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Results for “RNA Splicing”

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PubMed1997

Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization.

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Because it plays a key role in the in vitro dimerization of short HIV-1 RNA transcripts (M. Laughrea and L. Jette, Biochemistry 35:1589-1598, 1996, and references therein; M. Laughrea and L. Jette, Biochemistry 35:9366-9374, 1996, and references therein) and because dimeric RNAs may be preferably encapsidated into the HIV-1 virus, alterations of the kissing-loop hairpin might affect the in vivo dimerization and encapsidation processes. Accordingly, substitution and deletion mutations were introduced into the kissing-loop hairpin of an infectious HIV-1 molecular clone in order to produce viruses by transfection methods. The infectivity of the resulting viruses was decreased by at least 99%, the amount of genomic RNA packaged per virus was decreased by 50 to 75%, and the proportion of dimeric genomic RNA was reduced from >80 to 40 to 50%, but the dissociation temperature of the genomic RNA was unchanged. There is evidence suggesting that the deletion mutations moderately inhibited CAp24 production but had no significant effect on RNA splicing. These results are consistent with the kissing-loop model of HIV-1 RNA dimerization. In fact, because intracellular viral RNAs are probably more concentrated in transfected cells than in cells infected by one virus and because the dimerization and encapsidation processes are concentration dependent, it is likely that much larger dimerization and encapsidation defects would have been manifested within cells infected by no more than one virus.

Animals↗

PubMed1995

Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster ovary cells.

The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3-8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene.

Animals↗

PubMed1994

Aberrant rearrangements within the immunoglobulin heavy chain locus in hairy cell leukemia.

The cell surface expression of multiple immunoglobulin heavy chain (IGH) isotypes, preferentially observed in hairy cell leukemia (HCL), is incompatible with the deletion model of IGH class switching, and alternative models involving RNA-splicing have been proposed. To help discriminate between these possibilities we have examined the configuration of the IGH locus by DNA blot in 38 cases of HCL. Deletion of at least one allele of C mu/C delta was seen in 14 cases (37%). Of 12 cases in which IgG and/or IgA were expressed, three exhibited biallelic deletion of C mu/C delta compatible with deletional class-switching whereas the remaining 9 cases retained both alleles of C mu/C delta. These data indicate that both DNA deletion and RNA-splicing mechanisms of class-switching may operate in HCL. In a further 17 cases, other unexpected abnormalities of the IGH locus mapping to the JH-C mu intron in one of two patterns were observed. First, discordance between JH and C mu in BamHI DNA digests was found in 12 cases. Similar patterns were observed in only 4/91 cases of B-cell chronic lymphocytic leukemia and 0/15 of B-cell prolymphocytic leukemia. To preclude chromosomal translocations involving the JH-C mu intron, rearrangements of BCL1, BCL2, BCL3, and MYC were sought in 23 cases but none of the cases showed oncogene rearrangement to the intron. Second, abnormal-sized C mu fragments arising as a consequence of abnormalities of the JH-C mu intron were detected in HindIII, BgIII, and/or Xbal digests in 10/23 cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed2002

Point mutations causing the McLeod phenotype.

BACKGROUND: The McLeod phenotype is defined by absence of Kx, weakening of Kell system antigens, and acanthocytosis. Individuals with the McLeod phenotype usually develop late-onset neuromuscular abnormalities. Gene deletions, insertions, and point mutations that affect RNA splicing or that lead to premature stop codons have been reported to cause the McLeod phenotype. The McLeod phenotype may also be caused by mutations at a different splice site and by a novel mutation encoding an amino acid substitution that prevents transport to the cell surface. STUDY DESIGN AND METHODS: The coding and flanking intron regions of XK from four male, unrelated individuals with the McLeod phenotype and non-chronic granulomatous disease were sequenced and compared with the wild type sequence. Genomic DNA was amplified by PCR, and the products were sequenced. In one case, the mutant cDNA was expressed in a heterologous cell, and cell surface expression was determined. RESULTS: Three individuals with the McLeod phenotype had mutations that disrupted conserved GT sequences present at RNA splice sites. Two of them had G>C mutations at the 5' splice site of intron 1, and one had a G>A mutation at the 5' splice site of intron 2. One person with the McLeod phenotype had a 746C>G mutation in exon 3 encoding an R222G substitution. In a transfected cell, the expressed protein from the latter mutant did not travel to the cell surface. CONCLUSION: The McLeod phenotype may be caused by several different mutations.

