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Results for “RNA Splicing”

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PubMed1997

RNA splicing specificity determined by the coordinated action of RNA recognition motifs in SR proteins.

Pre-mRNA splicing requires a large number of RNA-binding proteins that have one or more RNA-recognition motifs (RRMs). Among these is the SR protein family, whose members are essential for splicing and are able to commit pre-mRNAs to the splicing pathway with overlapping but distinct substrate specificity. Some SR proteins, such as SC35, contain an N-terminal RRM and a C-terminal arginine/serine-rich (RS) domain, whereas others, such as SF2/ASF, also contain a second, atypical RRM. Although both the RRMs and the RS domain of SR proteins are required for constitutive splicing, it is unclear which domain(s) defines their substrate specificity, and whether two RRMs in a given SR protein function independently or act coordinately. Using domain swaps between SC35 and SF2/ASF and a functional commitment assay, we demonstrate that individual domains are functional modules, RS domains are interchangeable, and substrate specificity is defined by the RRMs. The atypical RRM of SF2/ASF does not appear to function alone in splicing, but can either activate or suppress the splicing specificity of an N-terminal RRM. Therefore, multiple RRMs in SR proteins act coordinately to achieve a unique spectrum of pre-mRNA substrate specificity.

Binding Sites↗

PubMed1987

RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression.

A systematic analysis of the RNA splice junction sequences of eukaryotic protein coding genes was carried out using the GENBANK databank. Nucleotide frequencies obtained for the highly conserved regions around the splice sites for different categories of organisms closely agree with each other. A striking similarity among the rare splice junctions which do not contain AG at the 3' splice site or GT at the 5' splice site indicates the existence of special mechanisms to recognize them, and that these unique signals may be involved in crucial gene-regulation events and in differentiation. A method was developed to predict potential exons in a bare sequence, using a scoring and ranking scheme based on nucleotide weight tables. This method was used to find a majority of the exons in selected known genes, and also predicted potential new exons which may be used in alternative splicing situations.

Animals↗

PubMed1987

Nuclear suppression of a mitochondrial RNA splice defect: nucleotide sequence and disruption of the MRS3 gene.

A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-101. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEp13 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-101 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5' end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.

Base Sequence↗

PubMed1987

Analysis of the gene and multiple messenger ribonucleic acids (mRNAs) encoding human gastrin-releasing peptide: alternate RNA splicing occurs in neural and endocrine tissue.

Gastrin-releasing peptide (GRP), the mammalian homolog of the amphibian peptide bombesin, is encoded in man by a single gene located on chromosome 18. Restriction enzyme and DNA sequence analyses establish that the gene is 10 kilobases in size with two introns of 4.8 and 3.9 kilobases. Exon 1 encodes the 5'-untranslated region, the signal peptide, and the first 23 amino acids of GRP. Exon 2 encodes the remaining three complete amino acids of GRP and the first 74 amino acids of the GRP carboxy-terminal extension peptide. Hence, intron 1 interrupts the coding region of the bioactive portion of GRP between the first and second nucleotides for Gly, the 24th amino acid of GRP. Exon 3 encodes the remainder of the GRP-extension peptide and the 3'-untranslated region. Two GC-rich, potential regulatory sequences and a sequence associated with regulation by cAMP lie between the CAAT and TATA boxes; the primary transcriptional start site is located 30 bases downstream from the TATA box. The second intron has an alternate donor site at its 5'-end and an alternate acceptor site at its 3'-end. S1 nuclease mapping demonstrates that differential RNA splicing using these sites results in the similar expression of three GRP mRNAs in GRP-containing neurons (in stomach and brain) as well as in GRP-containing neuroendocrine cells (fetal lung). In addition, the pattern of RNA splicing is similar between normal tissue and neoplastic tissue (small cell carcinoma of the lung and medullary carcinoma of the thyroid).

Base Sequence↗

PubMed1993

The role of small nuclear RNAs in RNA splicing.

