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Results for “RNA Splicing”

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PubMed1998

The molecular basis of disease variability among cystic fibrosis patients carrying the 3849+10 kb C-->T mutation.

Disease severity varies among cystic fibrosis (CF) patients carrying the same CFTR genotype. Here we studied the mechanism underlying disease variability in individuals carrying a splicing CFTR mutation, 3849+10 kb C-->T. This mutation was shown to produce both correctly and aberrantly spliced CFTR transcripts containing an additional cryptic exon. Semiquantitative nondifferential RT-PCR showed considerable variability in the level (0-28%) of aberrantly spliced RNA transcribed from the 3849+10 kb C-->T mutation in nasal epithelium from 10 patients. A significant inverse correlation was found between the level of the aberrantly spliced CFTR transcripts and pulmonary function, expressed as FEV1 (r = 0.92, P < 0.0001). Patients with normal pulmonary function (FEV1 > 80% predicted) had lower levels of aberrantly spliced CFTR RNA (0 to 3%) than those with FEV1 < 80%, (9 to 28% aberrantly spliced RNA). Only aberrantly spliced CFTR RNA was detected in the lung of a patient with severe lung disease who underwent lung transplantation. Our results show that the severity of CF lung disease correlates with insufficiency of normal CFTR RNA. Thus, the regulation of alternative splice site selection may be an important mechanism underlying partial penetrance in CF. Further understanding of this regulation will contribute to potential therapy for patients carrying splicing mutations in human disease genes.

Adolescent↗

PubMed2001

SF2/ASF binds to a splicing enhancer in the third HIV-1 tat exon and stimulates U2AF binding independently of the RS domain.

Splicing of a single HIV-1 primary transcript into more than 30 different mRNAs is regulated by a combination of suboptimal splice sites, cis-acting RNA splicing enhancers and silencers, and trans-acting factors. We have studied the splicing of the second tat intron (SD4 to SA7) and find that activation of splicing by SF2/ASF is mediated by a degenerate exon splicing enhancer (ESE3), consisting of at least three functionally independent sub-elements. One of these sub-elements appears to have both enhancing and silencing properties, depending on the context. SF2/ASF stimulates U2AF65 binding to the suboptimal tat polypyrimidine tract in an ESE3-dependent manner, whereas the exon splicing silencer (ESS3) that is located downstream of the ESE3 inhibits this step. Truncated SF2/ASF protein without the RS domain binds specifically to the ESE3 and retains almost full capacity to stimulate U2AF65 binding and activate splicing. This suggests that SF2/ASF can stimulate the recruitment of U2AF65 by an RS domain-independent mechanism.

Base Sequence↗

PubMed1990

A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA.

To explore the structural basis of alternative splicing, we have analyzed the splicing of pre-mRNAs containing an optional exon, E4, from the preprotachykinin gene. This gene encodes substance P and related tachykinin peptides by alternative splicing of a common pre-mRNA. We have shown that alternative splicing of preprotachykinin pre-mRNA occurs by preferential skipping of optional E4. The competing mechanism that incorporates E4 into the final spliced RNA is constrained by an initial block to splicing of the immediate upstream intervening sequence (IVS), IVS3. This block is relieved by sequential splicing, in which the immediate downstream IVS4 is removed first. The structural change resulting from the first splicing event is directly responsible for activation of IVS3 splicing. This structural rearrangement replaces IVS4 sequences with E5 and its adjacent IVS5 sequences. To determine how this structural change promoted IVS3 splicing, we asked what structural change(s) would restore activity of IVS3 splicing-defective mutants. The most significant effect was observed by a 2-nucleotide substitution that converted the 5' splice site of E4 to an exact consensus match, GUAAGU. Exon 5 sequences alone were found not to promote splicing when present in one or multiple copies. However, when a 15-nucleotide segment of IVS5 containing GUAAGU was inserted into a splicing-defective mutant just downstream of the hybrid exon segment E4E5, splicing activity was recovered. Curiously, the 72-nucleotide L2 exon of adenovirus, without its associated 5' splice site, activates splicing when juxtaposed to E4. Models for the activation of splicing by an RNA structural change are discussed.