Acanthocytes↗

PubMed2000

Molecular cloning and expression analysis of a putative nuclear protein, SR-25.

We cloned a full-length mouse cDNA and its human homologue encoding a novel protein designated as "SR-25." In Northern blot analysis, SR-25 mRNA was expressed in all organs tested, and relatively abundant in testis and thymus. Deduced amino acid sequences of mouse SR-25 and human SR-25 showed 77.7% identity. SR-25 has a serine-arginine repeat (SR repeat) and two types of amino acid clusters: a serine cluster and a highly basic cluster. Based on the presence of many nuclear localizing signals and a similarity to RNA splicing proteins, SR-25 is strongly suggested to be a nuclear protein and may contribute to RNA splicing.

Amino Acid Sequence↗

PubMed1999

Saccharomyces cerevisiae telomerase is an Sm small nuclear ribonucleoprotein particle.

Activation of the chromosome end-replicating enzyme telomerase can greatly extend the lifespan of normal human cells and is associated with most human cancers. In all eukaryotes examined, telomerase has an RNA subunit, a conserved reverse transcriptase subunit and additional proteins, but little is known about the assembly of these components. Here we show that the Saccharomyces cerevisiae telomerase RNA has a 5'-2,2,7-trimethylguanosine (TMG) cap and a binding site for the Sm proteins, both hallmarks of small nuclear ribonucleoprotein particles (snRNPs) that are involved in nuclear messenger RNA splicing. Immunoprecipitation of telomerase from yeast extracts shows that Sm proteins are assembled on the RNA and that most or all of the telomerase activity is associated with the Sm-containing complex. These data support a model in which telomerase RNA is transcribed by RNA polymerase II and 7-methylguanosine-capped, binds the seven Sm proteins, becomes TMG-capped and picks up the other protein subunits. We conclude that the functions of snRNPs assembled by this pathway are not restricted to RNA processing, but also include chromosome telomere replication.

Autoantigens↗

PubMed2005

Three- and four-repeat tau regulate the dynamic instability of two distinct microtubule subpopulations in qualitatively different manners. Implications for neurodegeneration.

The microtubule-associated protein tau is implicated in the pathogenesis of many neurodegenerative diseases, including fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), in which both RNA splicing and amino acid substitution mutations in tau cause dominantly inherited early onset dementia. RNA-splicing FTDP-17 mutations alter the wild-type approximately 50:50 3-repeat (3R) to 4-repeat (4R) tau isoform ratio, usually resulting in an excess of 4R tau. To examine further how splicing mutations might cause dysfunction by misregulation of microtubule dynamics, we used video microscopy to determine the in vitro behavior of individual microtubules stabilized by varying amounts of human 4R and 3R tau. At low tau:tubulin ratios (1:55 and 1:45), all 3R isoforms reduced microtubule growth rates relative to the no-tau control, whereas all 4R isoforms increased them; however, at a high tau:tubulin ratio (1:20), both 4R and 3R tau increased the growth rates. Further analysis revealed two distinct subpopulations of growing microtubules in the absence of tau. Increasing concentrations of both 4R and 3R tau resulted in an increase in the size of the faster growing subpopulation of microtubules; however, 4R tau caused a redistribution to the faster growing subpopulation at lower tau:tubulin ratios than 3R tau. This modulation of discrete growth rate subpopulations by tau suggests that tau causes a conformational shift in the microtubule resulting in altered dynamics. Quantitative and qualitative differences observed between 4R and 3R tau are consistent with a "microtubule misregulation" model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal death and dementia.

Alternative Splicing↗

PubMed1997

Folding of RNA involves parallel pathways.

Folding kinetics of large RNAs are just beginning to be investigated. We show that the Tetrahymena self-splicing RNA partitions into a population that rapidly reaches the native state, and a slowly folding population that is trapped in metastable misfolded structures. Transitions from the misfolded structures to the native state involve partial unfolding. The total yield of native RNA is increased by iterative annealing of the inactive population, and mildly denaturing conditions increase the rate of folding at physiological temperatures. These results provide the first evidence that an RNA can fold by multiple parallel paths.

Animals↗

PubMed2003

5' exon replacement and repair by spliceosome-mediated RNA trans-splicing.

