence↗
PubMed1987

Splicing of yeast nuclear pre-mRNA in vitro requires a functional 40S spliceosome and several extrinsic factors.

We have previously shown that extracts prepared from most of the yeast temperature-sensitive rna mutants are heat sensitive for pre-mRNA splicing in vitro, and that the products of the corresponding RNA genes are essential for the early stages of the splicing region. In this report, we demonstrate that most heat-inactivated mutant extracts do not form the spliceosome, suggesting that their gene products are likely to be involved in spliceosome formation. Heat-inactivated rna2 extracts, on the other hand, do form a splicing-dependent 40S complex containing uncleaved pre-mRNA exclusively. The pre-mRNA in the 40S complex can be converted to the splicing products in the presence of ATP and complementing extracts. These results demonstrate that: (1) the 40S complex formed in heat-inactivated rna2 extracts is a spliceosome (termed the rna2 delta spliceosome), (2) the spliceosome is a functional intermediate in the splicing pathway, and (3) the splicing process can be dissected into two steps, spliceosome formation and cleavage-ligation reactions. Additional results indicate that at least two extrinsic factors, as well as the RNA2 gene product, are required for complementation of the rna2 delta spliceosome. A three-step mechanism for nuclear pre-mRNA splicing in yeast is proposed.

Genes, Fungal↗
PubMed1997

A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer.

We have purified and cloned a new splicing factor, KSRP. KSRP is a component of a multiprotein complex that binds specifically to an intronic splicing enhancer element downstream of the neuron-specific c-src N1 exon. This 75-kD protein induces the assembly of five other proteins, including the heterogeneous nuclear ribonucleoprotein F, onto the splicing enhancer. The sequence of the KSRP cDNA indicates that the protein contains four K homology RNA-binding domains and an unusual carboxy-terminal domain. KSRP is similar to two proteins, FUSE-binding protein and P-element somatic inhibitor. KSRP is expressed in both neural and non-neural cell lines, although it is present at higher levels in neural cells. Antibodies specific for KSRP inhibit the splicing of the N1 exon in vitro. Moreover, this inhibition of N1 splicing can be rescued by the addition of purified KSRP. KSRP is likely to regulate splicing from a number of intronic splicing enhancer sequences.

Amino Acid Sequence↗
PubMed1998

A ribosomal function is necessary for efficient splicing of the T4 phage thymidylate synthase intron in vivo.

Splicing of the group I intron of the T4 thymidylate synthase (td) gene was uncoupled from translation by introducing stop codons in the upstream exon. This resulted in severe splicing deficiency in vivo. Overexpression of a UGA suppressor tRNA partially rescued splicing, suggesting that this in vitro self-splicing intron requires translation for splicing in vivo. Inhibition of translation by the antibiotics chloramphenicol and spectinomycin also resulted in splicing deficiency. Ribosomal protein S12, a protein with RNA chaperone activity, and CYT-18, a protein that stabilizes the three-dimensional structure of group I introns, efficiently rescued the stop codon mutants. We identified a region in the upstream exon that interferes with splicing. Point mutations in this region efficiently alleviate the effect of a nonsense codon. We infer from these results that the ribosome acts as an RNA chaperone to facilitate proper folding of the intron.

Bacteriophage T4↗
PubMed1999

A cis-acting element in the 3'-untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR.

We report a role for the 3'-untranslated region in control of mRNA splicing and show that human TNF-alpha 3' UTR harbors a cis-acting element that renders splicing of precursor transcripts dependent on activation of PKR, the RNA-activated protein kinase that phosphorylates eukaryotic initiation factor 2 (eIF2). When this element, designated 2-APRE, is present, splicing becomes sensitive to inhibition by the PKR inhibitor, 2-aminopurine, or by coexpression of transdominant-negative mutant PKR. Our results reveal that activation of PKR is required for splicing of mRNA when precursor transcripts contain the 2-APRE and that increased expression of wild-type PKR enhances their splicing efficiency. Thus, PKR responds as trans-acting factor to the 2-APRE. 2-APRE RNA forms a stable, 17-bp stem-loop structure and strongly activates PKR in vitro, inducing eIF2alpha phosphorylation. Despite its ability to activate PKR during splicing, the 2-APRE within the 3' UTR does not affect translation efficiency of the resulting TNF-alpha mRNA in transfected cells. PKR and the 3' UTR thus interact during mRNA splicing to confer a novel type of regulation on expression of the TNF-alpha gene.

2-Aminopurine↗
PubMed2005

Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila.

To gain insight into splicing regulation, we developed a microarray to assay all annotated alternative splicing events in Drosophila melanogaster and identified the alternative splice events controlled by four splicing regulators: dASF/SF2, B52/SRp55, hrp48, and PSI. The number of events controlled by each of these factors was found to be highly variable: dASF/SF2 strongly affects >300 splicing events, whereas PSI strongly affects only 43 events. Pairwise analysis also revealed many instances of splice site usage affected by multiple factors and provides the framework to understand the network controlling the alternatively spliced mRNA isoforms that compose the Drosophila transcriptome.

Animals↗