Base Sequence↗

PubMed1993

Expression of human cytomegalovirus immediate early protein IE1 in insect cells: splicing of RNA and recognition by CD4+ T-cell clones.

Recombinant baculoviruses containing the unspliced gene (Bac-IE1) and a truncated cDNA (Bac-EX4) of the immediate early protein 1 (IE1) of human cytomegalovirus (HCMV) were constructed. The recombinant proteins IE1 and EX4 were expressed in Sf 9 insect cells. Immunoblot analyses using a specific monoclonal antibody or human sera from HCMV seropositive subjects revealed that the IE1 protein had an apparent molecular mass of 71 kDa which was similar to that observed in both HCMV infected human fibroblasts and infected or transfected human astrocytoma cells. Furthermore, HCMV-specific CD4+ T cell clones proliferated in the presence of IE1 or of EX4 used as a control, and appropriate antigen presenting cells. Our data on the IE1 gene provide evidence that two introns can be properly spliced out in baculovirus infected insect cells. The expressed proteins should be useful in further studies on the immune response to the virus.

Animals↗

PubMed1989

An alternate promoter in the glucokinase gene is active in the pancreatic beta cell.

An alternate promoter in the glucokinase gene is active in the beta cell and produces a glucokinase mRNA which is longer and that has a different leader sequence and translation start site than the hepatic glucokinase mRNA. The glucokinase beta cell promoter is located at least 12 kilobases upstream from the glucokinase hepatic promoter. Transcription from the glucokinase beta cell promoter initiates over a region of 62 bases. The absence of a TATA box homology in the proximal promoter region may account for the diffuse transcriptional initiation. Translation of the beta cell glucokinase mRNA predicts a glucokinase isozyme that is different from the hepatic isozyme by 15 amino acids at the N terminus. The use of alternative promoters apparently enables the glucokinase gene to be regulated by insulin in the liver and by glucose in the beta cell, thus possibly constituting an important feedback control loop for maintaining glucose homeostasis. Alternate RNA splicing of the beta cell glucokinase mRNA predicts at least two beta cell glucokinase isoforms. An alternate splice acceptor site in the 4th exon of the glucokinase gene was identified in two glucokinase cDNAs from rat insulinoma tissue. Use of the alternate splice acceptor site results in a 51-nucleotide in frame deletion in the beta cell glucokinase mRNA and removal of 17 amino acids from a region of the protein situated between the putative glucose and ATP binding domains. Analysis of the pattern of RNA splicing in tissues containing beta cells indicates that the splice acceptor site utilized in producing hepatic glucokinase mRNA is also utilized in the beta cell.

Adenoma, Islet Cell↗

PubMed2002

Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing.

Spliceosome-mediated RNA trans-splicing (SMaRT) was investigated as a means for functionally correcting endogenous DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) transcripts using in vitro human cystic fibrosis (CF) polarized airway epithelia and in vivo human CF bronchial xenografts. Recombinant adenovirus (Ad.CFTR-PTM) encoding a pre-therapeutic molecule (PTM) targeted to CFTR intron 9 corrected transepithelial cyclic AMP (cAMP)-sensitive short-circuit current (Isc) in DeltaF508 homozygous epithelia to a level 16% of that observed in normal human bronchial epithelia. Molecular analyses using RT-PCR and western blotting confirmed SMaRT-mediated partial correction of endogenous DeltaF508 messenger RNA (mRNA) transcripts and protein. In an in vivo model of DeltaF508 CF airway epithelia, human CF bronchial xenografts infected with Ad.CFTR-PTM also demonstrated partial correction of CFTR-mediated Cl- permeability at a level 22% of that seen in non-CF xenografts. These results provide functional evidence for SMaRT-mediated repair of mutant endogenous CFTR mRNA in intact polarized CF airway epithelial models.

