Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Splicing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,153 records · Page 64Linked to original sources
PubMed1995

Species- and tissue-specific expression of the C-terminal alternatively spliced form of the tumor suppressor p53.

Alternative splicing of the p53 transcript which so far has been demonstrated only in the murine system has been proposed as a general regulatory mechanism for the generation of functionally different p53 proteins. We analyzed by RT-PCR the pattern of p53 mRNAs within the region spanning exons 10 and 11 of the p53 gene in 13 different tissues from two independent mouse strains, in 10 different rat tissues and in six different human tissues. PCR products of the expected sizes, corresponding to the normally spliced and the alternatively spliced p53 mRNAs, were detected in mice. Alternatively spliced mRNA was found at approximately 25-20% the level of the normally spliced p53 mRNA in most tissues analyzed. In spleen and kidney the proportion of alternatively spliced p53 mRNA was much lower. Surprisingly, examination of p53 mRNAs isolated from 10 different rat tissues and six human tissues within the same region of the p53 gene showed only products of normal size. Although a potential homologous alternative 3' splice site within intron 10 of the human p53 gene is present in the genomic sequence of human p53, the expected corresponding alternatively spliced p53 mRNA was undetectable. These findings imply that the generation of functionally different forms of p53 by alternative splicing of p53 transcripts is a species-specific event, possibly indicating species-specific mechanisms for regulating p53 activities.

Alternative Splicing↗
PubMed1996

During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles.

Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping.

Base Sequence↗
PubMed1997

Functional analysis of the polypyrimidine tract in pre-mRNA splicing.

The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing. Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing. In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection. However, there appears to be great flexibility in the specific sequence of a given tract. Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain. Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts. An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts. We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts. In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant. In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency. The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing. While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays.

Introns↗
PubMed2000

Novel splice variants of cyclin E with altered substrate specificity.

Cyclin E, a G(1) cyclin, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In this study we have examined the possibility that the shortened mRNA splice variants could give rise to tumor-specific cyclin E LMW proteins. We used the Splice Capture method to identify, enumerate and isolate known spliced mRNAs and to look for previously undetected mRNA forms of cyclin E that might be translated into the LMW proteins. We show that a new splice variant of cyclin E found in tumor cells isolated by the Splice Capture strategy, named Delta48, activates CDK2 more robustly than full-length cyclin E when assayed from transiently transfected cells with the natural substrate GST-Rb. We also found the Splice Capture method to be superior to the conventional RNase protection assay in analyzing the cyclin E mRNA present in normal and tumor cells. Splice Capture enumerated the relative abundance of known forms of cyclin E mRNA and easily discovered new splice variants in both normal and tumor cells. We conclude that the abundance of cyclin E splice variants in cells may represent a novel form of regulation of cyclin E, and if translated they show altered substrate specificity compared to the full length form of cyclin E.

Alternative Splicing↗
PubMed2000

Species-specific regulation of alternative splicing in the C-terminal region of the p53 tumor suppressor gene.

Alternative splicing occurs in the C-terminal region of the p53 tumor suppressor gene between two alternative 3' splice sites in intron 10. This alternative splicing event has been detected in murine cells, but not in rat or human tissues. In this paper, we have characterized the pattern of p53 alternative splicing in cell lines from five different species. Our results confirm that p53 alternative splicing is species-specific, being detected only in cell lines of rodent origin. Using transient transfection assays, we have established that the rat p53 gene undergoes efficient alternative splicing in both mouse and rat cell lines, thus demonstrating that it has all the necessary cis -acting sequences to be alternatively spliced. In contrast, we were unable to detect any usage of the human alternative 3' splice site under the same experimental conditions. Thus, the low levels or absence of alternatively spliced p53 mRNA in rat and human cell lines seems to be the result of different mechanisms. Our results support the hypothesis that there are species-specific mechanisms implicated in the regulation of p53 activity.

Alternative Splicing↗
PubMed2003

Conservation of human alternative splice events in mouse.

