Expanding the MYBPC1 phenotypic spectrum: a novel homozygous mutation causes arthrogryposis multiplex congenita.

Arthrogryposis multiplex congenita (AMC) is characterized by heterogeneous nonprogressive multiple joint contractures appearing at birth. We present a consanguineous Israeli-Druze family with several members presenting with AMC. A variable intra-familial phenotype and pected autosomal recessive inheritance prompted molecular diagnosis by whole-exome sequencing. Variant analysis focused on rare homozygous changes, revealed a missense variant in MYBPC1, NM_002465:c.556G>A (p.E286K), affecting the last nucleotide of Exon 8. This novel variant was not observed in the common variant databases and co-segregated as expected within the extended family. MYBPC1 encodes a slow skeletal muscle isoform, essential for muscle contraction. Heterozygous mutations in this gene are associated with distal arthrogryposis types 1b and 2, whereas a homozygous nonsense mutation is implicated in one family with lethal congenital contractural syndrome 4. We present a novel milder MYBPC1 homozygous phenotype.

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction.

Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem-loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem-loop IIa can participate in spliceosome assembly.

Internal carotid artery dissection causes hypoglossal nerve palsy: CT, MRI, and angiographic findings.

A case of unilateral XIIth nerve palsy due to the dissection of the internal carotid artery is reported. The clinical and radiological features are described. In this patient, cranial nerve palsy is probably the result of compression by an enlarging carotid artery due to mural hematoma. Diagnosis is discussed with emphasis on magnetic resonance imaging findings. Magnetic resonance imaging is also useful for follow-up of arterial lesions.

Antisense oligonucleotide binding to U5 snRNP induces a conformational change that exposes the conserved loop of U5 snRNA.

Conformational rearrangements of the spliceosomal small nuclear RNAs (U snRNAs) are essential for proper assembly of the active site prior to the first catalytic step of splicing. We have previously shown that conformational changes caused by binding of an antisense 2'-O-methyl RNA oligonucleotide (BU5Ae) to U5 snRNA nt 68-88 disrupted the U4/U5/U6 complex and induced formation of the U1/U4/U5 and U2/U6 complexes. Here we show that the conformational change induced by BU5Ae exposes the invariant loop of U5 that binds the 5'exon and also reorganizes internal loop 1 (IL1) and the top of stem 2. Interestingly, we have also previously found that the U1/U4/U5 complex induced by BU5Ae brings the invariant loop of U5 into close proximity with the 5'-end of U1. Taken together, these data suggest that U1 and U5 may both contribute to the ability of the U1/U4/U5 complex to bind the 5' splice site.

A novel U1/U5 interaction indicates proximity between U1 and U5 snRNAs during an early step of mRNA splicing.

The five spliceosomal snRNAs (U1, U2, U4, U5, and U6) undergo an ordered sequence of conformational changes as mRNA splicing progresses. We have shown that an antisense RNA oligonucleotide complementary to U5 snRNA induces a novel U1/U4/U5 complex that may be a transitional stage in the displacement of U1 from the 5' splice site by U5. Here we identify a novel site-specific crosslink between the 5' end of U1 and the invariant loop of U5 snRNA. This crosslink can be induced in nuclear extract by an antisense oligonucleotide directed against U5 snRNA, but can also be detected during an early step of the splicing reaction in the absence of oligonucleotide. Our data indicate proximity between U1 and U5 snRNPs before the first catalytic step of splicing, and may suggest that U1 helps to direct U5 to the 5' splice site.

A U1/U4/U5 snRNP complex induced by a 2'-O-methyl-oligoribonucleotide complementary to U5 snRNA.

Nuclear messenger RNA splicing involves multiple interactions between the five spliceosomal small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5, and U6 and numerous spliceosomal proteins. Here it is shown that binding of a 2'-O-methyl-oligoribonucleotide complementary to U5 small nuclear RNA (snRNA) nucleotides 68 to 88 (BU5Ae) disrupts the initial U4/U5/U6 tri-snRNP complex, enhances the U2/U6 interaction, and induces a Ul/U4/U5 snRNP complex. The Ul/U4/U5 snRNP complex interacts specifically with an RNA oligonucleotide containing the 5' splice site sequence and may therefore represent a transitional stage in the displacement of U1 from the 5' splice site by U5 snRNP.

Splicing components are excluded from the transcriptionally inactive XY body in male meiotic nuclei.

The study of the effect of programmed cessation of transcription in a large nuclear domain, on the distribution of elements of the pre-mRNA splicing machinery, is the main aim of this paper. To this end, we took advantage of the nuclear partitioning of mouse spermatocytes early in meiosis into autosomal transcribing and XY nontranscribing compartments. This system also allows to extend this study to stages in sperm differentiation that are accompanied by reduction and eventual cessation of transcription. We show by indirect immunofluorescence in spermatogenetic cells that 1) fluorescent signals of the pre-mRNA splicing factors SF53/4 and SC35, of the Sm antigens, and of RNA polymerase II, are largely absent from the nontranscribing, X-inactivated compartment, but are abundantly present in the transcribing autosomal compartment and 2) the presence, gradual reduction, and absence of transcriptive activity in nuclei undergoing the sperm formation sequence are positively correlated with the fluorescence patterns of the antibodies against SF53/4, SC35, and the Sm antigens. These data suggest that cessation of transcription during spermatogenesis is accompanied by exclusion of the splicing machinery from nontranscribing chromatin to its vicinity.

An autoantibody derived from mice with experimental systemic lupus erythematosus is directed against the essential splicing factor SF53/4--a possible role for large nuclear ribonucleoprotein particles in autoimmune disorders.

