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Results for “RNA Splicing”

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PubMed1988

RNA splicing and genes.

The splicing of long transcripts of RNA (copied from DNA in the cell nucleus) into smaller, specific mRNA (ready for export to the protein-producing machinery in the cytoplasm) is an important event in the regulation of gene expression in eukaryotic cells. The splicing reaction occurs as a late step in the nuclear pathway for synthesis of mRNAs. This pathway commences with initiation of transcription by RNA polymerase II and probably involves an integrated series of steps each dependent on previous events. Splicing of precursors to mRNAs involves the formation of a spliceosome complex containing the 5' and 3' splice sites. This complex contains the evolutionarily highly conserved small nuclear RNAs (snRNAs) U2, U4, U5, and U6. The most abundant snRNA, U1, is required to form the spliceosome and may be a part of the spliceosome. Analogues of these snRNAs have been identified in yeast. Assembly of the spliceosome probably involves the binding of a multi-snRNA complex containing U4, U5, and U6 snRNAs. Several observations suggest that the association of snRNAs in such complexes is quite dynamic. It is argued that the snRNAs in the spliceosome form a catalytic RNA structure that is responsible for the cleavage and ligation steps during splicing.

Animals↗

PubMed2001

Impaired RNA splicing of 5'-regulatory sequences of the astroglial glutamate transporter EAAT2 in human astrocytoma.

A loss of the glutamate transporter EAAT2 has been reported in the neoplastic transformation of astrocytic cells and astrocytoma. The RNA expression of EAAT2 and five 5'-regulatory splice variants was investigated to identify alterations of the post-transcriptional EAAT2 gene regulation in human astrocytic tumours. Three known (EAAT2, HBGTII, and HBGTIIC) and two novel (EAAT2/3 and EAAT2/31) EAAT2 transcripts originating from alternative splicing of 5'-regulatory sequences were subject to an RNA expression analysis using reverse transcription and competitive PCR. Specimens of astrocytoma World Health Organisation (WHO) grade I-IV in 14 patients and control brain tissue obtained from three normal persons were studied. The main EAAT2 RNA was found to be equally expressed in normal human brain and astrocytic tumour samples. By contrast, the expression pattern of four 5'-variants of the transporter transcript was altered in the investigated series of astrocytoma compared with normal brain. HBGTII, HBGTIIC, and EAAT2/3 were amplified from seven and four tumours and one sample, respectively. EAAT2/31 was expressed in none of the tumour specimens studied. In conclusion, in astrocytic tumours of different histopathological grades there was a substantial reduction of RNA splicing events in EAAT2. The impairment of EAAT2 splicing indicates an altered expression which is not primarily involved in the tumorigenesis but may contribute to some biological properties of astrocytoma such as oedema, necrosis, and tumour related seizures.

Alternative Splicing↗

PubMed1986

Sequence and structure of the dopa decarboxylase gene of Drosophila: evidence for novel RNA splicing variants.

In Drosophila, dopa decarboxylase (DDC) serves a dual role in neurotransmitter production and sclerotization of the cuticle. The Ddc gene is under complex hormonal and tissue-specific control and several sizes of Ddc RNA are observed at embryonic hatching, pupariation and adult eclosion. We present here the complete nucleotide sequence of the Drosophila dopa decarboxylase gene and the partial sequence of two corresponding Ddc cDNAs. The sequence allows us to account for the detailed structure of four of the five major Ddc RNA species observed. The cDNA sequence reveals the existence of previously undetected splicing events and provides evidence for two RNA splicing alternatives which appear to encode two protein isoforms. The structure, processing and developmental regulation of the Ddc transcripts and putative protein isoforms are discussed. Interestingly, the pyridoxal-binding peptide of porcine DDC matches the Drosophila sequence perfectly suggesting considerable selective pressure on at least portions of the sequence. This is the first available Ddc gene sequence from any organism and should serve as a basis of comparison for the related proteins of other species.

Amino Acid Sequence↗

PubMed1992

Two unusual forms of human immunoglobulin E encoded by alternative RNA splicing of epsilon heavy chain membrane exons.