Adenoviridae↗

PubMed1991

cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis.

Adult rabbit smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2 which are generated through alternative RNA splicing from a single gene (Nagai, R., Kuro-o, M., Babij, P. & Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). We previously reported that the expression of SM1 and SM2 during vascular development is differentially regulated at the level of RNA splicing, whereby SM1 is constitutively expressed from early development but SM2 appear after birth (Kuro-o, M., Nagai, R., Tsuchimochi, H., Katoh, H., Yazaki, Y., Ohkubo, A. & Takaku, F. (1989) J. Biol. Chem. 264, 18272-18275). We also demonstrated that embryonic vascular smooth muscles contain a third type of MHC isoform, referred to as SMemb in this report, which comigrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SM2. In the present study we have isolated and characterized a cDNA clone (FSMHC34) for SMemb. FSMHC34 encodes the light meromyosin region including the carboxyl terminus and showed 70% amino acid sequence identity with SM1 or SM2. SMemb is a nonmuscle-type MHC and identical with brain MHC, but clearly distinct from 196-kDa nonmuscle MHC in cultured smooth muscle cells. The expression of SMemb was predominant in embryonic and perinatal aortas, but down-regulated with vascular development. Interestingly SMemb was reexpressed in proliferating smooth muscle cells of arteriosclerotic neointimas. These results suggest that smooth muscle proliferation is coupled to the expression of SMemb and that dedifferentiation of smooth muscles toward the embryonic phenotype is involved in the mechanisms underlying atherosclerosis.

Aging↗

PubMed1991

MRS3 and MRS4, two suppressors of mtRNA splicing defects in yeast, are new members of the mitochondrial carrier family.

When present in high copy number plasmids, the nuclear genes MRS3 and MRS4 from Saccharomyces cerevisiae can suppress the mitochondrial RNA splicing defects of several mit- intron mutations. Both genes code for closely related proteins of about Mr 32,000; they are 73% identical. Sequence comparisons indicate that MRS3 and MRS4 may be related to the family of mitochondrial carrier proteins. Support for this notion comes from a structural analysis of these proteins. Like the ADP/ATP carrier protein (AAC), the mitochondrial phosphate carrier protein (PiC) and the uncoupling protein (UCP), the two MRS proteins have a tripartite structure; each of the three repeats consists of two hydrophobic domains that are flanked by specific amino acid residues. The spacing of these specific residues is identical in all domains of all proteins of the family, whereas spacing between the hydrophobic domains is variable. Like the AAC protein, the MRS3 and MRS4 proteins are imported into mitochondria in vitro and without proteolytic cleavage of a presequence and they are located in the inner mitochondrial membrane. In vivo studies support this mitochondrial localization of the MRS proteins. Overexpression of the MRS3 and MRS4 proteins causes a temperature-dependent petite phenotype; this is consistent with a mitochondrial function of these proteins. Disruption of these genes affected neither mitochondrial functions nor cellular viability. Their products thus have no essential function for mitochondrial biogenesis or for whole yeast cells that could not be taken over by other gene products. The findings are discussed in relation to possible functions of the MRS proteins in mitochondrial solute translocation and RNA splicing.

Amino Acid Sequence↗

PubMed2004

Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays.

