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PubMed2000

Variable number tandem repeat in exon/intron border of the cystathionine beta-synthase gene: a single nucleotide substitution in the second repeat prevents multiple alternate splicing.

We studied a large number of individuals with respect to the 31-bp variable number tandem repeat (VNTR) in the cystathionine beta-synthase (CBS) gene. The number of repeats varies from 15-20, with 17 repeats the most common allele. Significantly, we found that the first repeat of the 31-bp VNTR originates 12 bp from the 5' end of exon 13 and extends 19 bp into intron 13. Since this VNTR spans across the exon-intron border, it can theoretically create multiple alternate splice sites. However, a substitution of g-->a at the exon-intron border is uniquely present in the second repeat, preventing alternate splicing at that site. While the g-->a substitution is absent from all subsequent 31-bp repeats, alternate splicing probably does not occur at those distal sites due to the lack of exon 13 sequences not contained in the repeats but needed for the binding of spliceosomes. Investigation of five individuals with normal plasma total homocysteine (tHcy) and five individuals with mild hyper-homocysteinemia shows that all have the g-->a substitution in the second repeat. Nonetheless, we speculate that the absence of this substitution may be found in rare individuals with normal CBS cDNA and unexplained hyperhomocysteinemia. Gene scanning and direct nucleotide sequencing were used to characterize the VNTR in 398 patients with premature coronary artery disease and 137 controls. Five alleles and 10 genotypes were found; 17/17 is the most prevalent genotype in our study population. The two other prevalent genotypes, 16/17 and 17/18, are associated with significantly decreased tHcy levels as compared to the 17/ 17 genotype, suggesting that the 16 and 18 repeats haplotype may be in linkage disequilibrium with regulatory elements which upregulate CBS gene transcription.

Adolescent↗

PubMed2001

Deficient brain snRNP70K in patients with Down syndrome.

The small nuclear ribonucleoprotein 70K (snRNP 70K; U1-70 kDa) is an integral part of the spliceosome, a large RNA-protein complex catalyzing the removal of introns from nuclear pre-mRNA. snRNP is one of the best-studied essential subunits of snRNPs, is highly conserved and its inactivation was shown to result in complete inhibition of splicing. Applying subtractive hybridization, we found a sequence with 100% identity to snRNP absent in fetal Down syndrome (DS) brain. This observation made us determine snRNP-mRNA steady-state levels and protein levels in brains of adult patients with DS. snRNP-mRNA and protein levels of five individual brain regions of DS and controls each, were determined by blotting techniques. snRNP-mRNA steady state levels were significantly decreased in DS brain. Performing Western blots with monoclonal and human antibodies, snRNP protein levels were decreased in several regions of DS brain, although one monoclonal antibody did not reveal different snRNP-immunoreactivity. Although decreased snRNP-protein could be explained by decreased mRNA-steady state levels, another underlying mechanism might be suggested: snRNP is one of the death substrates rapidly cleaved during apoptosis by interleukin-1-beta-converting enzyme-like (ICE) proteases, which was well-documented by several groups. As apoptosis is unrequivocally taking place in DS brain leading to permanent cell loses, decreased snRNP-protein levels may therefore reflect decreased synthesis and increased apoptosis-related proteolytic cleavage.

Adult↗

PubMed2001

Systemic lupus erythematosus in adults is associated with previous Epstein-Barr virus exposure.