Human and mouse genomes share similar long-range sequence organization, and have most of their genes being homologous. As alternative splicing is a frequent and important aspect of gene regulation, it is of interest to assess the level of conservation of alternative splicing. We examined mouse transcript data sets (EST and mRNA) for the presence of transcripts that both make spliced-alignment with the draft mouse genome sequence and demonstrate conservation of human transcript-confirmed alternative and constitutive splice junctions. This revealed 15% of alternative and 67% of constitutive splice junctions as conserved; however, these numbers are patently dependent on the extent of transcript coverage. Transcript coverage of conserved splice patterns is found to correlate well between human and mouse. A model, which extrapolates from observed levels of conservation at increasing levels of transcript support, estimates overall conservation of 61% of alternative and 74% of constitutive splice junctions, albeit with broad confidence intervals. Observed numbers of conserved alternative splicing events agreed with those expected on the basis of the model. Thus, it is apparent that many, and probably most, alternative splicing events are conserved between human and mouse. This, combined with the preservation of alternative frame stop codons in conserved frame breaking events, indicates a high level of commonality in patterns of gene expression between these two species.

Alternative Splicing↗
PubMed2003

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth.

The chloroplast-encoded psbA gene encodes the D1 polypeptide of the photosystem II reaction center, which is synthesized at high rates in the light. In Chlamydomonas reinhardtii, the psbA gene contains four self-splicing group I introns whose rates of splicing in vivo are increased at least 6-10-fold by light. However, because psbA is an abundant mRNA, and some chloroplast mRNAs appear to be in great excess of what is needed to sustain translation rates, the developmental significance of light-promoted splicing has not been clear. To address this and other questions, potentially destabilizing substitutions were made in several predicted helices of the fourth psbA intron, Cr.psbA4, and their effects on in vitro and in vivo splicing assessed. Two-nucleotide substitutions in P4 and P7 were necessary to substantially reduce splicing of this intron in vivo, although most mutations reduced self-splicing in vitro. The P7-4,5 mutant, whose splicing was completely blocked, showed no photoautotrophic growth and synthesis of a truncated D1 (exons 1-4) polypeptide from the unspliced mRNA. Most informative was the P4'-3,4 mutant, which exhibited a 45% reduction in spliced psbA mRNA, a 28% reduction in synthesis of full-length D1, and an 18% reduction in photoautotrophic growth. These results indicate that psbA mRNA is not in great excess, and that highly efficient splicing of psbA introns, which is afforded by light conditions, is necessary for optimal photosynthetic growth.

Animals↗
PubMed2006

A novel multifunctional factor involved in trans-splicing of chloroplast introns in Chlamydomonas.

In the chloroplast of Chlamydomonas reinhardtii, psaA mRNA is spliced in trans from three separate precursors which assemble to form two group II introns. A fourth transcript, tscA, completes the structure of the first intron. Of the fourteen nucleus-encoded factors involved in psaA splicing, only two are required for splicing of both introns. We cloned and characterized the first of these more general factors, Raa1. Consistently with its role in psaA splicing, Raa1 is imported in the chloroplast where it is found in a membrane fraction and is part of a large ribonucleoprotein complex. One mutant, raa1-L137H, is defective for splicing of both introns, but another allelic mutant, raa1-314B, still expresses the 3' part of the Raa1 gene and is deficient only in splicing of intron 2. This observation and a deletion analysis indicate the presence of two domains in Raa1. The C-terminal domain is necessary and sufficient for processing of tscA RNA and splicing of the first intron, while the central domain is essential for splicing of the second intron. The combination of these two functional domains in Raa1 suggests that this new factor may coordinate trans-splicing of the two introns to improve the efficiency of psaA maturation.

Algal Proteins↗
PubMed2006

Comprehensive splice-site analysis using comparative genomics.

We have collected over half a million splice sites from five species-Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana-and classified them into four subtypes: U2-type GT-AG and GC-AG and U12-type GT-AG and AT-AC. We have also found new examples of rare splice-site categories, such as U12-type introns without canonical borders, and U2-dependent AT-AC introns. The splice-site sequences and several tools to explore them are available on a public website (SpliceRack). For the U12-type introns, we find several features conserved across species, as well as a clustering of these introns on genes. Using the information content of the splice-site motifs, and the phylogenetic distance between them, we identify: (i) a higher degree of conservation in the exonic portion of the U2-type splice sites in more complex organisms; (ii) conservation of exonic nucleotides for U12-type splice sites; (iii) divergent evolution of C.elegans 3' splice sites (3'ss) and (iv) distinct evolutionary histories of 5' and 3'ss. Our study proves that the identification of broad patterns in naturally-occurring splice sites, through the analysis of genomic datasets, provides mechanistic and evolutionary insights into pre-mRNA splicing.