BACKGROUND: The activity of synthetic antisense oligonucleotides (splicomers) designed to block pre-mRNA splicing at specific exons has been demonstrated in a number of model systems, including constitutively spliced exons in mouse dystrophin RNA. Splicomer reagents directed to Duchenne muscular dystrophy (DMD) RNAs might thus circumvent nonsense or frame-shifting mutations, leading to therapeutic expression of partially functional dystrophin, as occurs in the milder, allelic (Becker) form of the disease (BMD). METHODS: Functional and hybridisation array screens have been used to select optimised splicomers directed to exon 23 of dystrophin mRNA which carries a nonsense mutation in the mdx mouse. Splicomers were transfected into cultured primary muscle cells, and dystrophin mRNA assessed for exon exclusion. Splicomers were also administered to the muscles of mdx mice. RESULTS: Oligonucleotide array analyses with dystrophin pre-mRNA probes revealed strong and highly specific hybridisation patterns spanning the exon 23/intron 23 boundary, indicating an open secondary structure conformation in this region of the RNA. Functional screening of splicomer arrays by direct analysis of exon 23 RNA splicing in mdx muscle cultures identified a subset of biologically active reagents which target sequence elements associated with the 5' splice site region of dystrophin intron 23; splicomer-mediated exclusion of exon 23 was specific and dose-responsive up to a level exceeding 50% of dystrophin mRNA, and Western blotting demonstrated de novo expression of dystrophin protein at 2-5% of wild-type levels. Direct intramuscular administration of optimised splicomer reagents in vivo resulted in the reappearance of sarcolemmal dystrophin immunoreactivity in > 30% of muscle fibres in the mdx mouse CONCLUSIONS: These results suggest that correctly designed splicomers may have direct therapeutic value in vivo, not only for DMD, but also for a range of other genetic disorders.

Alleles↗

PubMed1996

The localization of sites containing nascent RNA and splicing factors.

Sites of transcription in the HeLa nucleus are not diffusely spread throughout euchromatin but are concentrated in approximately 2000 discrete sites or "transcription factories." These sites can be immunolabeled after allowing permeabilized cells to elongate nascent RNA chains in the presence of BrUTP. Splicing factors are also concentrated in a few tens of nuclear domains known as "speckles"; they are also more diffusely spread throughout euchromatin. As there has been some controversy whether the speckles are sites of transcription, we investigated the relative distributions of transcription factories and the speckles (detected using Sm autoimmune antibodies). We used conditions that minimize the redistribution of both the nascent RNA and the antigens during permeabilization and fixation and imaged the sites with high resolution using cryosections and a "confocal" microscope. The speckles contained little-if any-nascent RNA and so were usually not transcriptionally active, but they were often surrounded by a few transcription sites. Whether they are active sites of processing of RNA or merely sites where the machinery is stored remains to be established.

Binding Sites↗

PubMed1995

Alternatively spliced isoforms of the NMDARI receptor subunit.

Molecularly diverse forms of the NMDA-receptor subunit NRI are formed by alternative RNA splicing. Differential splicing of three exons generates as many as eight NRI splice variants, seven of which have been identified in cDNA libraries. The alternatively spliced exons encode a 21 amino acid sequence in the N-terminus domain (termed NI), and adjacent sequences of 37 and 38 amino acids in the C-terminus domain (termed C1 and C2, respectively). Splicing out the exon segment that encodes the C2 cassette removes the first stop codon, resulting in a new open reading frame that encodes an unrelated sequence of 22 amino acids (C2') before a second stop codon is reached. Differential RNA splicing alters the structural, physiological and pharmacological properties of receptors that comprise NRI subunits. Diversity of NMDA receptors is also caused by differential association with members of the NR2 gene family. The finding of cell-specific expression and developmental regulation of NRI splice variants, and of the NR2 subunits, provides an explanation for the diversity of properties of NMDA receptors in different neuronal populations.

Amino Acid Sequence↗

PubMed1992

Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus.

In retroviral integration, the viral integration protein (integrase) mediates a concerted DNA cleavage-ligation reaction in which the target DNA is cleaved and the resulting 5' ends of target DNA are joined to the 3' ends of viral DNA. Through an oligonucleotide substrate that mimics the recombination intermediate formed by this initial cleavage-ligation reaction, the purified integrase of human immunodeficiency virus was shown to promote the same reaction in reverse, a process called disintegration. Analysis of a set of structurally related substrates showed that integrase could promote a range of DNA cleavage-ligation reactions. When the viral DNA component of the disintegration substrate was single-stranded, integrase could mediate a DNA splicing reaction analogous to RNA splicing.