Spliceosome-mediated RNA trans-splicing (SMaRT) has been used previously to reprogram mutant endogenous CFTR and factor VIII mRNAs in human epithelial cell and tissue models and knockout mice, respectively. Those studies used 3' exon replacement (3'ER); a process in which the distal portion of RNA is reprogrammed. Here, we also show that the 5' end of mRNA can be completely rewritten by 5'ER. For proof-of-concept, and to test whether 5'ER could generate functional CFTR, we generated a mutant minigene target containing CFTR exons 10-24 (deltaF508) and a mini-intron 10, and a pretrans-splicing molecule (targeted to intron 10) containing CFTR exons 1-10 (+F508), and tested these two constructs in 293T cells for anion efflux transport. Cells cotransfected with target and PTM showed a consistent increase in anion efflux, but there was no response in control cells that received PTM or target alone. Using a LacZ reporter system to accurately quantify trans-splicing efficiency, we tested several unique PTM designs. These studies provided two important findings as follows: (1) efficient trans-splicing can be achieved by binding the PTM to different locations in the target, and (2) relatively few changes in PTM design can have a profound impact on trans-splicing activity. Tethering the PTM close to the target 3' splice site (as opposed to the donor site) and inserting an intron in the PTM coding resulted in a 65-fold enhancement of LacZ activity. These studies demonstrate that (1) SMaRT can be used to reprogram the 5' end of mRNA, and (2) efficiency can be improved substantially.

Cell Line↗

PubMed1993

Evidence for an essential non-Watson-Crick interaction between the first and last nucleotides of a nuclear pre-mRNA intron.

Nuclear pre-messenger RNA splicing requires the action of five small nuclear (sn) RNAs, U1, U2, U4, U5 and U6, and more than 50 proteins. The mechanistic similarity of nuclear pre-mRNA splicing and group II self-splicing suggests that many of the central processes of nuclear pre-mRNA splicing are based on RNA-RNA interaction. To understand the mechanism of pre-mRNA splicing, the interactions, and their temporal relationships, that occur between the snRNAs and the pre-mRNA during splicing must be identified. Several snRNA-snRNA and snRNA-intron interactions have been demonstrated but the putative RNA-based interactions that recognize the AG dinucleotide at the 3' splice site during 3' cleavage and exon ligation are unknown. We report here the reciprocal suppression between 5' and 3' splice site mutations in the yeast actin intron, and propose that the 3' splice site is positioned for 3' cleavage and exon ligation, at least in part, through a non-Watson-Crick interaction between the guanosines at the 5' and 3' splice sites.

Base Composition↗

PubMed1992

Moloney murine sarcoma virus MuSVts110 DNA: cloning, nucleotide sequence, and gene expression.

We have cloned Moloney murine sarcoma virus (MuSV) MuSVts110 DNA by assembly of polymerase chain reaction (PCR)-amplified segments of integrated viral DNA from infected NRK cells (6m2 cells) and determined its complete sequence. Previously, by direct sequencing of MuSVts110 RNA transcribed in 6m2 cells, we established that the thermosensitive RNA splicing phenotype uniquely characteristic of MuSVts110 results from a deletion of 1,487 nucleotides of progenitor MuSV-124 sequences. As anticipated, the sequence obtained in this study contained precisely this same deletion. In addition, several other unexpected sequence differences were found between MuSVts110 and MuSV-124. For example, in the noncoding region upstream of the gag gene, MuSVts110 DNA contained a 52-nucleotide tract typical of murine leukemia virus rather than MuSV-124, suggesting that MuSVts110 originated as a MuSV-helper murine leukemia virus recombinant during reverse transcription rather than from a straightforward deletion within MuSV-124. In addition, both MuSVts110 long terminal repeats contained head-to-tail duplications of eight nucleotides in the U3 region. Finally, seven single-nucleotide substitutions were found scattered throughout MuSVts110 DNA. Three of the nucleotide substitutions were in the gag gene, resulting in one coding change in p15 and one in p30. All of the remaining nucleotide changes were found in the noncoding region between the 5' long terminal repeat and the gag gene. In NIH 3T3 cells transfected with the cloned MuSVts110 DNA, the pattern of viral RNA expression conformed with that observed in cells infected with authentic MuSVts110 virus in that viral RNA splicing was 30 to 40% efficient at growth temperatures between 28 and 33 degrees C but reduced to trace levels above 37 degrees C.

3T3 Cells↗

PubMed1999

The stability and fate of a spliced intron from vertebrate cells.