Recent genetic and biochemical experiments have revealed an intimate and dynamic role for small nuclear RNAs (snRNAs) in multiple steps of RNA-splicing reactions. Both snRNA-substrate and snRNA-snRNA interactions are involved. These interactions concern not only splice site and branch point definition, but also the catalytic reactions of the first and second steps of splicing. Studies reveal a striking conservation between snRNA interactions and interactions found in RNAs encoded by genes with group II self-splicing introns.

Animals↗

PubMed1990

The self-splicing RNA of Tetrahymena is trapped in a less active conformation by gel purification.

When the circular form of the self-splicing intervening sequence of Tetrahymena thermophila was purified by denaturing polyacrylamide gel electrophoresis by standard methods, the rate of its reaction with tetrauridylate decreased 150-fold at 30 degrees C and at least 1000-fold at 0 degrees C. The activity of the self-splicing RNA was restored by heating it to high temperature and letting it renature in the presence of Mg2+. The rate of reaction of tetrauridylate with the self-splicing RNA flanked by exons was also greatly decreased by gel purification. The difference in activation energies for the reaction of native and denatured intervening sequences suggests that a substantial conformational rearrangement of the gel-purified RNA occurs prior to reaction.

Animals↗

PubMed2004

Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements.

Alternative RNA splicing is now known to be pervasive throughout the genome and a target of human disease. We evaluated if targeting intronic splicing regulatory sequences with antisense oligonucleotides could be used to correct aberrant exon skipping. As a model, we targeted the intronic silencing sequence (ISS) elements flanking the alternatively spliced alpha-exon of the endogenous fibroblast growth factor receptor 1 (FGFR1) gene, which is aberrantly skipped in human glioblastoma. Antisense morpholino oligonucleotides targeting either upstream or downstream ISS elements increased alpha-exon inclusion from 10% up to 70% in vivo. The effect was dose dependent, sequence specific and reproducible in several human cell lines, but did not necessarily correlate with blocking of protein association in vitro. Simultaneous targeting of the ISS elements had no additive effect, suggesting that splicing regulation occurred through a shared mechanism. Broad applicability of this approach was demonstrated by similar targeting of the ISS elements of the human hnRNPA1 gene. The correction of FGFR1 gene splicing to >90% alpha-exon inclusion in glioblastoma cells had no discernable effect on cell growth in culture, but was associated with an increase in unstimulated caspase-3 and -7 activity. The ability to manipulate endogenously expressed mRNA variants allows exploration of their functional relevance under normal and diseased physiological states.

Alternative Splicing↗

PubMed2004

CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing.

We have shown that steroid hormones coordinately control gene transcriptional activity and splicing decisions in a promoter-dependent manner. Our hypothesis is that a subset of hormonally recruited coregulators involved in regulation of promoter transcriptional activity also directly participate in alternative RNA splicing decisions. To gain insight into the molecular mechanisms by which transcriptional coregulators could control splicing decisions, we focused our attention on a recently identified coactivator, CoAA. This heterogeneous nuclear ribonucleoprotein (hnRNP)-like protein interacts with the transcriptional coregulator TRBP, a protein recruited to target promoters through interactions with activated nuclear receptors. Using transcriptional and splicing reporter genes driven by different promoters, we observed that CoAA mediates transcriptional and splicing effects in a promoter-preferential manner. We compared the activity of CoAA to the activity of other hnRNP-related proteins that, like CoAA, contain two N-terminal RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain and either have or have not been implicated in transcriptional control. By swapping either CoAA RRMs or the CoAA auxiliary domain with the corresponding domains of the proteins selected, we showed that depending on the promoter, the RRMs and the auxiliary domain of CoAA are differentially engaged in transcription. This contributes to the promoter-preferential effects mediated by CoAA on RNA splicing during the course of steroid hormone action.

Alternative Splicing↗

PubMed1996

Biochemical properties of a novel U2AF65 protein isoform generated by alternative RNA splicing.