Animals↗
PubMed2003

High frequency of alternative splicing of human genes participating in the HIV-1 life cycle: a model using TSG101, betaTrCP, PPIA, INI1, NAF1, and PML.

Alternative splicing may generate splice forms with different biologic roles or missing protein domains implicated in the interaction with HIV-1. To address this issue, 6 human genes were investigated-tumor suppressor gene 101 (TSG101), beta-transducin repeats containing protein (betaTrCP), peptidyl-proly cis-trans isomerase, cyclophilin A (PPIA), integrase interactor 1 protein (INI1), Nef-associated factor 1 (NAF1), and promyelacytic leukemia (PML)-involved in the viral life cycle and HIV-1 pathogenesis. All 6 genes presented alternative splicing, and a combined bioinformatic and reverse transcription polymerase chain reaction (RT-PCR) analysis identified 27 new variants for a total of 53 splice forms (an average of 9 variants per gene). The predicted frequency of the various splice forms based on expressed sequence tags (EST) analysis corresponded to the semiquantitative findings on RT-PCR analysis for the cell culture systems and for native CD4 cells investigated. Interindividual variation in the frequencies of various splice forms in CD4 T cells from blood donors was observed for INI1. Cell type-specific variation of splice pattern was observed for NAF1. Eight splice forms lacked or modified motifs implicated in the interaction with HIV-1, underscoring the potential interest of assessing alternative splicing when investigating viral cell biology and pathogenesis.

Alternative Splicing↗
PubMed1993

Feline immunodeficiency virus gene expression: analysis of the RNA splicing pattern and the monocistronic rev mRNA.

The transcription pattern of the feline immunodeficiency virus (FIV) genome in a feline CD4+ cell line was examined. In addition to the genomic RNA (9.2 kb), at least five FIV-specific transcripts [5.2, 4.4 (doublet), 1.7 and 1.4 kb] were detected by using subgenomic restriction enzyme fragments of an FIV molecular clone or FIV-specific oligonucleotides as probes. Among these transcripts, the 9.2, 5.2 and 4.4 (doublet) kb mRNAs were not expressed in the cytoplasm of cells transfected with a rev- mutant. To determine the location of splice junctions in the FIV genome, we used PCR to amplify and clone cDNAs corresponding to the viral mRNAs from infected cells. The region between pol and env was found to contain at least two splice donor and three splice acceptor sites. Two splice acceptor sites were detected in the 3' region of env. By hybridization analysis and sequencing of cDNA clones, it was revealed that the medium sized mRNAs are derived from a single splice event, with different splice acceptor sites, and that the two smaller transcripts are doubly or triply spliced mRNAs. Our results demonstrate a complex pattern of alternative splicing of FIV mRNAs. Furthermore, we identified monocistronic rev mRNA species that employ a unique splice acceptor site.

Amino Acid Sequence↗
PubMed2000

Authentic Borna disease virus transcripts are spliced less efficiently than cDNA-derived viral RNAs.

Borna disease virus (BDV) is a non-segmented, negative-strand RNA virus that replicates and transcribes its genome in the nucleus of infected cells. It uses the cellular splicing machinery to generate a set of alternatively spliced mRNAs from the 2.8 and 7.1 kb primary transcripts, each harbouring two introns. To determine whether splicing of these transcripts is regulated by viral factors, the extent of splicing was studied in infected cells and COS-7 cells transiently transfected with plasmids encoding the 2.8 kb RNA of BDV. Unspliced RNA was found to be the most abundant RNA species in infected cells, whereas viral transcripts lacking both introns were only found in minute amounts. In sharp contrast, plasmid-derived 2.8 kb RNA was predominantly intron 1-spliced and double-spliced. Co-expression of the BDV proteins P, N and X did not influence splicing of plasmid-expressed 2.8 kb RNA. Furthermore, the splicing pattern did not change when the 2.8 kb RNA was expressed in BDV-infected cells. Based on these results we speculate that splicing of authentic BDV transcripts is tightly linked to transcription by the viral polymerase.