Introns constitute most of the length of typical pre-mRNAs in vertebrate cells. Thus, the turnover rate of introns may significantly influence the availability of ribonucleotides and splicing factors for further rounds of transcription and RNA splicing, respectively. Given the importance of intron turnover, it is surprising that there have been no reports on the half-life of introns from higher eukaryotic cells. Here, we determined the stability of IVS1Cbeta1, the first intron from the constant region of the mouse T-cell receptor-beta, (TCR-beta) gene. Using a tetracycline (tet)-regulated promoter, we demonstrate that spliced IVS1Cbeta1 and its pre-mRNA had half-lives of 6.0+/-1.4 min and 3.7+/-1.0 min, respectively. We also examined the half-lives of these transcripts by using actinomycin D (Act.D). Act.D significantly stabilized IVS1Cbeta1 and its pre-mRNA, suggesting that Act.D not only blocks transcription but exerts rapid and direct posttranscriptional effects in the nucleus. We observed that in vivo spliced IVS1Cbeta1 accumulated predominantly as lariat molecules that use a consensus branchpoint nucleotide. The accumulation of IVS1Cbeta1 as a lariat did not result from an intrinsic inability to be debranched, as it could be debranched in vitro, albeit somewhat less efficiently than an adenovirus intron. Subcellular-fractionation and sucrose-gradient analyses showed that most spliced IVS1Cbeta1 lariats cofractionated with pre-mRNA, but not always with mRNA in the nucleus. Some IVS1Cbeta1 also appeared to be selectively exported to the cytoplasm, whereas TCR-beta pre-mRNA remained in the nucleus. This study constitutes the first detailed analysis of the stability and fate of a spliced nuclear intron in vivo.

Animals↗

PubMed2005

Human hHpr1/p84/Thoc1 regulates transcriptional elongation and physically links RNA polymerase II and RNA processing factors.

Cotranscriptional loading of RNA processing factors onto nascent RNA facilitates efficient gene expression. Mechanisms responsible for coupling transcription and RNA processing are not well defined, but the Saccharomyces cerevisiae TREX complex provides an example. TREX is composed of the subcomplex THO that associates with RNA polymerase II and is required for normal transcriptional elongation. THO associates with proteins involved in RNA splicing and export to form the larger TREX complex. Hence, assembly of TREX physically couples transcriptional elongation with RNA processing factors. Whether metazoan species with long, intron-containing genes utilize a similar mechanism has not been established. Here we show that human hHpr1/p84/Thoc1 associates with elongating RNA polymerase II and the RNA splicing and export factor UAP56 in intact cells. Depletion of hHpr1/p84/Thoc1 causes transcriptional elongation defects and associated cellular phenotypes similar to those observed in THO-deficient yeast. We conclude that hHpr1/p84/Thoc1 regulates transcriptional elongation and may participate in a protein complex functionally analogous to yeast TREX, physically linking elongating RNA polymerase II with RNA processing factors.

Cell Cycle Proteins↗

PubMed2002

Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR.

Alternative RNA splicing generates extensive proteomic diversity in the nervous system, yet few neural-specific RNA binding proteins have been implicated in splicing control. Here we show that the biochemical properties and spatial expression of mouse neuroblastoma apoptosis-related RNA-binding protein (NAPOR; also called NAPOR-1) are consistent with its roles in the regulation of the exon 5 and exon 21 splicing events of the N-methyl-D-aspartate (NMDA) receptor R1 transcript. NAPOR, which is closely related to CUG binding protein 2 (CUG-BP2), promotes exon 21 and represses exon 5 splicing in functional coexpression assays. These NMDA mRNA isoforms are distributed, in vivo, in a region-specific manner in rat brain, such that high levels of exon 21 selection and exon 5 skipping coincide with high NAPOR mRNA expression in the forebrain. Within the forebrain, this spatial correspondence is most striking in the visual cortex. In contrast, low NAPOR expression coincides with the reciprocal pattern of alternative splicing in the hindbrain. Complementary experiments demonstrate a tissue-specific distribution of NAPOR, CUG-BP, and other highly related proteins within the nervous system as assayed by probing forebrain and hindbrain nuclear extracts with monoclonal antibody, mAb 3B1. Thus, NAPOR may be one of a group of closely related proteins involved in splicing regulation within the brain. An intronic RNA element responsible for the silencing of exon 21 splicing is identified by mutational analysis and shown to bind directly to recombinant NAPOR protein, suggesting a model in which exon 21 selection is positively regulated by an antirepression mechanism of action.

Alternative Splicing↗

PubMed1996

Fibronectin mRNA splice variant in articular cartilage lacks bases encoding the V, III-15, and I-10 protein segments.