A variety of RNA binding proteins with one or more RNA recognition (RNP-CS) motifs play essential roles in the pre-mRNA splicing process. One such factor, the U2 snRNP auxiliary factor large subunit (U2AF65), contains three RNP-CS motifs each of which is required for high affinity binding to polypyrimidine tracts. Here we report the isolation of a natural cDNA variant of human U2AF65, U2AF65 (S), which is shortened by a 12 nucleotide in frame deletion between RNP-CS2 and -CS3 motifs. A portion of the U2AF65 (S) cDNA was reported previously but was not characterized further. We observe that the U2AF65 (S) variant predominates in a variety of tissues and cell lines, and is generated together with the U2AF65 (L) form (2) by alternative 5' splice site selection from a single gene. The corresponding histidine-tagged recombinant proteins bind with similar affinities to model RNA substrates containing strong or weak polypyrimidine tracts. Both U2AF65 (S) and (L) protein isoforms reconstitute splicing activity with similar kinetic profiles in U2AF-depleted (splicing-deficient) HeLa nuclear extracts. Finally, the thermal stabilities of the protein isoforms are essentially equivalent. Thus, the presence or absence of the peptide segment, VSPP (residues 345-348), in the linker region between RNP-CS2 and -CS3 does not detract from the intrinsic RNA binding and splicing properties of the U2AF65 protein. The biological implications of alternative splicing for the function and evolution of RNA binding proteins are discussed.

Alternative Splicing↗

PubMed1988

Defective E beta expression in three mouse H-2 haplotypes results from aberrant RNA splicing.

The molecular basis for the defective expression of the mouse class II E beta genes in the H-2w17, H-2q, and H-2f haplotypes has been examined. The results of nuclear run-on transcription and S1 nuclease digestion assays demonstrate that E beta transcription is normal in these haplotypes. Northern blot analyses reveal reduced amounts of E beta RNA of both normal and aberrant size in the w17 and q haplotypes; an even more reduced level of E beta RNA of normal size was detected in the f haplotype. In the preceding study, we reported that the only defect detected in the E beta w17 gene is a single nucleotide insertion in the 5' RNA splice site of the first intervening sequence. S1 nuclease analysis of E beta w17 RNA indicates that splicing at this site is aberrant. One major cryptic RNA splice site is used, leading to reduced amounts of aberrantly processed RNA. Limited use of the mutated splice site and of a second cryptic site also is detected. In all three cases, stop codons in the resulting RNA would prevent their translation. The molecular defect in E beta q appears identical to that of E beta w17. In the f haplotype, even more reduced levels of E beta RNA of both normal and aberrant sizes are found. We thus show that in the three defective E beta alleles, two distinct defects are responsible for the absence of E beta protein synthesis; both of these defects affect RNA processing.

Animals↗

PubMed1984

RNA splicing in Neurospora mitochondria: nuclear mutants defective in both splicing and 3' end synthesis of the large rRNA.

We have identified nuclear mutants of Neurospora that are defective in splicing the mitochondrial large rRNA and that accumulate unspliced pre-rRNA (35S RNA). In cyt-4 mutants, the unspliced pre-rRNA contains short 3' end extensions (110 nucleotides) that are not present in pre-rRNAs from the other mutants. This and other characteristics suggest that the cyt-4 mutants may be primarily defective in 3' end synthesis and the RNA splicing defect occurs secondarily as a result of impaired RNA folding. The cyt-4 mutants also accumulate a "short" intron RNA and small exon RNAs that may reflect aberrant RNA cleavages. The 5' end of the short intron is about 285 nucleotides downstream from the 5' splice site at or near the base of the "central hairpin", a putative intermediate in folding of the pre-rRNA. Furthermore, the aberrant cleavage sites are immediately after a six nucleotide sequence (GAUAAU) homologous to the final splice junction (GAU/AAC).

Base Sequence↗

PubMed1983

Inactivation of an acceptor RNA splice site by a short deletion in beta-thalassemia.

