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PubMed2002

p68 RNA helicase is an essential human splicing factor that acts at the U1 snRNA-5' splice site duplex.

Modulation of the interaction between U1 snRNP and the 5' splice site (5'ss) is a key event that governs 5'ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5'ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5'ss during early stage of splicing. However, dissociation of the U1 from the 5'ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5'ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.

5' Untranslated Regions↗
PubMed1992

A base substitution at the splice acceptor site of intron 5 of the COL1A2 gene activates a cryptic splice site within exon 6 and generates abnormal type I procollagen in a patient with Ehlers-Danlos syndrome type VII.

The dermal type I collagen of a patient with Ehlers-Danlos type VIIB (EDS-VIIB) contained normal alpha 2(I) chains and mutant pN-alpha 2(I)' chains in which the amino-terminal propeptide (N-propeptide) remained attached to the alpha 2(I) chain. Similar alpha 2(I) chains were produced by cultured dermal fibroblasts. Amino acid sequencing of tryptic peptides, prepared from the mutant amino-terminal pN-alpha 2(I) CB1' peptide, indicated that five amino acids, including the N-proteinase (the specific proteinase that cleaves the procollagen N-propeptide) cleavage site, had been deleted from the junction of the N-propeptide and the N-telopeptide (the nonhelical domain at the amino-terminus of the alpha chains of fully processed type I polypeptide chains) of the mutant pro-alpha 2(I)' chain. The corresponding 15 nucleotides, which were deleted from approximately half of the alpha 2(I) cDNA polymerase chain reaction products, of the alpha 2(I) cDNA polymerase chain reaction products, were encoded by the +1 to +15 nucleotides of exon 6 of the normal alpha 2(I) gene (COL1A2). These 15 nucleotides were deleted in the splicing of alpha 2(I) pre-mRNA to mRNA as a result of inactivation of the 3' splice site of intron 5 by an AG to AC mutation and the activation of a cryptic AG splice acceptor site corresponding to positions +14 and +15 of exon 6. Loss of the N-proteinase cleavage site explained the persistence of the pN-alpha 2(I)' chains in the dermis and in fibroblast cultures. Collagen production by cultured dermal fibroblasts was doubled, possibly due to reduced feedback inhibition by the N-propeptides. In contrast to previously reported cases of EDS-VIIB, Lys5 of the N-telopeptide was not deleted and appeared to take part in the formation of intramolecular cross-linkages. However, increased collagen solubility and abnormal extraction profiles of the mutant type I collagen molecules indicated that collagen cross-linking was abnormal in the dermis. The proband and her son were heterozygous for the mutation. It is likely that the heterozygous loss of the N-proteinase cleavage site, with persistence of a shortened N-propeptide, was the major factor responsible for the EDS-VIIB phenotype.

Adult↗
PubMed1990

RNA splicing in Chlamydomonas chloroplasts. Self-splicing of 23 S preRNA.

The 23 S rRNA gene of the Chlamydomonas reinhardtii chloroplast contains an 888-base pair intron with structural features characteristic of Group I introns. The nuclear, chloroplast ribosome-deficient mutant of C. reinhardtii, ac20, overaccumulates a approximately 3.6-kilobase unspliced 23 S preRNA compared to wild-type cells. We have used [alpha-32P]GTP labeling of total RNA preparations from ac20 to rapidly determine that 23 S preRNA is capable of self-splicing. The ability of the 23 S intron (with flanking exon sequences) to correctly catalyze its own splicing was confirmed using RNA produced by in vitro transcription of cloned DNA. These results identify the first example of a self-splicing RNA of chloroplast origin.

Base Sequence↗
PubMedDate not supplied

[Splicing. I. Splicing of tRNA, rRNA and mRNA in organelles].

Mosaic structure of genes is shown for three out of four known types of RNA: transfer, ribosomal and messenger. At least three different mechanisms are involved in maturation of transcripts from these genes. The peculiarity of tRNA splicing is connected with the possibility of existence of tRNA molecules free of ribonucleoprotein complexes. The mode of Tetrahymena pre-tRNA self-splicing may also take place during maturation of other RNAs (rRNA of protozoa, ribosomal and messenger rRNA of lower fungi, plant messenger rRNA), which share the similar structure. The third type of mechanism is involved in splicing messenger RNA in eukaryotic cell nuclei.

Animals↗
PubMedDate not supplied

[Splicing. 2. Splicing of mRNA in the cell nucleus].

The mosaic structure of genes is shown for three out of four known types of RNAs, namely transfer, ribosomal and messenger. At least three different splicing mechanisms are involved in maturation of transcripts of these genes, two of them had been described in the previous review. Well-developed effective in vitro splicing systems permit to provide a scheme for the nuclear pre-mRNA maturation processes which take place presumably in specific ribonucleoprotein complex particles. Alternative splicing character of these pre-mRNAs, yields in increased variety of the encoded proteins.