OBJECTIVE: The possible molecular mimicry of the Epstein-Barr virus (EBV) peptide PPPGRRP by the peptide PPPGMRPP from Sm B'/B of the human spliceosome is consistent with the possibility that EBV infection is related to the origin of systemic lupus erythematosus (SLE) in some patients. Association of EBV exposure with SLE was therefore tested for and subsequently found in children and adolescents (odds ratio [OR] 49.9, 95% confidence interval [95% CI] 9.3-1,025, P < 10(-11)). These results were confirmed at the level of EBV DNA (OR > 10, 95% CI 2.53-infinity, P < 0.002). Much smaller seroconversion rate differences were found against 4 other herpes viruses. Herein, we extend these studies to adults and test the hypothesis that EBV infection is associated with adult SLE. METHODS: We selected 196 antinuclear antibody-positive adult SLE patients (age > or =20 years) and 2 age-, race-, and sex-matched controls per patient. SLE patients and matched controls were tested for evidence of previous infection with EBV, cytomegalovirus (CMV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), or varicella-zoster virus (VZV) by standardized enzyme-linked immunosorbent assays. RESULTS: Of the 196 lupus patients tested, all but 1 had been exposed to EBV, while 22 of the 392 controls did not have antibodies consistent with previous EBV exposure (OR 9.35, 95% CI 1.45-infinity, P = 0.014). No differences were observed between SLE patients and controls in the seroconversion rate against CMV, HSV-2, or VZV. CONCLUSION: These new data from adults, along with the many suggestive features of EBV infection, are consistent with the contribution of this infection to the etiology of SLE.

Adult↗

PubMed1998

Imprinted genes in the Prader-Willi deletion.

Parent-of-origin-specific deletions of proximal chromosome 15q cause either the Prader-Willi syndrome (paternal deletion) or the Angelman syndrome (maternal deletion), two distinct neurodevelopmental disorders. In contrast to the Angelman syndrome, which can also be caused by mutations in a single gene (UBE3A, encoding a ubiquitin ligase), the Prader-Willi syndrome is caused by deletions in about two-thirds of cases and by maternal uniparental disomy in the remaining third. The consequence of both mechanisms, in addition to rare microdeletions or so-called 'imprinting mutations', is lack of the products of multiple genes in the region that are normally expressed only from the paternal chromosome. One gene that is consistently silent in the Prader-Willi syndrome is SNRPN, which encodes the small nuclear ribonucleoprotein particle-associated polypeptide N that forms part of the spliceosomes in the brain. A systematic search for other imprinted genes in the Prader-Willi syndrome region revealed a paternally expressed transcript (IPW, for imprinted in the Prader-Willi region) and a similarly imprinted mouse homologue (Ipw) in the conserved syntenic region on mouse chromosome 7. Ipw is highly expressed in the brain and alternatively spliced to generate different transcripts. Since there is no open reading frame that is conserved in the human and mouse IPW genes, they are postulated to function as untranslated RNAs, possibly regulating transcription in cis in the region.

Animals↗

PubMed2026

Prominent Movement Disorders in RNU2-2-Related Spliceosomopathy.

Pediatric movement disorders often overlap with neurodevelopmental diseases, suggesting shared molecular mechanisms. Variants in small nuclear RNA (snRNA) genes encoding spliceosome components have recently been associated with neurodevelopmental disorders, termed "RNUopathies." We analyzed genome sequencing data from 14 patients with undiagnosed pediatric movement disorders for pathogenic variants in snRNA genes. We identified recurrent de novo RNU2-2 variants (n.35A > G and n.4G > A) in two patients with intellectual disability, epilepsy, and hyperkinetic movement disorders. RNA sequencing of fibroblasts in one patient showed no characteristic transcriptomic signature. Spliceosomopathies should be considered in neurodevelopmental disorders and developmental and epileptic encephalopathies with hyperkinetic features.

Humans↗

PubMed1990

Three structures associated with the nucleolus in male rat germinal cells: round body, coiled body, and "nubecula" and general presence of round body at male meiosis.

In addition to chromosomes and nucleoli, three structures, i.e., round body, coiled body, and nubecula, are encountered in the nucleus during the meiotic prophase in male rats. These structures have been examined by electron microscopy in random and serial sections. The round body is a finely fibrillar, proteinaceous structure closely associated with the granular component of a nucleolus in rat spermatocytes and young spermatids. A similar structure has been observed in man, the monkey Macaca mulatta, the gastropod Achatina fulica, and the insect Locusta migratoria. Together with evidence from the literature, these results support the view that the round body is of general occurrence in the male meiocytes of eukaryotes and may, therefore, play a role in meiosis. The coiled body is a group of electron-dense elements called "coils", which average 35 nm in width, except after mid-pachytene when their size almost doubles. The coils are composed of 2-nm-wide filaments and 8 to 10-nm-wide granules, both of which are ribonucleoprotein. The coiled bodies are interpreted to be groups of "spliceosomes", that is, structures containing heterogeneous RNA and small nuclear RNA. A remarkable feature of the coiled body is its temporary disappearance at early pachytene and its reappearance at late pachytene, possibly due to drastic changes in the turnover rate of its component RNAs. The nubecula is a newly identified nuclear inclusion, composed of weakly staining threads loosely organized into a 560 nm-wide spheroid. It has been observed only in early pachytene nuclei.