Fibronectin is an extracellular matrix glycoprotein encoded by a single gene. Alternative RNA splicing has been reported at three sites, ED (extra type III domain)-A, ED-B, and the variable or V region. Articular cartilage fibronectin monomers are rarely (ED-A)+, but approximately 25% are (ED-B)+. RNA gel electrophoresis and Northern blot analysis identified two (ED-B)+ and two (ED-B)- fibronectin transcripts in cartilage, each pair differing by approximately 750 bases. This difference results from a previously unreported RNA splicing pattern that eliminates not only the V region but also nucleotides encoding protein segments III-15 and I-10. This new splice variant, which we designate (V+C)-, represents the majority of fibronectin transcripts in equine, canine, and rabbit articular cartilage but is absent in the liver. Reverse transcriptase-polymerase chain reaction analyses of 11 additional equine tissues failed to detect the (V+C)- splice variant, except for very low levels in lymph node, bone, aorta, and skin. Furthermore, chondrocytes grown in monolayer culture maintain high levels of fibronectin expression but stop expressing (V+C)- transcripts over time. The tissue-specific expression pattern of this novel fibronectin isoform suggests that it may have an important function in the matrix organization of cartilage.

Alternative Splicing↗

PubMed1994

Maintenance of an open reading frame as an additional level of scrutiny during splice site selection.

Although nonsense mutations have been associated with the skipping of specific constitutively spliced exons in selected genes, notably the fibrillin gene, the basis for this association is unclear. Now, using chimaeric constructs in a model in vivo expression system, premature termination codons are identified as determinants of splice site selection. Nonsense codon recognition prior to RNA splicing necessitates the ability to read the frame of precursor mRNA in the nucleus. We propose that maintenance of an open reading frame can serve as an additional level of scrutiny during exon definition. This process may have pathogenic and evolutionary significance.

Base Sequence↗

PubMed2001

RNA-RNA noncovalent interactions investigated by microspray ionization mass spectrometry.

Electrospray ionization mass spectrometry is playing an increasing role in the study of noncovalent interactions involving biomolecules. RNA-RNA complexes are important in many areas of biology, including RNA catalysis, RNA splicing, ribosome function, and gene regulation. Here, microelectrospray mass spectrometry (microESI-MS) is used to study noncovalent base-pairing interactions between RNA oligonucleotides, an area not previously explored by this technique. Using a set of complementary RNA oligonucleotides, we demonstrate the formation of the expected double-helical RNA complexes composed of three distinct oligonucleotides. The ability to study specific RNA noncovalent interactions by microESI-MS has the potential to provide a unique method by which to analyze and assign precise molecular masses to RNA-RNA complexes.

Base Sequence↗

PubMed2002

Implication of alternative splicing for expression of a variant NAD(P)H:quinone oxidoreductase-1 with a single nucleotide polymorphism at 465C>T.

Three alleles of the human reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase-1 (NQO1) gene are known; wild-type, 609C>T variant, and 465C>T variant; designated as NQO1*1 and NQO1*2, and NQO1*3, respectively. Previously, we found NQO1*3 in one allele of HCT-116 cells, and in both alleles of the mitomycin C-resistant subline, HCT-116R30A. The NQO1 protein content of HCT-116R30A is 5% that of HCT-116. RT-PCR revealed an exon-4-deleted NQO1 mRNA in both cell lines indicating alternative splicing. However, the cause of the lower expression of NQO1 in HCT-116R30A is unknown. Current data show that HCT-116R30A cells are able to express NQO1 protein from transfected cDNA constructs when RNA splicing is omitted. The ratio of full-length to exon-4-deleted mRNA measured by semiquantitative PCR shows that HCT-116 and HCT-116R30A have ratios of 64 : 36 and 34 : 66, respectively. All other cell lines tested have a ratio of 90 : 10, including HT-29, NIH-125 and NCI-H1688 (homozygous NQO1*1); MCF-7 and HL-60 (heterozygous NQO1*1/*2); and MDA-MB231 (homozygous NQO1*2/*2). Alternative splicing of NQO1 at the 5'-splice site of intron-4 increased in cells with NQO1*3. The 465C>T single nucleotide polymorphism (SNP) disrupts the consensus sequence at the 5'-splice site, which is required for binding by U1 small nuclear RNA (U1 snRNA) in spliceosomes. This defective RNA splicing was partially corrected by transfecting HCT-116R30A cells with U1 snRNA constructs, containing base changes to compensate for the 465 SNP. NQO1 protein and enzymatic activity increased with corrected splicing. The 465 SNP was the major cause of increased alternative splicing and decreased expression of NQO1 protein in HCT-116R30A cells.

Alternative Splicing↗