We present evidence for RNA transcripts encoding two forms of human epsilon immunoglobulin (Ig) heavy chain that differ significantly from those of other isotypes. We previously demonstrated three human epsilon mRNA species, instead of the two, corresponding to membrane and secreted proteins, seen with other heavy chain transcripts. In human genomic DNA downstream of the C epsilon gene, we identified sequences homologous to the two putative murine exons M1 (encoding a hydrophobic, presumably transmembrane region) and M2 (encoding hydrophilic residues). To determine the structures of epsilon transcripts containing these sequences, we amplified epsilon-related RNAs with the reverse transcriptase polymerase chain reaction. RNA was examined from fresh human B cells stimulated to IgE production by interleukin 4 plus anti-CD40, as well as from the human IgE-producing line AF10. Instead of the single CH4-M1-M2 splice product predicted for murine membrane IgE, we found two other RNA species. One form has the structure CH4-M1'-M2, in which M1' includes the human sequence homologous to the murine M1 as well as a unique segment of 52 codons further upstream in the genomic sequence; this RNA species apparently encodes the IgE expressed on the membrane of IgE-producing lymphocytes. The other RNA has the structure CH4-M2', in which M2' is spliced in an alternative reading frame that includes an additional 109 codons downstream of the termination codon of the CH4-M1'-M2 form. Because the CH4-M2' mRNA form does not encode a hydrophobic segment, its translated product should be secreted. A secreted epsilon protein of approximately the size predicted for this form was identified by Western blotting. This novel IgE protein could play a significant and distinctive role in allergic disorders.

Amino Acid Sequence↗

PubMed2005

Regulation of Cdc2p and Cdc13p is required for cell cycle arrest induced by defective RNA splicing in fission yeast.

Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants.

Alternative Splicing↗

PubMed1988

RNA splicing in yeast mitochondria: DNA sequence analysis of mit- mutants deficient in the excision of introns aI1 and aI2 of the gene for subunit I of cytochrome c oxidase.

We have characterized two yeast mutants deficient in the splicing of transcripts of the mitochondrial gene for cytochrome c oxidase subunit I (coxI). Both map to the first intron (aI1). RNA blot analysis shows that in addition to a reduced (mutant M15-190) or blocked (mutant M12-193) excision of the mutated intron aI1, the mutants are unable to excise the adjacent aI2 intron, the reading frame of which displays an amino acid sequence similarity to aI1. Splicing of the downstream introns is not affected, however. Sequence analysis of the first mutant DNA (M12-193) reveals a premature termination of the intron-encoded open reading frame, followed by two alterations at a short distance downstream. The other (M15-190) contains 11 separate changes. Although these occur in the intron reading frame, their main effect on RNA splicing may be exerted through the disturbance of intron secondary structure proposed for the 5' end of several group II introns. The implications of these findings in relation to maturase function and structure of intron aI1 are discussed.

Base Sequence↗

PubMed2000

Identification and characterization of a structural protein of hepatitis B virus: a polymerase and surface fusion protein encoded by a spliced RNA.

The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using (32)P-labeling of an introduced protein kinase A site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22-nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins. This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with a deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF. This finding is consistent with the result obtained from an immunobiochemical study. Mutations at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. These mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase-surface fusion protein encoded by a spliced RNA. Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle.

Aspartic Acid Endopeptidases↗

PubMed2003

Regulation of adenovirus alternative RNA splicing correlates with a reorganization of splicing factors in the nucleus.

Subcellular compartmentalization of splicing factors has been proposed to play a role in the regulation of alternative splicing. In the present work we have addressed this issue using adenovirus-infected cells to visualize an alternative splicing switch at the single cell level. The adenovirus gene expression program requires the activation of distal alternative 3' splice sites during the late phase of infection in major late transcripts. We have established in situ hybridization conditions that allow for the specific detection of 3' alternatively spliced adenoviral mRNAs from the L1 transcript family. Results show that the switch from proximal to distal 3' splice sites correlates with a massive reorganization of splicing factors in the cell nucleus, involving their recruitment from nuclear speckles to sites of viral transcription. This observation raises the possibility that the subnuclear organization of splicing factors may be regulated in response to events that lead to the activation of alternative splicing programs.

Adenoviridae↗

PubMed1999

RNA splicing: What has phosphorylation got to do with it?

Many pre-mRNA splicing factors are phosphorylated in vivo, but the role of this modification has been unclear. Recent observations suggest that phosphorylation modulates protein-protein interactions within the spliceosome, thereby contributing to dynamic structural reorganization of the spliceosome during splicing.

Animals↗

PubMed1994

RNA splicing. U2 fulfils a commitment.

Genetic and biochemical studies of pre-mRNA splicing have recently converged to elucidate an early step in the process: the targeting of the U2 small nuclear ribonucleoprotein particle to the pre-mRNA.

Alternative Splicing↗

PubMed1986

A self-splicing RNA excises an intron lariat.

