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At least 829 records · Page 46Linked to original sources
PubMed1999

Regulation of alternative splicing by RNA editing.

The enzyme ADAR2 is a double-stranded RNA-specific adenosine deaminase which is involved in the editing of mammalian messenger RNAs by the site-specific conversion of adenosine to inosine. Here we identify several rat ADAR2 mRNAs produced as a result of two distinct alternative splicing events. One such splicing event uses a proximal 3' acceptor site, adding 47 nucleotides to the ADAR2 coding region, changing the predicted reading frame of the mature ADAR2 transcript. Nucleotide-sequence analysis of ADAR2 genomic DNA revealed the presence of adenosine-adenosine (AA) and adenosine-guanosine (AG) dinucleotides at these proximal and distal alternative 3' acceptor sites, respectively. Use of the proximal 3' acceptor depends upon the ability of ADAR2 to edit its own pre-mRNA, converting the intronic AA to an adenosine-inosine (AI) dinucleotide which effectively mimics the highly conserved AG sequence normally found at 3' splice junctions. Our observations indicate that RNA editing can serve as a mechanism for regulating alternative splicing and they suggest a novel strategy by which ADAR2 can modulate its own expression.

Adenosine↗
PubMed2001

Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing.

The functional repertoire of the human genome is amplified by the differential assortment of exons. Spliceosome-mediated RNA trans-splicing can mobilize these packets of genetic information to reprogram mRNAs. In principle, this process could repair defective transcripts in loss-of-function genetic disorders in humans. We developed a tractable lacZ repair system to serve as a model for these genetic disorders. Targeted pre-trans-splicing RNA molecules efficiently and specifically repaired mutated lacZ transcripts and restored enzymatic activity in human cells. The development of this model confirms the potential for spliceosome-mediated RNA trans-splicing in genetic repairs and provides a powerful tool for rational design and in vitro evolution of pre-trans-splicing molecules.

Cell Fractionation↗
PubMed1989

Scanning from an independently specified branch point defines the 3' splice site of mammalian introns.

During pre-messenger RNA splicing the lariat branch point in mammalian introns is specified independently of the 3' splice site by the sequence surrounding the branch point and by an adjacent downstream polypyrimidine tract. The 3' splice site is dispensable for spliceosome assembly and cleavage at the 5' splice site, and is itself determined by a scanning process that recognizes the first AG located 3' of the branch point/polypyrimidine tract, irrespective of distance or sequence environment.

Animals↗
PubMed1993

A general two-metal-ion mechanism for catalytic RNA.

A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA. The mechanism postulates that chemical catalysis is facilitated by two divalent metal ions 3.9 A apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I. One metal ion activates the attacking water or sugar hydroxyl, while the other coordinates and stabilizes the oxyanion leaving group. Both ions act as Lewis acids and stabilize the expected pentacovalent transition state. The symmetry of a two-metal-ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P. The role of the RNA is to position the two catalytic metal ions and properly orient the substrates via three specific binding sites.

Catalysis↗
PubMed1997

Common themes in the function of transcription and splicing enhancers.

Regulation of both transcription and RNA splicing requires enhancer elements, that is, cis-acting DNA or RNA sequences that promote the activities of linked promoters or splice sites, respectively. Both types of enhancer associate with regulatory proteins to form multicomponent enhancer complexes that recruit the necessary enzymatic machinery to promoter or splice site recognition sequences. This recruitment occurs as a result of direct interactions between regulatory proteins in the enhancer complexes and components of the basic enzymatic machineries. Recent advances suggest that the high degree of regulatory specificity observed for both transcription and splicing is due, in large part, to the multicomponent nature of enhancer complexes and to their cooperative assembly.

Animals↗
PubMed1989

Alteration of substrate specificity for the endoribonucleolytic cleavage of RNA by the Tetrahymena ribozyme.

A shortened form of the intervening sequence of the self-splicing RNA from Tetrahymena thermophila catalyzes sequence-specific cleavage of RNA. Cleavage site selection involves a base-pairing interaction between the substrate RNA and a binding site within the intervening sequence. Single-base changes in this binding site were previously shown to alter substrate specificity in a predictable manner. To examine the generality with which substrate specificity can be altered, six variant catalytic RNAs (ribozymes) have been produced with two- or three-base changes in the active site. Each ribozyme cleaves its predicted substrate. The conditions required for good reactivity and for discrimination against cleavage at mismatched sites vary and were independently determined for each ribozyme.