We have previously shown that nuclear transcripts of several pre-mRNAs can be released from nuclei of mammalian cells in a form of large nuclear ribonucleoprotein (InRNP) particles. These particles, which invariably sediment at the 200S region in sucrose gradient, contain all U small nuclear RNPs required for pre-mRNA splicing and a multitude of heterogeneous nuclear RNP proteins. From a panel of mAbs raised against the InRNP particles, a specific mAb (53/4) identified a nuclear protein of 88 kDa as an essential splicing factor (SF53/4). In a parallel independent study, mAbs were established in mice with experimental systemic lupus erythematosus (SLE), that had been induced by immunization with a murine mAb against a human anti-DNA mAb bearing the common 16/6 idiotype. One of the produced mAbs (2C5/3) recognized an 88 kDa RNP protein. In the present study we have used the following criteria to demonstrate that mAb 2C5/3 and mAb 53/4 recognize the same protein. First, mAb 2C5/3 inhibited splicing by direct addition. Second, the 88 kDa polypeptide that had been immunodepleted from HeLa cells nuclear extract by mAb 2C5/3 was recognized by mAb 53/4 in protein blots. Third, the HeLa nuclear extract depleted by mAb 2C5/3 was devoid of splicing activity and could not assemble into splicing complexes with exogenous pre-mRNA; however, splicing and spliceosome assembly activities were restored to such a defective extract by adding back the 88 kDa protein that had been purified by immunoaffinity binding to immobilized mAb 53/4.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous dissection of the internal carotid artery in 68 patients.

OBJECTIVES: Our purpose was to know more about the symptoms and clinical and radiological outcome of spontaneous dissections of the internal carotid artery in a retrospective study of 68 patients aged 20 to 71 (mean 46). METHODS: The diagnosis of dissection was based on angiographic findings. Nine percent of patients had minor symptoms such as a subjective bruit or painful Horner's syndrome, without an ischaemic event. Cerebral ischaemia was present in 90% of cases and occurred within a month of the initial event in all cases but one and was the first symptom in 53% of cases. RESULTS: Magnetic resonance imaging performed in 21 cases showed haemorrhage in the vessel wall. Resolution of the angiographic appearances occurred in 65% of cases after 3 months. In cases of stroke, more than half of the patients had poor functional outcome, factors conveying poor prognosis were massive stroke, embolic mechanism and lack of local recanalization. CONCLUSION: Spontaneous dissection of the internal carotid artery is not a rare cause of cerebral ischaemia and can present with minor symptoms without an ischaemic event. Doppler ultrasonography and magnetic resonance imagery are helpful in diagnosis and follow-up.

A novel splicing factor is an integral component of 200S large nuclear ribonucleoprotein (InRNP) particles.

In previous studies we have shown that nuclear transcripts of several pre-mRNAs can be released from nuclei of mammalian cells in the form of large nuclear ribonucleoprotein (InRNP) particles. By electron microscopy, these particles appeared as compact composite structures, 50 nm in diameter, which invariably sedimented at the 200S region in sucrose gradients. In order to identify putative protein splicing factors associated with the 200S InRNP particles, a panel of monoclonal antibodies directed against these particles were screened for their ability to inhibit splicing of pre-mRNA in vitro. In this study we have focused on a nuclear protein of 88 kd in molecular weight, which is an integral component of the InRNP complex and is recognized by monoclonal antibodies from a specific clone. This protein has been identified here as a novel splicing factor by, (i) antibody inhibition of splicing in vitro and (ii) depletion of splicing activity from HeLa cell nuclear extract after removing the 88 kd polypeptide by immunoadsorption, and complementation of the depleted activity with an affinity-purified 88 kd antigen. This splicing factor has further been shown to be required for the assembly of an active splicing complex.

[Schistosomiasis of the spinal cord. Contribution of magnetic resonance imaging]. / Schistosomiase médullaire. Apport de l'imagerie par résonance magnétique.

The authors report a case of spinal cord schistosomiasis presenting as myelitis, with rapidly developing deficit, signs of severe cerebrospinal fluid inflammation, normal myelography and computerized tomography. The patient's country of origin suggested schistosomiasis, and the diagnosis was confirmed by serology and rectal biopsy which showed eggs of Schistosoma mansoni. Magnetic resonance imaging was helpful as it confirmed the absence of spinal cord compression and showed a lesion of the conus medullaris, this region being the most frequent site of schistosomial myelitis.

NMR study of the complexes between a synthetic peptide derived from the B subunit of cholera toxin and three monoclonal antibodies against it.

The contact interactions between a synthetic peptide and three different anti-peptide monoclonal antibodies have been studied by nuclear magnetic resonance (NMR). The synthetic peptide is CTP3 (residues 50-64 of the B subunit of cholera toxin) suggested as a possible epitope for synthetic vaccine against cholera. The hybridoma cell lines TE33 and TE32 derived after immunization with CTP3 produce antibodies cross-reactive with the native toxin. The cell line TE34 produces anti-CTP3 antibodies that do not bind the toxin. Selective deuteriation of the antibodies has been used to simplify the proton NMR spectra and to assign resonances to specific types of amino acids. The difference spectra between the proton NMR spectrum of the peptide-Fab complex and that of Fab indicate that the combining site structures of TE32 and TE33 are very similar but differ considerably from the combining site structure of TE34. By magnetization transfer experiments with selectively deuteriated Fab fragment of the antibody, we have found that in TE32 and TE33 the histidine residue of the peptide is buried in a hydrophobic pocket of the antibody combining site, formed by a tryptophan and two tyrosine residues. The hydrophobic nature of the pocket is further demonstrated by the lack of any pH titration effect on the chemical shift of the C4H of the bound peptide histidine. In contrast, for TE34 we have found only one tyrosine residue in contact with the histidine of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)