The cloned beta-globin gene of an Indian patient with beta-thalassemia revealed a 25-nucleotide deletion at the 3'-end of the first intervening sequence, including the acceptor RNA splicing site. RNA transcripts of this mutant gene produced following transfection into HeLa cells remained unspliced at both the first intervening sequence donor and acceptor sites. This beta-thalassemic gene is the first in which critical sequences of an acceptor splice junction are mutated and associated with abnormal RNA processing.

Base Sequence↗

PubMed1992

RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes.

Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors (AChRs) on muscle fibers. A region near the carboxyl terminus of the protein exists in four forms that are generated by alternative RNA splicing. All four alternatively spliced forms of agrin are active in inducing AChR clusters on rat primary and C2-derived muscle fibers. In contrast, only two forms of the protein, each containing an 8 amino acid insert, are capable of inducing clusters on myotubes of S27 cells, a C2 variant that has defective proteoglycans. These two forms are also most active in inducing clusters on chick myotubes. This pattern of differential activity suggests that RNA splicing of agrin transcripts and interactions with proteoglycans or other components of basal lamina have important roles in regulating the localization of neurotransmitter receptors at synaptic sites.

Agrin↗

PubMed1983

An RNA ligase from wheat germ which participates in transfer RNA splicing in vitro.

Transfer RNA half-molecules are intermediates in the splicing of tRNA precursors containing intervening sequences. We have utilized yeast tRNA half-molecules to identify and partially purify an ATP-dependent RNA ligase activity from extracts of wheat germ. This activity can complement a yeast tRNA endonuclease in vitro to efficiently splice 10 different yeast tRNA precursors. The products of in vitro splicing are a covalently joined tRNA and a circular intervening sequence RNA. The internucleotide bond formed at the splice junction is a 2'-phosphomonoester, 3',5'-phosphodiester structure. The 2'-phosphate originates from the 2',3'-cyclic phosphate at the 3' terminus of the 5' half-tRNA. The phosphodiester phosphate is derived from the gamma-phosphate of ATP.

Phosphorus Radioisotopes↗

PubMed1984

RNA splicing as an error-screening mechanism.

Eukaryote nuclear genes are generally split into coding (exon) and noncoding (intron) regions. During the formation of messenger RNA the introns are precisely excised and the exons religated. A widely accepted explanation for the split structure of eukaryotic genes is that proposed by Gilbert who hypothesized that the division of coding information into small units speeded up the rate of protein evolution by allowing for the recombination of the independent peptide domains encoded by these units ("exon shuffling"). However it has recently become clear that the exon:intron structure of genes most likely preceded the uninterrupted form. This makes it difficult to accept Gilbert's argument as it applies to the origin(s) of split genes since early genes were very inaccurately copied and a highly error-prone system needs less variation not more. Here I propose that split genes and the concomitant process of RNA splicing arose as a mechanism for maintaining the stability of the genetic information in the face of a high level of noise in the gene copier mechanism.

Base Sequence↗

PubMed1995

Alternative RNA splicing generates diversity of neuropeptide expression in the brain of the snail Lymnaea: in situ analysis of mutually exclusive transcripts of the FMRFamide gene.

In the CNS of the snail Lymnaea stagnalis, Phe-Met-Arg-Phe-amide (FMRFamide)-like and additional novel neuropeptides are encoded by a common, multi-exon gene. This complex locus, comprising at least five exons, is subject to post-transcriptional regulation at the level of alternative RNA splicing. Our aim was first to analyse the pattern by which exons of this neuropeptide locus combine during splicing of the primary RNA transcript, and second to investigate the functional significance of splicing by mapping the expression and neuronal localization in the CNS of the alternative mRNA transcripts, in the context of defined neuronal networks and single identified neurons. The approach was a combination of comparative in situ hybridization and immunocytochemistry, using a battery of exon-specific oligonucleotides and anti-peptide antisera. The analysis illustrated that exons III, IV and V were always coexpressed and colocalized whereas the expression of exon II was always differential and mutually exclusive. Both sets of exons were, however, coexpressed with exon I: the total number of exon I-expressing neurons was equal to the combined number of neurons expressing exon III/IV/V and neurons expressing exon II. In addition, it was revealed that the extreme 5' of exon II, encoding a potential hydrophobic leader signal, was not expressed in the CNS of Lymnaea but was apparently spliced out during RNA processing. Both mRNA transcripts of the FMRFamide locus, type 1 (exons I/II) and type 2 (exons I/III/IV/V), were translated in the CNS and the resulting protein precursors were also expressed in a mutually exclusive fashion, as were their respective transcripts. The expression of alternative transcripts within identified networks or neuronal clusters was heterogeneous, as exemplified by the cardiorespiratory network. On the basis of this work and a previous cDNA analysis, we put forward a revised model of differential splicing and expression of the FMRFamide gene in the CNS of Lymnaea.