Base Sequence↗

PubMed2001

Osteocalcin is incompletely spliced in non-osseous tissues.

Osteocalcin (OC) is known to be a bone tissue-specific protein, expression of which is believed to be controlled by the OC promoter. In this communication, we provided evidence to demonstrate that tissue-specific expression of OC was also regulated at the RNA splicing level. We identified incompletely spliced variants of human OC mRNA, which retain one or more introns during RNA splicing, existing dominantly in non-osseous organs. Northern blot analysis identified two OC RNA transcripts expressed in normal human tissues, but the expression level of the transcripts varied between the tissues. Most non-osseous tissues expressed transcripts with higher molecular weight, prominent in ovary, kidney, pancreas, spleen, thymus, prostate, and testis, than the expected size of OC mRNA as seen in bone marrow. RT-PCR analysis identified up to six OC transcripts in most tissues tested except bone marrow. Sequence analysis showed that four of five RNA variants contained intron 1 in common and the dominant one contained all three introns. MG63, an osteoblastic osteosarcoma cell, expressed only the completely-spliced form of OC, whereas incompletely spliced RNA was dominant in most prostate tumor cells. Combined study of in situ hybridization and immunohistochemistry revealed that OC RNA was highly expressed in prostate tumor epithelial cells while only very low levels of protein were detected, which confirms that there are OC RNA variants in non-osseous tissues. In conclusion, we demonstrated that OC mRNA is also expressed in several non-osseous tissues. However, only bone preferentially underwent the complete splicing event of all three introns. The function of other splicing variants of OC mRNA needs to be further investigated.

Alternative Splicing↗

PubMed1984

Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro.

To study the mechanisms of RNA splicing we have analyzed the products generated by in vitro processing of a truncated 32P-labeled human beta-globin RNA precursor that contains the first two exons and the first intervening sequence (IVS1). Six major RNA products were detected and characterized. The first detectable RNA processing event is cleavage at the 5' GT of IVS1. Subsequently, accurately spliced RNA and the excised, intact IVS1 are simultaneously observed. The IVS1-containing RNA processing products have several unusual properties, which include: anomalous electrophoretic mobilities on polyacrylamide gels; a block to reverse transcription near the 3' end of IVS1; the presence of a nuclease-resistant component within IVS1. The block to reverse transcription and the nuclease-resistant component map to the same site near the 3' end of IVS1. The nuclease-resistant component appears to be a modified adenosine residue that contains an RNA branch. Based upon these and other structural studies we propose that the 5' end of IVS1 is joined by a 2'-5' phosphodiester linkage to the A residue in the RNAase T1 oligonucleotide ACTCTCTCTG located 28-37 nucleotides upstream from the IVS1 3' end. The IVS1 is therefore in the form of a lariat. These results imply that sequences within IVS1 actively participate in splicing.

Base Sequence↗

PubMed1990

Alternative splicing of hepatitis B virus RNAs in HepG2 cells transfected with the viral DNA.

We identified a novel spliced RNA of 2.6 kb from a human hepatoma cell line HepG2 transfected with the hepatitis B virus (HBV) genome. The splicing acceptor site of the novel 2.6-kb RNA (position 489) was shown to be common to that of the previously described 2.1-kb spliced RNA which codes for an altered core antigen lacking the carboxy-terminal amino acid, cysteine. However, the donor site of the 2.6-kb RNA is different from any of the spliced RNA reported and located at 538 nucleotides (nt) downstream of the donor site of the 2.1-kb RNA. Introduction of single-base change mutations in the consensus sequence of the donor site of the 2.1-kb RNA maintained the splicing by using the cryptic donor site. The amount of the 2.6-kb spliced RNA was unchanged by these mutations. These results suggest independent regulations for the synthesis of the 2.1- and 2.6-kb spliced RNAs.