Animals↗

PubMed1997

Heterogenous nuclear RNP C1 and C2 core proteins are targets for an autoantibody found in the serum of a patient with systemic sclerosis and psoriatic arthritis.

OBJECTIVE: To determine a target recognized by anti-Bh autoantibody, found in the serum of a patient with the unusual coexistence of systemic sclerosis (SSc) and psoriatic arthritis (PsA). METHODS: Antigens recognized by the anti-Bh serum were characterized by indirect immunofluorescence on HeLa cells, by conventional immunoblotting using nuclear extract or partially purified preparation of heterogenous nuclear RNP (hnRNP) proteins, and by 2-dimensional immunoblotting. For the analysis of cross-reactivity and immunofluorescence patterns, autoantibodies were affinity-purified by blot elution and then retested. RESULTS: Comparison of the reactivity of the anti-Bh antibody with the monoclonal antibody 4F4 against both the hnRNP C proteins, together with the determination of biochemical properties of the autoantigens, led to the identification of C1 and C2 core proteins as the targets for the anti-Bh autoantibody. CONCLUSION: Several essential components of the spliceosome are targeted by autoantibodies that are present in the sera of patients with systemic rheumatic diseases. We also found that the hnRNP core proteins C1 and C2 are recognized by the autoantibody present in the serum of a patient with SSc and PsA. C1 and C2 hnRNP proteins should be added to the several intracellular autoantigens recently shown to be cleaved by interleukin-1beta-converting enzyme-like enzymes during apoptosis.

Aged↗

PubMed2004

Intramolecular T cell spreading in unprimed MRL/lpr mice: importance of the U1-70k protein sequence 131-151.

OBJECTIVE: To analyze spontaneous T cell spreading against determinants of the U1-70K protein in young autoimmune MRL/lpr lupus mice, in comparison with the T cell spreading occurring in normal BALB/c mice immunized with peptide 131-151 of this protein. METHODS: Peripheral blood lymphocytes (PBLs) from both unprimed MRL/lpr mice and immunized BALB/c mice were tested for their ability to proliferate ex vivo in response to 18 overlapping peptides of the U1-70K spliceosomal protein, using assays for lymphocyte proliferation and secretion of interleukin-2. RESULTS: The proliferative response to peptides of the U1-70K protein evolved rapidly in MRL/lpr mice tested at different ages. At least 5 peptides were recognized by PBLs from 8-week-old autoimmune mice, whereas a different peptide was recognized by PBLs from MRL/lpr mice at 12 weeks of age. At 15 weeks, the proliferative response was weak or negative when assessed with any of the test peptides. At least 2 major peptides recognized by MRL/lpr PBLs were also recognized by PBLs generated in the BALB/c mice primed with peptide 131-151. We further demonstrated that, in preautoimmune MRL/lpr mice, repeated administration of phosphorylated peptide 131-151 (called P140), which was shown previously to be protective, transiently abolished T cell intramolecular spreading to other regions of the 70K protein. CONCLUSION: This is the first study to demonstrate that intramolecular T cell spreading effectively occurs in MRL/lpr mice with lupus, and that region 131-151 is important in the cascade of events observed in the murine lupus response. This sequence might originate a mechanism of tolerance spreading that leads to the beneficial effect observed in MRL/lpr mice after treatment with the phosphorylated peptide 131-151.

Animals↗

PubMed1995

Rubisco rules fall; gene transfer triumphs.