Animals↗
PubMed1995

Lineage-specific alternative splicing of the human Fc gamma RIIA transmembrane exon requires sequences near the 3' splice site.

The human Fc gamma RIIA gene produces multiple transcripts, including those with (Fc gamma RIIa1) and without (Fc gamma RIIa2) the single exon encoding the transmembrane domain (TM). Previously, a fluorescence-based RT-PCR assay showed lineage-specific differences in Fc gamma RIIA transcript ratios (Fc gamma RIIa2/Fc gamma RIIa1). The mechanism of this lineage-specific expression was investigated in this study. Differential transcript stability does not play a major role, because transcript ratios remained constant in cells with both low (K562) and high (Dami) ratios following actinomycin D treatment. Transient expression studies in K562 and Dami cells using a minigene construct containing a 5.0 kb genomic fragment including the TM exon and adjacent intron and exon sequences showed recapitulation of endogenous transcript ratios. The TM exon was efficiently spliced in by the constitutive splicing machinery in HeLa cells, an Fc gamma RIIA-negative cell line. Lineage-specific TM exon skipping was markedly diminished by two independent minigene mutations: a point mutation of the first nucleotide of the TM exon, and a five basepair intronic deletion near a putative branchpoint. These data demonstrate that cis-acting sequences in or near the TM exon 3' splice acceptor site contribute to lineage-specific differences in Fc gamma RIIA transcript ratios.

Alternative Splicing↗
PubMed1998

Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor.

The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gsalpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gsalpha (GsalphaL) binds GDP with lower affinity than the short splice variant (GsalphaS), but the impact of this difference on the interaction of Gsalpha with the beta2AR is not known. We studied the beta2AR/Gsalpha interaction using receptor/G-protein fusion proteins (beta2ARGsalphaS and beta2ARGsalphaL) expressed in Sf9 cells. Fusion of the beta2AR to Gsalpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2AR or Gsalpha. The beta2AR in beta2ARGsalphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2AR in beta2ARGsalphaS. The apparent constitutive activity of the beta2AR in beta2ARGsalphaL may be due to the lower GDP affinity of GsalphaL compared with GsalphaS, i.e. GsalphaL is more often nucleotide-free than GsalphaS and, therefore, more frequently available to stabilize the beta2AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

Adenylyl Cyclases↗
PubMed1998

Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The Beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor.

The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gs alpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gs alpha (Gs alphaL) binds GDP with lower affinity than the short splice variant (Gs alphaS), but the impact of this difference on the interaction of Gs alpha with the beta 2AR is not known. We studied the beta2 AR/Gs alpha interaction using receptor/G-protein fusion proteins (beta2 AR Gs alphaS and beta2 AR Gs alphaL) expressed in Sf9 cells. Fusion of the beta2 AR to Gs alpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2 AR or Gs alpha. The beta2 AR in beta2 AR Gs alphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2 AR in beta2 AR Gs alphaS. The apparent constitutive activity of the beta2 AR in beta2 AR Gs alphaL may be due to the lower GDP affinity of Gs alphaL compared with Gs alphaS, i.e. Gs alphaL is more often nucleotide-free than Gs alphaS and, therefore, more frequently available to stabilize the beta2 AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

Adrenergic beta-Agonists↗
PubMed1999

Reduced stability of regularly spliced but not alternatively spliced p53 protein in PARP-deficient mouse fibroblasts.

The interaction between poly(ADP-ribose) polymerase (PARP) and the product of the tumor suppressor gene p53 has been described previously. Here, we have investigated whether PARP deficiency may affect the expression and regulation of wild-type (wt) p53. For this purpose, we have used immortalized cells derived from wt and PARP knockout mice. We have found a clearly reduced basal level of PAb421 immunoreactive wt p53 protein in PARP-deficient cells. The monoclonal antibody PAb421 is known to recognize an epitope in the COOH terminus of normally spliced p53 protein. Under indirect immunofluorescence, this antibody stained nuclei in normal but not in PARP-deficient cells. Despite marked reduction of wt p53 protein in PARP knockout cells, no significant difference of the p53 transcription rate was observed between wt and PARP-deficient cells. Interestingly, in both cell types, an additional p53 transcript representing the alternatively spliced (AS) p53 form was detected. Because of its reactivity with different specific anti-p53 antibodies, we have determined that the p53 protein present in PARP knockout mouse cells possesses characteristic features of AS p53. Our results clearly show that PARP-deficient cells constitutively express the AS form of wt p53 and indicate that the regularly spliced p53 is extremely unstable in the absence of PARP. Moreover, PARP-/- cells fail to transactivate p53-responsive genes. Treatment of PARP-/- cells with genotoxic agents primarily leads to the activation of AS p53 protein.