Amino Acid Sequence↗

PubMed2004

A group II intron-encoded maturase functions preferentially in cis and requires both the reverse transcriptase and X domains to promote RNA splicing.

Mobile group II introns encode proteins with both reverse transcriptase activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active structure of the intron RNA. Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein binds first to a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of its own coding region, and then makes additional contacts with conserved catalytic core regions to stabilize the active RNA structure. Here, we developed an Escherichia coli genetic assay that links the splicing of the Ll.LtrB intron to the expression of green fluorescent protein and used it to study the in vivo splicing of wild-type and mutant introns and to delineate regions of the maturase required for splicing. Our results show that the maturase functions most efficiently when expressed in cis from the same transcript as the intron RNA. In agreement with previous in vitro assays, we find that the high-affinity binding site in DIVa is required for efficient splicing of the Ll.LtrB intron in vivo, but in the absence of DIVa, 6-10% residual splicing occurs by the direct binding of the maturase to the catalytic core. Critical regions of the maturase were identified by statistically analyzing ratios of missense to silent mutations in functional LtrA variants isolated from a library generated by mutagenic PCR ("unigenic evolution"). This analysis shows that both the reverse transcriptase domain and domain X, which likely corresponds to the reverse transcriptase thumb, are required for RNA splicing, while the C-terminal DNA-binding and DNA endonuclease domains are not required. Within the reverse transcriptase domain, the most critical regions for maturase activity include parts of the fingers and palm that function in template and primer binding in HIV-1 reverse transcriptase, but the integrity of the reverse transcriptase active site is not required. Biochemical analysis of LtrA mutants indicates that the N terminus of the reverse transcriptase domain is required for high-affinity binding of the intron RNA, possibly via direct interaction with DIVa, while parts of domain X interact with conserved regions of the catalytic core. Our results support the hypothesis that the intron-encoded protein adapted to function in splicing by using, at least in part, interactions used initially to recognize the intron RNA as a template for reverse transcription.

Base Sequence↗

PubMed1992

Role of TAR RNA splicing in translational regulation of simian immunodeficiency virus from rhesus macaques.

The untranslated leader sequences of rhesus macaque simian immunodeficiency virus mRNAs form a stable secondary structure, TAR. This structure can be modified by RNA splicing. In this study, the role of TAR splicing in virus replication was investigated. The proportion of viral RNAs containing a spliced TAR structure is high early after infection and decreases at later times. Moreover, proviruses containing mutations which prevent TAR splicing are significantly delayed in replication. These mutant viruses require approximately 20 days to achieve half-maximal virus production, in contrast to wild-type viruses, which require approximately 8 days. We attribute this delay to the inefficient translation of unspliced-TAR-containing mRNAs. The molecular basis for this translational effect was examined in in vitro assays. We found that spliced-TAR-containing mRNAs were translated up to 8.5 times more efficiently than were similar mRNAs containing an unspliced TAR leader. Furthermore, these spliced-TAR-containing mRNAs were more efficiently associated with ribosomes. We postulate that the level of TAR splicing provides a balance for the optimal expression of both viral proteins and genomic RNA and therefore ultimately controls the production of infectious virions.

Animals↗