The most common form of the CO2-fixing enzyme rubisco is a form I enzyme, heretofore found universally in oxygenic phototrophs (cyanobacteria and plastids) and widely in proteobacteria. Two groups, however (1-4), now report that in dinoflagellate plastids the usual form I rubisco has been replaced by the distantly related form II enzyme, known previously only from anaerobic proteobacteria. This raises the important question of how such an oxygen-sensitive rubisco could function in an aerobic organism. Moreover, the dinoflagellate rubisco has unusual molecular properties: it is encoded as a polyprotein, by nuclear (rather than plastid) genes, and these genes contain noncanonical spliceosomal introns. The nuclear location and alpha-proteobacterial affinity of dinoflagellate rubisco genes hint at a possible mitochondrial origin and highlight the extraordinary richness of lateral gene transfers, both between and within organisms, that have occurred during rubisco evolution.

Animals↗

PubMed1997

Good things in small packages: the tiny genomes of chlorarachniophyte endosymbionts.

Chlorarachniophytes are amoeboflagellate, marine protists that have acquired photosynthetic capacity by engulfing and retaining a green alga. These green algal endosymbionts are severely reduced, retaining only the chloroplast, nucleus, cytoplasm and plasma membrane. The vestigial nucleus of the endosymbiont, called the nucleomorph, contains only three small linear chromosomes and has a haploid genome size of just 380 kb--the smallest eukaryotic genome known. Initial characterisation of nucleomorph DNA has revealed that all chromosomes are capped with inverted repeats comprising a telomere and a single ribosomal RNA operon. The nucleomorph genome is the quintessence of compactness; average space between genes is a mere 65 bp,- some genes overlap, others are cotranscribed. Intense reductive pressures upon nucleomorph genes have apparently squeezed their spliceosomal-type introns down to only 18, 19 or 20 bases in length. Studies to date indicate the nucleomorph--essentially a stripped-down eukaryotic genome--encodes principally genetic housekeeping functions such as translation, transcription and sing.

Animals↗

PubMed2005

Alterations of pre-mRNA splicing in cancer.

Recent genomewide analyses of alternative splicing (AS) indicate that up to 70% of human genes may have alternative splice forms, suggesting that AS together with various posttranslational modifications plays a major role in the production of proteome complexity. Splice-site selection under normal physiological conditions is regulated in the developmental stage in a tissue type-specific manner by changing the concentrations and the activity of splicing regulatory proteins. Whereas spliceosomal errors resulting in the production of aberrant transcripts rarely occur in normal cells, they seem to be an intrinsic property of cancer cells. Changes in splice-site selection have been observed in various types of cancer and may affect genes implicated in tumor progression (for example, CD44, MDM2, and FHIT) and in susceptibility to cancer (for example, BRCA1 and APC). Splicing defects can arise from inherited or somatic mutations in cis-acting regulatory elements (splice donor, acceptor and branch sites, and exonic and intronic splicing enhancers and silencers) or variations in the composition, concentration, localization, and activity of regulatory proteins. This may lead to altered efficiency of splice-site recognition, resulting in overexpression or down-regulation of certain splice variants, a switch in splice-site usage, or failure to recognize splice sites correctly, resulting in cancer-specific splice forms. At least in some cases, changes in splicing have been shown to play a functionally significant role in tumorigenesis, either by inactivating tumor suppressors or by gain of function of proteins promoting tumor development. Moreover, cancer-specific splicing events may generate novel epitopes that can be recognized by the host's immune system as cancer specific and may serve as targets for immunotherapy. Thus, the identification of cancer-specific splice forms provides a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention.

Exons↗

PubMed2002

cAMP-dependent reorganization of the Cajal bodies and splicing machinery in cultured Schwann cells.