We have investigated the in vitro self-splicing of a class II mitochondrial intron. A model pre-mRNA containing intron 5 gamma of the oxi 3 gene of yeast mitochondrial DNA undergoes an efficient intramolecular rearrangement reaction in vitro. This reaction proceeds under conditions distinct from those optimal for self-splicing of class I introns, such as the Tetrahymena nuclear rRNA intron. Intron 5 gamma is excised as a nonlinear RNA indistinguishable from the in vivo excised intron product by gel electrophoresis and primer extension analysis. Studies of the in vitro excised intron product strongly indicate that it is a branched RNA with a circular component joined by a linkage other than a 3'-5' phosphodiester. Two other products, the spliced exons and the broken form of the lariat, were also characterized. These results show that the class II intron products are similar to those of nuclear pre-mRNA splicing.

DNA, Mitochondrial↗

PubMed1997

Cloning of Caenorhabditis U2AF65: an alternatively spliced RNA containing a novel exon.

The U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF, is an essential splicing factor required for recognition of the polypyrimidine tract and subsequent U2 snRNP assembly at the branch point. Because Caenorhabditis elegans introns lack both polypyrimidine tract and branch point consensus sequences but have a very highly conserved UUUUCAG/R consensus at their 3' splice sites, we hypothesized that U2AF might serve to recognize this sequence and thus promote intron recognition in C. elegans. Here we report the cloning of the gene for the large subunit of U2AF, uaf-1. Three classes of cDNA were identified. In the most abundant class the open reading frame is similar to that for the U2AF65 from mammals and flies. The remaining two classes result from an alternative splicing event in which an exon containing an in-frame stop codon is inserted near the beginning of the second RNA recognition motif. However, this alternative mRNA is apparently not translated. Interestingly, the inserted exon contains 10 matches to the 3' splice site consensus. To determine whether this feature is conserved, we sequenced uaf-1 from the related nematode Caenorhabditis briggsae. It is composed of six exons, including an alternatively spliced third exon interrupting the gene at the same location as in C. elegans. uaf-1 is contained in an operon with the rab-18 gene in both species. Although the alternative exons from the two species are not highly conserved and would not encode related polypeptides, the C. briggsae alternative exon has 18 matches to the 3' splice site consensus. We hypothesize that the array of 3' splice site-like sequences in the pre-mRNA and alternatively spliced exon may have a regulatory role. The alternatively spliced RNA accumulates at high levels following starvation, suggesting that this RNA may represent an adaption for reducing U2AF65 levels when pre-mRNA levels are low.

Alternative Splicing↗

PubMed1982

Abnormal RNA splicing causes one form of alpha thalassemia.

The alpha 2-globin gene from a patient with alpha thalassemia contains a pentanucleotide deletion in intron 1 immediately adjacent to exon 1. We tested the functional consequences of this mutation by introducing the thalassemic gene, along with its normal counterpart as a control, into cultured monkey cells on SV40 plasmid vectors. Both genes are expressed, at similar levels, into globin RNA with the correct 5' and 3' ends. However, while most of the normal transcripts are appropriately processed, the thalassemic transcripts are abnormally spliced from a 5' donor site in the middle of exon 1 to the normal 3' acceptor site. This results in the synthesis of a truncated RNA incapable of encoding a normal globin polypeptide. The alternative donor, also used at a low level in monkey cells transfected with the normal gene, shows strong homology to the consensus donor sequence characteristic of many eucaryotic splice junctions. No unspliced or partially spliced thalassemic RNA was detected, indicating that recognition of this site is efficient and does not block removal of intron 2. The alternatively spliced RNA was also found in bone marrow RNA from the alpha-thalassemic patient, although not in that from a normal individual or a beta +-thalassemic patient. The thalassemic phenotype of the patient therefore results from abnormal RNA splicing owing to the deletion of the first splice donor signal.

Base Sequence↗

PubMed1998

Oncoprotein TLS interacts with serine-arginine proteins involved in RNA splicing.

The gene encoding the human TLS protein, also termed FUS, is located at the site of chromosomal translocations in human leukemias and sarcomas where it forms a chimeric fusion gene with one of several different genes. To identify interacting partners of TLS, we screened a yeast two-hybrid cDNA library constructed from mouse hematopoietic cells using the C-terminal region of TLS in the bait plasmid. Two cDNAs encoding members of the serine-arginine (SR) family of proteins were isolated. The first SR protein is the mouse homolog of human splicing factor SC35, and the second SR member is a novel 183-amino acid protein that we term TASR (TLS-associated serine-arginine protein). cDNA cloning of human TASR indicated that mouse and human TASR have identical amino acid sequences. The interactions between TLS and these two SR proteins were confirmed by co-transfection and immunoprecipitation studies. In vivo splicing assays indicated that SC35 and TASR influence splice site selection of adenovirus E1A pre-mRNA. TLS may recruit SR splicing factors to specific target genes through interaction with its C-terminal region, and chromosomal translocations that truncate the C-terminal region of TLS may prevent this interaction. Thus TLS translocations may alter RNA processing and play a role in malignant transformation.