Animals↗
PubMed2004

CRS1, a chloroplast group II intron splicing factor, promotes intron folding through specific interactions with two intron domains.

Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1's specificity for the atpF intron in vivo can be explained by CRS1's intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1's intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments.

Binding Sites↗
PubMed1992

Retroviral insertions downstream of the heterogeneous nuclear ribonucleoprotein A1 gene in erythroleukemia cells: evidence that A1 is not essential for cell growth.

A large number of novel cellular proto-oncogenes have been identified and cloned by analysis of common integration sites in retrovirally induced malignancies. In the multistage erythroleukemias induced by the various strains of Friend leukemia virus, the analysis of proviral-integration events has led to the identification of two genes, Fli-1 and Spi-1, both novel members of the ets oncogene family of transcription factors. In this report, we describe the identification of another integration site, designated Fli-2 (Friend leukemia virus integration-2), in an erythroleukemia cell line induced by Friend murine leukemia virus (F-MuLV). Rearrangements at the Fli-2 locus were found in two erythroleukemia cell lines independently induced by F-MuLV and one leukemic cell line derived from the spleen of a mouse infected with the polycythemia strain of Friend leukemia virus. The deduced amino acid sequence of a cDNA corresponding to a transcript originating from genomic DNA adjacent to Fli-2 is identical to that of the human heterogeneous nuclear ribonucleoprotein A1 gene, a member of the gene family of RNA-binding proteins involved in RNA splicing. In one erythroleukemia cell line, A1 expression was undetectable as a result of F-MuLV integration in one allele and loss of the other allele. These results suggest that perturbations in RNA splicing mechanisms may contribute to malignant transformation and provide direct evidence that the A1 protein is not required for cell growth.

Amino Acid Sequence↗
PubMed1997

The molecular basis of partial penetrance of splicing mutations in cystic fibrosis.

The splicing variant, 5T allele, in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was shown to be associated with partial penetrance of the clinical expression. This splicing variant leads to two possible transcripts: one normal and the other aberrantly spliced that lacks exon 9. The aim of this study was to analyze the molecular basis of the partial penetrance in individuals carrying the 5T allele. We analyzed the level of the correctly spliced RNA transcribed from the 5T allele in nasal and epididymal epithelium and correlated it with disease expression. Semiquantitative nondifferential reverse-transcriptase-PCR showed a considerable variability (6%-37%) in the total level of correctly spliced RNA transcribed from the 5T allele in nasal epithelium from 11 patients. A significant nonlinear correlation (r = .82, P = .002) between the level of the normal CFTR transcripts and the severity of lung disease was shown. No individuals with normal lung function and minimal or no lung disease (FEV1 >80% predicted) had <25% of normal transcripts, and individuals with <15% of normal transcripts did not have FEV1 >80%. The level of normal transcripts in epididymal epithelial cells from four infertile males with congenital bilateral absence of the vas deferens was low (6%-24%). In infertile males with normal lung function the level of correctly spliced transcripts in the nasal epithelium was higher than the level in the epididymal epithelium. These results indicate that there is variability in the efficiency of the splicing mechanism, among different individuals and between different organs of the same individual. This variability provides the molecular basis of the partial penetrance of cystic fibrosis disease in patients carrying the 5T allele.

Adolescent↗
PubMed2003

The prodomain of interleukin 1alpha interacts with elements of the RNA processing apparatus and induces apoptosis in malignant cells.

Interleukin 1alpha (IL-1alpha), a 33 kDa precursor, is cleaved releasing the 17 kDa carboxyl-terminal cytokine IL-1alpha to which all of the biological properties of IL-1alpha have been attributed. We investigated the potential independent properties of the remaining 16 kDa IL-1alpha amino-terminal propiece by expression in human tumor and primary human cell lines. The IL-1alpha propiece produced apoptosis in malignant but not normal cell lines. A minimal fragment comprised of amino acids 55-108 was required for apoptosis. Deletion and mutation studies identified an extended nuclear localization sequence required for nuclear localization, induction of apoptosis and concentration of the IL-1alpha propiece in interchromatin granule clusters, concentrations of proteins in the RNA splicing and processing pathways. The IL-1alpha propiece interacted with five known components of the RNA splicing/processing pathway, suggesting that the mechanism of action may involve changes in RNA splicing or processing. Expression of the IL-1alpha propiece caused a shift in the ratio of Bcl-Xl/Bcl-Xs toward the apoptotic direction. Our findings indicate that the IL-1alpha propiece induces apoptosis in a range of tumor cells and likely operates through a mechanism involving the RNA processing apparatus and the alternate splicing of apoptosis regulatory proteins.