It is well established that forskolin-induced elevation of cAMP results in activation of DNA synthesis in Schwann cell cultures. This promitotic response is partially mediated by the Cdk2, which is required for the transition from the G1 to the S phase of the cell cycle. In the present study, we analyze the effects of cAMP elevation in cultured Schwann cells on the transcriptional activity and on the organization of two nuclear compartments involved in pre-mRNA processing: Cajal bodies (CBs) and splicing factor compartments. Our immunofluorescence and quantitative studies show that forskolin treatment induces a 5.6-fold increase in the proportion of S phase Schwann cells, detected by a short pulse (20 min) of BrdU incorporation. This increase in DNA synthesis correlates with an activation of global transcription, as is indicated by the higher nuclear incorporation of BrU in nascent RNA. Forskolin treatment significantly increases the percentage of Schwann cells containing typical CBs, which concentrate spliceosomal snRNPs and the survival motor neuron (SMN) protein. This increase in the number of CBs closely correlates with the activation of transcription. Moreover, the occurrence of CBs is significantly higher in BrdU (+) cells than in BrdU (-) cells, indicating that entry in the S phase promotes the formation of CBs. During the S phase, Schwann cell nuclei display higher Cdk2 nuclear staining and concentrate this kinase in CBs. Forskolin also induces a redistribution of the pre-mRNA splicing factors in Schwann cells. Primary cultures of Schwann cells provide an excellent physiological model to demonstrate that the assembly of CBs is a transcription- and replication-dependent cellular event. Moreover, the S phase accumulation of Cdk2 observed in Schwann cells supports a functional link between CBs and DNA replication, which is mediated by the possible participation of CBs in the regulatory control of histone gene expression.

Animals↗

PubMed1988

Antisense RNA inhibits splicing of pre-mRNA in vitro.

Antisense RNAs complementary to human beta-globin pre-mRNA or to a chimeric globin/adenovirus E2a pre-mRNA specifically and efficiently inhibit pre-mRNA splicing in vitro. The level of inhibition depends on the length, position and concentration of the antisense RNA relative to the pre-mRNA substrate. Antisense RNAs complementary to sequences greater than 80 nucleotides downstream of the globin 3' splice site inhibit at least as efficiently as those extending across the splice sites. Thus splicing is sensitive to perturbations involving exon sequences some distance from the splice sites. Inhibition is mediated by factors which affect the annealing of antisense and substrate RNAs. Direct analysis of RNA duplex formation demonstrates the presence of an activity in HeLa cell nuclear extract which promotes the rapid annealing of complementary RNAs in an ATP-independent manner. Both annealing and inhibition are greatly reduced when antisense RNA is added to the splicing reaction greater than or equal to 5 min after substrate. This result may reflect a transition between an open structure, in which annealing of antisense RNA with pre-mRNA is facilitated, and a closed complex in which pre-mRNA is sequestered at an early stage of spliceosome assembly.

Adenoviridae↗

PubMed1989

Pre-mRNA splicing mutants of Schizosaccharomyces pombe.

A collection of temperature sensitive (ts-) mutants was prepared by chemical mutagenesis of a wild type Schizosaccharomyces pombe strain. To screen the ts- mutants for pre-mRNA splicing defects, an oligodeoxynucleotide that recognizes one of the introns of the beta-tubulin pre-mRNA was used as a probe in a Northern blot assay to detect accumulation of intron sequences. This screening procedure identified three pre-mRNA splicing mutants from 100 ts- strains. The three mutants are defective in an early step of the pre-mRNA splicing reaction; none accumulate intermediates. The precursors that accumulate at 37 degrees C are polyadenylated. Analysis of the splicing of another pre-mRNA showed that the mutations are not specific for beta-tubulin. The total RNA pattern in the three splicing mutants appears to be normal. In addition, the amounts of the spliceosomal snRNAs are not drastically changed compared to the wild type and splicing of pre-tRNAs is not blocked. Genetic analyses demonstrate that all three splicing mutations are tightly linked to the ts- growth defects and are recessive. Crosses among the mutants place them in three complementation groups. The mutants have been named prp1, prp2 and prp3.

Crosses, Genetic↗

PubMed1989

Evidence from complementation assays in vitro that U5 snRNP is required for both steps of mRNA splicing.