Amino Acid Sequence↗

PubMed1993

Evolutionary relationships among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function.

Many group II introns encode reverse transcriptase-like proteins that potentially function in intron mobility and RNA splicing. We compared 34 intron-encoded open reading frames and four related open reading frames that are not encoded in introns. Many of these open reading frames have a reverse transcriptase-like domain, followed by an additional conserved domain X, and a Zn(2+)-finger-like region. Some open reading frames have lost conserved sequence blocks or key amino acids characteristic of functional reverse transcriptases, and some lack the Zn(2+)-finger-like region. The open reading frames encoded by the chloroplast tRNA(Lys) genes and the related Epifagus virginiana matK open reading frame lack a Zn(2+)-finger-like region and have only remnants of a reverse transcriptase-like domain, but retain a readily identifiable domain X. Several findings lead us to speculate that domain X may function in binding of the intron RNA during reverse transcription and RNA splicing. Overall, our findings are consistent with the hypothesis that all of the known group II intron open reading frames evolved from an ancestral open reading frame, which contained reverse transcriptase, X, and Zn(2+)-finger-like domains, and that the reverse transcriptase and Zn(2+)-finger-like domains were lost in some cases. The retention of domain X in most proteins may reflect an essential function in RNA splicing, which is independent of the reverse transcriptase activity of these proteins.

Amino Acid Sequence↗

PubMed1994

A tertiary interaction in the Tetrahymena intron contributes to selection of the 5' splice site.

The utilization of cryptic splice sites has been observed in a number of RNA splicing reactions. In the self-splicing group I intron of Tetrahymena thermophila, point mutations of either A57 or A95 promote cleavage at two sites other than the normal 5' splice site, suggesting that these nucleotides are involved in a common tertiary interaction. These results are unusual since A57 and A95 are neither at nor near the 5' splice site in the sequence or secondary structure. Cleavage at the alternative sites appears to occur by intron cyclization, a reaction with well-established structural and mechanistic similarities to the first step of RNA self-splicing. Alternative docking of P1 (the helix containing the 5' splice site paired to the internal guide sequence of the intron) into the catalytic core accounts for cleavage at the cryptic reaction sites. We propose that the A57/A95 interaction, along with an element implicated previously (J1/2), provide structural connectivity from the reaction site in P1 to the catalytic core of the Tetrahymena intron. It seems likely that RNA splicing in general will require such tertiary interactions to position RNA helices.

Adenine↗

PubMed1989

Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms.

Two types of smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, were recently identified to have different carboxyl termini (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). SM1 and SM2 are considered to be generated from a single gene through alternative RNA splicing. In this study we investigated expression of vascular MHC isoforms during development in rabbits at the mRNA, protein, and histological levels. In adults, all smooth muscle cells reacted with both anti-SM1 and anti-SM2 antibodies on immunofluorescence, suggesting the coexpression of SM1 and SM2 in a single cell. In fetal and perinatal rabbits, however, only anti-SM1 antibody consistently reacted with smooth muscles. Reactivity with anti-SM2 antibody was negative in the fetal and neonatal blood vessels and gradually increased during 30 days after birth. These developmental changes in SM1 and SM2 expression at the histological level coincided with mRNA expression of each MHC isoform as determined by S1 nuclease mapping, indicating that expression of SM1 and SM2 is controlled at the level of RNA splicing. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin from fetal and perinatal aortas revealed the presence of large amount of SM2. Interestingly, fetal SM2 did not cross-react with our anti-SM2 antibody on immunoblotting. We conclude that expression of SM1 and SM2 are differentially regulated during development and that a third type of MHC isoform may exist in embryonic and perinatal vascular smooth muscles.

Animals↗