Animals↗
PubMed1996

Spliced leader RNA trans-splicing in metazoa.

Spliced leader trans-splicing is a form of RNA processing originally described and studied in parasitic kinetoplastida. This mechanism of gene expression also occurs in parasitic and free-living metazoa. In this review, Dick Davis describes current knowledge of the distribution, substrates, specificity and functional significance of trans-splicing in metazoa.

Journal Article↗
PubMed1999

Supravalvular aortic stenosis: a splice site mutation within the elastin gene results in reduced expression of two aberrantly spliced transcripts.

We have screened the elastin gene for mutations responsible for supravalvular aortic stenosis (SVAS) in two large, independently collected families with isolated (nonsyndromic) SVAS. By single-strand conformation polymorphism and heteroduplex analysis, we have identified a C to G transversion within the acceptor splice site of exon 16 in SVAS patients from both families. This mutation segregates in both families with high penetrance of SVAS, and all affected individuals carry the mutation. Haplotype analysis by using closely linked polymorphisms, including a previously unreported BfaI restriction fragment length polymorphism within the 3'-UTR of the elastin gene, indicates that the mutations found in the two apparently non-overlapping kindreds are identical by descent. To study the effect of the mutation on the expression of the mutant allele, we have established a primary skin fibroblast culture from one of the affected individuals. Reverse transcription/polymerase chain reaction analysis of elastin mRNA species indicates that the mutation results in two abnormal elastin mRNA species. One mutant elastin mRNA is generated by the activation of a cryptic splice site that lies within intron 15 and that adds 44 bp of intronic sequence to the sequence encoded by exon 16. This insertion creates a frame shift that results in a 59-amino-acid-long abnormal protein sequence and leads to a termination codon in the mRNA sequence encoded by exon 17. The smaller abnormal mRNA species arises as a consequence of the skipping of exon 16. This study demonstrates, for the first time, the expression of mutant alleles of the elastin gene in patients with isolated SVAS.

Alternative Splicing↗
PubMed2004

Mutation of the major 5' splice site renders a CMV-driven HIV-1 proviral clone Tat-dependent: connections between transcription and splicing.

Efficient transcription from the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) promoter is dependent on the viral transactivator Tat. To generate a Tat-independent proviral plasmid, we replaced the promoter in the HIV-1 LTR with the immediate early promoter of cytomegalovirus. Transfection of this plasmid yielded Tat-independent production of infectious HIV-1. Tat-independent expression was lost, however, when the major 5' splice site in the HIV-1 genome was mutated and no HIV-1-specific RNA or protein was detected. This defect was restored when a Tat expression plasmid was cotransfected. Our results support recent reports indicating an influence of the recognition of splice sites on efficient transcriptional elongation.

5' Untranslated Regions↗
PubMed2001

Cryptic splicing involving the splice site mutation in the canine model of Duchenne muscular dystrophy.

Golden retriever muscular dystrophy arises from a mutation in the acceptor splice site of intron 6 of the dystrophin gene. Skipping of exon 7 disrupts the mRNA reading frame and results in premature termination of translation. We are using this animal model to evaluate treatments for Duchenne muscular dystrophy, including gene repair induced by chimeric oligonucleotides. After injection of golden retriever muscular dystrophy (GRMD) muscle with a chimeric oligonucleotide to repair the lesion, immunostaining revealed a modest increase in the number of dystrophin-positive fibres at the injection sites. Dystrophin gene transcripts containing exon 7 were detected by reverse transcription-polymerase chain reaction, suggesting that low levels of splice site correction may have occurred. However, DNA sequencing of these apparently normal dystrophin gene transcripts revealed that the first five bases of exon 7 were missing. It will be important to be aware of this phenomenon with respect to further gene correction studies in the canine model.

Alternative Splicing↗
PubMed2003

A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia.

This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (</= 10% of control) platelet content of GPIIb-IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3'-UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb-mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb-mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady-state level of GPIIb-mRNA. The reduced availability of platelet GPIIb-mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in-frame STOP codon with the result of activation of nonsense-mediated mRNA decay, or both.

Alternative Splicing↗
PubMed2002

A conserved Drosophila transportin-serine/arginine-rich (SR) protein permits nuclear import of Drosophila SR protein splicing factors and their antagonist repressor splicing factor 1.