Animals↗
PubMed2003

Human cytomegalovirus UL37 immediate early target minigene RNAs are accurately spliced and polyadenylated.

The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for virus growth. The UL37 IE promoter drives production of differentially spliced and unspliced RNAs. To study their post-transcriptional processing, we generated target minigenes encoding each UL37 RNA splicing substrate. Target 1 RNA, spanning UL37 exon 1 (x1) donor and 2 (x2) acceptor as well as adjacent intronic sequences, but not the UL38 gene, accurately reproduced UL37 x1/x2 RNA splicing in transfected permissive cells. Surprisingly, deletion of distal intronic sequences nt -82 to -143 from the UL37x2 acceptor resulted in aberrant splicing to an upstream non-consensus exonic donor. Target 1 RNAs carry the UL37x1 polyadenylation (PA) signal and site as well as a downstream SV40 early PA signal. Both the UL37x1 and SV40 PA signals are used in wild-type target 1 RNAs but inhibited in UL37x1 PA signal mutants. Alternative RNA splicing of UL37 exons 2 to 3 or 3A as well as exons 3 to 4, observed in HCMV mature UL37 and UL36 spliced RNAs, is accurately reproduced with target minigene RNAs carrying the corresponding UL37 exonic and intronic sequences. Moreover, alternative splicing using two novel UL37 exon 3 consensus splice donors (di and dii) was found in target and in HCMV-infected cell RNA. These results demonstrate that: (i) target minigene RNAs accurately recapitulate the processing of UL37 IE RNAs in the HCMV-infected cell; (ii) precise UL37x1 donor selection is modulated by 3'-distal UL37 intronic sequences; and (iii) UL37 exon 3 contains multiple alternative consensus splice donors.

Base Sequence↗
PubMed1987

Activation of a cryptic 5' splice site in the upstream exon of the phage T4 td transcript: exon context, missplicing, and mRNA deletion in a fidelity mutant.

A collection of 100 td mutants defective in phage T4 thymidylate synthase (TS) production was screened for splicing impairments. Splicing-defective mutants were identified by a rapid assay developed to detect imbalances in the td protein products (TS, the exon ligation product, and NH2TS, encoded by the pre-mRNA). Thirteen selected mutants, confirmed to be splicing defective by an RNA-oligodeoxynucleotide hybridization assay, were all shown to be inhibited in the first step of the group I splicing pathway, cleavage at the 5' splice site. Of these, only one, SC99, appeared to be a specificity mutant. Whereas the 12 other mutants had sequence changes within the functionally important 5' and 3' domains of the intron, SC99 was shown to be an exon mutant. The G----A change at residue -3 of the upstream exon of SC99 resulted in loss of normal 5' splice site recognition. Furthermore, activation of a remote cryptic splice site at residue -29 of the upstream exon and missplicing of mRNA that is deleted for 29 nucleotides of the 5' exon are characteristic for this mutant. These results underscore the role of exon sequences in guiding the fidelity of the splicing reaction and they raise provocative questions about the alignment of introns within exon contexts that are consistent with accurate splicing and synthesis of an intact gene product.

Base Sequence↗
PubMed1999

Metal ion catalysis during group II intron self-splicing: parallels with the spliceosome.

The identical reaction pathway executed by the spliceosome and self-splicing group II intron ribozymes has prompted the idea that both may be derived from a common molecular ancestor. The minimal sequence and structural similarities between group II introns and the spliceosomal small nuclear RNAs, however, have left this proposal in question. Mechanistic comparisons between group II self-splicing introns and the spliceosome are therefore important in determining whether these two splicing machineries may be related. Here we show that 3'-sulfur substitution at the 5' splice site of a group II intron causes a metal specificity switch during the first step of splicing. In contrast, 3'-sulfur substitution has no significant effect on the metal specificity of the second step of cis-splicing. Isolation of the second step uncovers a metal specificity switch that is masked during the cis-splicing reaction. These results demonstrate that group II intron ribozymes are metalloenzymes that use a catalytic metal ion for leaving group stabilization during both steps of self-splicing. Furthermore, because 3'-sulfur substitution of a spliceosomal intron has precisely the same effects as were observed during cis-splicing of the group II intron, these results provide striking parallels between the catalytic mechanisms employed by these two systems.