Amino Acid Sequence↗
PubMed1994

Cloning, expression, and distribution of functionally distinct Ca(2+)-activated K+ channel isoforms from human brain.

We have cloned and expressed nine Ca(2+)-activated K+ channel isoforms from human brain. The open reading frames encode proteins ranging from 1154 to 1195 amino acids, and all possess significant identity with the slowpoke gene products in Drosophila and mouse. All isoforms are generated by alternative RNA splicing of a single gene on chromosome 10 at band q22.3 (hslo). RNA splicing occurs at four sites located in the carboxy-terminal portion of the protein and gives rise to at least nine ion channel constructs (hbr1-hbr9). hslo mRNA is expressed abundantly in human brain, and individual isoforms show unique expression patterns. Expression of hslo mRNA in Xenopus oocytes produces robust voltage and Ca(2+)-activated K+ currents. Splice variants differ significantly in their Ca2+ sensitivity, suggesting a broad functional role for these channels in the regulation of neuronal excitability.

Alternative Splicing↗
PubMed1994

A unique p53 intragenic deletion flanked by short direct repeats results in loss of mRNA expression in a human esophageal carcinoma.

A 45 base pair (bp) intragenic deletion of the p53 gene from a human esophageal cancer was analyzed in detail. This deletion contained all RNA splice consensus sequences at the 3' end of intron 7, including the RNA splice branch point, the pyrimidine-rich region and the 3' splice acceptor site. Northern blotting revealed a total lack of p53 mRNA expression in this tumor. Short direct repeats (TACTG) were found at the 5' and 3' breakpoints of the deletion and it removed one complete repeat as well as the entire region between the repeats. These results suggest that a 'slipped mispairing' mechanism occurring during DNA replication may generate p53 intragenic deletion in human esophageal cancer, leading to abolished p53 mRNA expression.

Base Sequence↗
PubMed1994

[Presence of alternatively spliced messenger RNA for HLA-A, -B and -C in platelets].

Recently we demonstrated that platelets contain specific messenger RNA (mRNA) encoding for the transmembrane HLA class I molecule (44 kDa). In this study we analyzed lymphocytes as well as platelets for the presence of alternatively spliced mRNA with deleted transmembrane domain resulting in soluble HLA class I antigens (39 kDa). PCR amplification of lymphocyte RNA led to a product specific for alternatively spliced cDNA corresponding to the presence of soluble HLA in plasma, while this product could not be obtained from platelet mRNA. These results suggest that platelets in contrast to lymphocytes are unable to synthesize soluble HLA antigens via alternative splicing.

Alternative Splicing↗
PubMed2004

Multifocal transitional cell carcinoma of the bladder and upper urinary tract: molecular screening of clonal origin by characterizing CD44 alternative splicing patterns.

PURPOSE: CD44 is a widely expressed cell surface adhesion molecule in which various isoforms arise from alternative RNA splicing mechanism during cancer initiation. We assessed whether multifocal transitional cell carcinoma of the urothelium is due to field change and/or intraluminal seeding and implantation. MATERIALS AND METHODS: In a series of 24 patients with synchronous and/or metachronous multiple urothelial cancers we performed reverse transcription-polymerase chain reaction analysis using a set of primers capable of amplifying all CD44 splice variant isoforms. After polymerase chain reaction products were electrophoresed band intensities with areas corresponding to the major isoforms (that is CD44s, CD44v10 and CD44v8-10) were quantified, and CD44v10-to-CD44s and CD44v8-10-to-CD44s ratios were calculated. Moreover, p53 gene mutations in exons 4 to 11 were screened by direct DNA sequencing. RESULTS: Of these 24 cases 18 showed similar CD44v10-to-CD44s and CD44v8-10-to-CD44s ratios in among multiple urothelial cancers in each case. However, in the remaining 6 cases these ratios were quite different among multiple cancer lesions. Furthermore, different types of p53 mutation were detected among multiple cancer lesions in only 2 of 24 cases, which also indicated different patterns of CD44 alternative splicing. CONCLUSIONS: These findings suggest that at least some multiple transitional cell carcinomas of the urothelium seem to be of independent origin based on the analysis of alternative RNA splicing of CD44. Moreover, this hypothesis was further supported by the evaluation of p53 gene mutation.