We have established an in vitro complementation system that has allowed us to investigate the role of individual purified snRNPs in the splicing of pre-mRNA molecules. For the preparation of snRNP-depleted nuclear extracts we have first removed the majority of endogenous snRNPs from the nuclear extracts by one passage over an anti-m3G column and then degraded the remaining snRNPs with micrococcal nuclease. The mixture of snRNPs U1, U2, U4/U6 and U5, obtained by anti-m3G immuno-affinity chromatography, was functionally active and able to restore the splicing of snRNP-depleted nuclear extracts. Mono-Q chromatography was used for further fractionation of the snRNPs U1-U6. This produced three fractions that were highly enriched in snRNPs U1 and U2, U5 and U4/U6 respectively. Conditions were found where addition of the [U1, U2] and the U4/U6 snRNP fractions to the snRNP-depleted nuclear extracts gave rise to the formation of splice intermediates in the absence of any 3' cleavage/exon 1-exon 2 product formation. Only when purified 20S U5 snRNPs were added did both steps of the splicing reaction occur efficiently. Our data suggest that U5 snRNP is absolutely required for the second step of splicing and is needed further for efficient initiation of the splicing reaction. The requirement for U5 snRNPs for splicing was corroborated by glycerol gradient sedimentation analysis of the respective reconstituted pre-mRNP complexes. Stable and efficient formation of 50-60S spliceosomes was observed only in the presence of all snRNPs.

Centrifugation, Density Gradient↗

PubMed1990

Splicing factor SF4 is dispensable for the assembly of a functional splicing complex and participates in the subsequent steps of the splicing reaction.

The splicing of nuclear messenger RNA precursors (pre-mRNA) can be reconstituted in vitro with factors partially purified from HeLa cell nuclear extracts. Splicing complexes are assembled in the presence of the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6 and the protein factors SF1, SF2, SF3 and U2AF. However, the complexes thus formed are inactive, i.e. they only contain unprocessed pre-mRNA. The intermediates and products of the splicing reaction are generated after addition of SF4. This splicing factor is a heat-labile protein which requires sulfhydryl groups for its activity. SF4 appears to participate, directly or indirectly, in the conversion of a functional but inactive splicing complex to the active spliceosome.

Cell Nucleus↗

PubMed1990

Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes.

U6 snRNA sequences required for assembly of U4/U6 snRNP and splicing complexes were determined by in vitro reconstitution of snRNPs. Both mutagenesis and chemical modification/interference assays identify a U6 snRNA domain required for U4/U6 snRNP formation. The results support the existence of a U4/U6 snRNA interaction domain previously proposed on the basis of phylogenetic evidence. In addition, two short U6 snRNA regions flanking the U4/U6 interaction domain are essential to assemble the U4/U6 snRNP into splicing complexes. These two regions may represent binding sites for splicing factors or may facilitate the formation of an alternative U6 snRNA secondary structure during spliceosome assembly.

Base Sequence↗

PubMed1990

A mutation in a single gene of Schizosaccharomyces pombe affects the expression of several snRNAs and causes defects in RNA processing.

A bank of temperature sensitive (ts-) mutants of Schizosaccharomyces pombe was screened for snRNA expression mutants using an oligodeoxynucleotide that recognizes U2 RNA. One mutant with a novel phenotype was identified that has reduced steady-state levels of the spliceosomal snRNAs U1, U2, U4, U5 and U6. In addition, the mutant exhibits a temperature-dependent accumulation of aberrant U2 and U4 transcripts elongated at their 3' end. The steady-state concentration of the RNA component of RNase P is also reduced in the mutant, whereas the amount of U3 RNA, 7SL RNA, tRNA, rRNA and mRNA are the same as wild-type. Pre-mRNA, pre-tRNA and U6 RNA precursor processing are impaired in the mutant. Genetic analysis demonstrates that the snRNA defects are tightly linked to the ts- growth defect and are recessive. We have named this mutant snm1 to indicate a defect in snRNA maintenance. The data on snm1 suggest that a single trans-acting factor is essential for the maintenance of steady-state levels of several snRNAs and for proper 3' end formation of U2 and U4 RNAs.

Base Sequence↗