Allosteric Regulation↗
PubMed1988

Alternative splicing of SV40 early pre-mRNA is determined by branch site selection.

Splicing of SV40 early pre-mRNA to alternative large-T and small-t mRNAs involves the utilization of multiple lariat branch sites. To determine the functional significance of these sites, we constructed and analyzed a set of base substitution mutants in which the major branch acceptors were altered, either singly or in combination. The ratio of large-T to small-t mRNAs produced in vivo was found to vary by over 100-fold between different mutants. When splicing was assayed in vitro with a standard pre-RNA, which results in splicing almost exclusively to large-T mRNA, the patterns of branch site utilization were altered dramatically, although the mutations were essentially without effect on splicing efficiency. However, use of a 5' truncated pre-RNA, which results in a splicing pattern that reflects the in vivo alternative splicing potential of this pre-RNA, revealed a strong correlation between the effects of the base substitutions on branch site selection and alternative splice-site utilization. An RNase protection analysis of factor interactions with the 5' splice sites and branch sites in wild-type and mutant pre-RNAs suggests that a competition for different branch sites plays a crucial role in the assembly of 'alternative' spliceosomes, thereby controlling alternative splice-site selection.

Base Sequence↗
PubMed1991

The Neurospora mitochondrial tyrosyl-tRNA synthetase is sufficient for group I intron splicing in vitro and uses the carboxy-terminal tRNA-binding domain along with other regions.

Neurospora mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by nuclear gene cyt-18, functions in splicing of group I introns in mitochondria. Here, we overproduced functional cyt-18 protein in Escherichia coli and purified it to near homogeneity. The purified protein has splicing and tyrRS activities similar to those of cyt-18 protein isolated from mitochondria and is by itself sufficient to splice the mitochondrial large rRNA intron in vitro. Structure-function relationships in the cyt-18 protein were analyzed by in vitro mutagenesis. We confirmed that a small amino-terminal domain not found in bacterial tyrRSs is required for splicing activity, but not tyrRS activity. Two linker insertion mutations, which disrupt the predicted ATP-binding site, completely inhibit tyrRS activity but leave substantial splicing activity. Finally, deletions or linker insertion mutations in the putative carboxy-terminal tRNA-binding domain inhibit both tyrRS and splicing activities, although some have differential effects on the two activities. Our results show that the normal catalytic activity of the cyt-18 protein is not required for splicing and are consistent with the hypothesis that the protein functions by binding to the precursor RNA and facilitating formation of the correct RNA structure. Regions required for splicing are distributed throughout the cyt-18 protein and overlap, but are not identical to, regions required for tyrRS activity. The finding that the putative carboxy-terminal tRNA-binding domain is required for both tyrRS and splicing activities suggests that the mechanism for binding the intron has similarities to the mechanism for binding tRNA(Tyr).

Adenosine Triphosphate↗
PubMed1993

Coupling of poly(A) site selection and trans-splicing in Leishmania.

Intergenic regions of polycistronic pre-mRNAs of trypanosomatid protozoans are the sites of two processing reactions: polyadenylation of the upstream gene and trans-splicing of the capped miniexon to the downstream gene. Their close proximity and the lack of consensus motifs at poly(A) sites led us to test whether poly(A) site selection is governed by the location of the downstream splice acceptor in the DHFR-TS locus of Leishmania major. Whenever the position of the downstream splice site was altered, the poly(A) site was shifted 400-500 nucleotides upstream of the new splice site. In contrast, when the wild-type poly(A) site was eliminated, the downstream splice site was unaffected, and polyadenylation was maintained 200-500 nucleotides upstream of the splice site. In a second set of experiments, T7 RNA polymerase expressed in Leishmania was used to direct the synthesis of artificial pre-RNAs in vivo whose expression was found to require the presence of a downstream splice acceptor. We conclude that poly(A) site selection in Leishmania is specified by the position of the downstream splice acceptor and propose a scanning model for poly(A) site selection after splice site recognition.

Animals↗