Members of the highly conserved serine/arginine-rich (SR) protein family are nuclear factors involved in splicing of metazoan mRNA precursors. In mammals, two nuclear import receptors, transportin (TRN)-SR1 and TRN-SR2, are responsible for targeting SR proteins to the nucleus. Distinctive features in the nuclear localization signal between Drosophila and mammalian SR proteins prompted us to examine the mechanism by which Drosophila SR proteins and their antagonist repressor splicing factor 1 (RSF1) are imported into nucleus. Herein, we report the identification and characterization of a Drosophila importin beta-family protein (dTRN-SR), homologous to TRN-SR2, that specifically interacts with both SR proteins and RSF1. dTRN-SR has a broad localization in the cytoplasm and the nucleus, whereas an N-terminal deletion mutant colocalizes with SR proteins in nuclear speckles. Far Western experiments established that the RS domain of SR proteins and the GRS domain of RSF1 are required for the direct interaction with dTRN-SR, an interaction that can be modulated by phosphorylation. Using the yeast model system in which nuclear import of Drosophila SR proteins and RSF1 is impaired, we demonstrate that complementation with dTRN-SR is sufficient to target these proteins to the nucleus. Together, the results imply that the mechanism by which SR proteins are imported to the nucleus is conserved between Drosophila and humans.

Active Transport, Cell Nucleus↗
PubMed2002

The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5' splice sites.

The U1 small nuclear ribonucleoprotein (U1 snRNP) binds to the pre-mRNA 5' splice site (ss) at early stages of spliceosome assembly. Recruitment of U1 to a class of weak 5' ss is promoted by binding of the protein TIA-1 to uridine-rich sequences immediately downstream from the 5' ss. Here we describe a molecular dissection of the activities of TIA-1. RNA recognition motifs (RRMs) 2 and 3 are necessary and sufficient for binding to the pre-mRNA. The non- consensus RRM1 and the C-terminal glutamine-rich (Q) domain are required for association with U1 snRNP and to facilitate its recruitment to 5' ss. Co-precipitation experiments revealed a specific and direct interaction involving the N-terminal region of the U1 protein U1-C and the Q-rich domain of TIA-1, an interaction enhanced by RRM1. The results argue that binding of TIA-1 in the vicinity of a 5' ss helps to stabilize U1 snRNP recruitment, at least in part, via a direct interaction with U1-C, thus providing one molecular mechanism for the function of this splicing regulator.

Alternative Splicing↗
PubMed1999

spp42, identified as a classical suppressor of prp4-73, which encodes a kinase involved in pre-mRNA splicing in fission yeast, is a homologue of the splicing factor Prp8p.

We have identified two classical extragenic suppressors, spp41 and spp42, of the temperature sensitive (ts) allele prp4-73. The prp4(+) gene of Schizosaccharomyces pombe encodes a protein kinase. Mutations in both suppressor genes suppress the growth and the pre-mRNA splicing defect of prp4-73(ts) at the restrictive temperature (36 degrees ). spp41 and spp42 are synthetically lethal with each other in the presence of prp4-73(ts), indicating a functional relationship between spp41 and spp42. The suppressor genes were mapped on the left arm of chromosome I proximal to the his6 gene. Based on our mapping data we isolated spp42 by screening PCR fragments for functional complementation of the prp4-73(ts) mutant at the restrictive temperature. spp42 encodes a large protein (p275), which is the homologue of Prp8p. This protein has been shown in budding yeast and mammalian cells to be a bona fide pre-mRNA splicing factor. Taken together with other recent genetic and biochemical data, our results suggest that Prp4 kinase plays an important role in the formation of catalytic spliceosomes.

Fungal Proteins↗
PubMed1997

Differential expression of the regularly spliced wild-type p53 and its COOH-terminal alternatively spliced form during epidermal differentiation.

In the present study, we investigated the role of p53 in the differentiation of epidermal keratinocyte cells. The interrelationship between p53 expression and the various stages of epidermal differentiation and the role of the COOH terminus of the p53 molecule in this process were determined by comparing the expression of the regularly spliced p53 (RSp53) molecule and that of the COOH-terminal alternatively spliced (ASp53) form. p53 mRNA distribution was studied by in situ analysis of frozen skin sections and by reverse transcription-PCR analysis of the various wild-type p53 forms expressed in neonatal skin cell fractions separated by Percoll gradient. p53 protein levels were measured by fluorescence-activated cell sorting analysis and immunohistochemistry, using antibodies that recognize either the COOH terminus of RSp53 or ASp53. The results show that although less mature keratinocyte cells predominantly express the RSp53 form, the more mature cells preferentially express the ASp53 form. Therefore, it is possible that the two p53 forms are associated with different functions required at the various stages of keratinocyte differentiation. The results suggest that the COOH-terminal domain of the p53 molecule is important for its activity in the process of keratinocyte differentiation.

Alternative Splicing↗