Alternative Splicing↗
PubMed2001

fusilli, an essential gene with a maternal role in Drosophila embryonic dorsal-ventral patterning.

The Drosophila fusilli (fus) gene was identified in a genetic screen for dominant maternal enhancers of an unusual dorsalizing mutation in the cactus gene, cact(E10). While females that are heterozygous for the cact(E10) allele produce embryos with wild-type dorsal-ventral patterning, more than 90% of the embryos produced by females that are heterozygous for both cact(E10) and the fus(1) mutation are weakly dorsalized. Loss of fusilli activity causes lethality during embryogenesis but not dorsal-ventral patterning defects, indicating that fusilli is important in more than one developmental process. The fusilli gene encodes a protein with RNA binding motifs related to those in mammalian hnRNP F and H, which play roles in regulated RNA splicing. The fusilli RNA is not present in the oocyte or early embryo, and germ-line clones of fusilli mutations have no maternal effect on dorsal-ventral patterning, indicating that the fusilli maternal effect does not depend on germ-line expression of the gene. Because the fusilli RNA is present in ovarian follicle cells, we propose that fusilli acts downstream of the Drosophila EGF receptor to control the biogenesis of follicle cell transcripts that control the initial dorsal-ventral asymmetry of the embryo.

Amino Acid Sequence↗
PubMed1997

Essential promoter elements are located within the 5' untranslated region of human insulin-like growth factor-I exon I.

Multiple mechanisms regulate insulin-like growth factor-I (IGF-I) gene expression in mammals, including transcription from two promoters, alternative RNA splicing, and differential RNA polyadenylation. In previous studies we demonstrated that IGF-I promoter 1, the major human promoter, initiated transcription within a dispersed 158 nt segment of exon 1, and showed that full promoter activity required the entire 322 nt 5' untranslated region (UTR) of exon 1. We now have examined the functional significance of this highly conserved region by testing the activity of hybrid promoter-reporter genes containing various portions of the 5' UTR after transient transfection into the IGF-I-producing SK-N-MC cell line. Recombinant plasmids containing the entire 322 nt 5' UTR of exon 1 and a 1630 nt segment of 5' flanking sequence stimulated luciferase activity nearly 70 times higher than a promoterless control plasmid. Truncation to +198 had little effect on promoter function, while subsequent 3' deletions (to +111. +51, and +5) led to a stepwise decrease in reporter gene expression. Internal deletions of nt +6 to +50, +52 to +110, and +112 to +197 led to 65, 25, and less than 10% decreases in promoter function, respectively. Removal of the entire segment from +6 to +197 caused a complete loss of activity. Analysis for DNA-protein interactions by in vitro DNase-I footprinting identified a broad region of protection extending from nt -12 to +38. Further characterization by gel mobility shift assays indicated that several specific DNA-protein complexes could be formed in this region with nuclear protein extracts from SK-N-MC cells. Substitution mutations within the footprinted segment had deleterious effects on promoter function, and one mutation involving nt +10 to +20 resulted in a greater than 70% decline in reporter gene expression. Our results demonstrate that the initial portion of the 5' UTR of human IGF-I exon 1 is required for high level basal transcription of promoter 1 and provide a starting point for defining the nuclear factors regulating this component of IGF-I gene expression.

Base Sequence↗
PubMed2000

Systematic and large-scale two-hybrid screens.

The increasing rate at which complete genome sequences become available necessitates rapid and robust methods for investigating the functions of their encoded proteins. Efforts have been made to study protein function by systematically screening large sets of proteins using the two-hybrid method. Analyses of the complete proteomes of baceriophage T7, the mammalian viruses hepatitis C and vaccinia, as well as of several protein complexes including RNA splicing proteins and RNA polymerase III from yeast, have been undertaken. Saccharomyces cerevisiae has been studied extensively by two-hybrid methods, with more than 2500 protein-protein interactions described. Systematic studies on metazoan proteomes are, however, still in their infancy.

Animals↗