Amino Acid Sequence↗

PubMed1984

A novel mechanism of alternative RNA splicing for the developmentally regulated generation of troponin T isoforms from a single gene.

Troponin T (TnT) is a major regulatory protein of the striated muscle that exhibits developmental and tissue-specific structural heterogeneity. The molecular basis for this heterogeneity was studied at the level of TnT structural gene organization and RNA expression. Two tissue-specific and developmentally regulated TnT mRNAs, alpha and beta, are derived from a single fast skeletal muscle TnT gene. Although otherwise structurally identical from amino acid 70 to the end of the 3' untranslated region, the alpha and beta TnT mRNAs differ by a small internal oligonucleotide coding for amino acids 229 to 242. These isoform-specific oligopeptides, both spanning the same internal portion of the TnT protein, are encoded by two distinct and adjacent miniexons in the TnT gene. Alternative and mutually exclusive splicing of these two miniexons results in the incorporation of either exon into the mature TnT mRNA and argues persuasively against a processive scanning model of RNA splice site selection.

Animals↗

PubMed1999

Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer.

Alternative mRNA splicing of the fibronectin EDI exon is controlled by a purine-rich exonic splicing enhancer (ESE), postulated as a binding site for SR proteins. By using a transient expression alternative splicing assay combined with promoter swapping, we have demonstrated that the promoter can also control EDI splicing, arguing for coupling between the transcription and splicing machineries. We now report that the SR proteins SF2/ASF and 9G8 stimulate EDI splicing in vivo and that their effect requires an intact EDI ESE. Most importantly, we show that sensitivity to these SR proteins critically depends on the promoter structure, suggesting that the transcription machinery modulates their recruitment to the ESE.

Alternative Splicing↗

PubMed1991

Induction of human immunodeficiency virus type 1 expression in chronically infected cells is associated primarily with a shift in RNA splicing patterns.

We have analyzed the kinetics of human immunodeficiency virus type 1 (HIV-1) RNA induction in chronically infected T cells and promonocytes. A substantial amount of spliced mRNAs and assembled virions was found in resting cells. Induction increased the steady-state level of total HIV-1 RNA by 4-fold but increased the level of unspliced transcripts by 25-fold. This increase in unspliced RNA was reflected in the amount of virus seen by electron microscopy. These data suggest a mechanism for the induction of HIV-1 RNA in chronically infected cells involving a shift in splicing greatly favoring the stability of unspliced viral RNA with only a modest increase in total viral RNA. Analysis of the relative abundance of transcript classes is critical to the measurement of HIV-1 viral replication kinetics.

Base Sequence↗

PubMed1993

Temperature sensitivity of aberrant RNA splicing with a mutation in the G+5 position of intron 37 of the gene for type III procollagen from a patient with Ehlers-Danlos syndrome type IV.

A single-base mutation in intron 37 of the gene for type III procollagen (COL3A1) was found in a proband with the type IV variant of Ehlers-Danlos syndrome. Probe-protection experiments with S1 nuclease and RNA from fibroblasts incubated at 37 degrees C demonstrated that about 35% of the total mRNA or about 70% of the mRNA from mutated allele was spliced by exon skipping. The effects of the mutation were temperature-sensitive in that the amount of RNA from the mutated allele that was spliced by exon skipping was 87.1 +/- 7.7% at 31 degrees C, 70.1 +/- 6.5% at 37 degrees C, and 85.4 +/- 11.1% at 42 degrees C. The effects of temperature on aberrant RNA splicing were, therefore, the reverse of those reported for four previous mutants in collagen genes. The increase in abnormal RNA splicing when the temperature was raised from 31 degrees to 37 degrees C seen with previously reported mutants suggested that RNA-RNA hybridization of U1snRNA to the 5'-splice site in the substrate may be limiting in the processing of transcripts from the mutated alleles, since RNA-RNA hybridizations become less favorable at higher temperatures. The decrease in abnormal RNA splicing seen here when the temperature was raised from 31 degrees to 37 degrees C suggested that protein-RNA or protein-protein binding steps become rate limiting with the G+5 mutation in intron 37 of the COL3A1 gene.

Adolescent↗