Putative Splicing Factor Research Articles

SCNM1, a putative RNA splicing factor that modifies disease severity in mice.

The severity of many inherited disorders is influenced by genetic background. We describe a modifier interaction in C57BL/6Jmice that converts a chronic movement disorder into a lethal neurological disease. The primary mutation (medJ) changes a splice donor site of the sodium channel gene Scn8a (Nav1.6). The modifier mutation is characteristic of strain C57BL/6Jand introduces a nonsense codon into sodium channel modifier 1 (SCNM1), a zinc finger protein and a putative splice factor. An internally deleted SCNM1 protein is also predicted as a result of exon skipping associated with disruption of a consensus exonic splicing enhancer. The effect of the modifier mutation is to reduce the abundance of correctly spliced sodium channel transcripts below the threshold for survival. Our finding that genetic variation in a putative RNA splicing factor influences disease susceptibility in mice raises the possibility that a similar mechanism modifies the severity of human inherited disorders.

Two Neuronal, Nuclear-Localized RNA Binding Proteins Involved in Synaptic Transmission

While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using Caenorhabditis elegans as a model for studying neuron/target communication, we report that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. We show that unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. We found that human orthologs of UNC-75, whose splicing activity has recently been documented in vitro [1, 2], are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, we report that the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, we identified a new neuronal, putative pre-mRNA splicing factor, UNC-75, and show that UNC-75, as well as the C. elegans homolog of ELAV, is required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function.

SF4 and SFRS14, two related putative splicing factors on human chromosome 19p13.11

The splicing of nascent mRNA precursors is an essential step for the expression of all intron-containing eukaryotic genes. Removal of intron sequences from nascent transcripts is mediated by the spliceosome, a large multicomponent complex. We describe here the identification of two genes encoding related, putative splicing factors on human chromosome 19p13.11, SF4 (splicing factor 4) and SFRS14 (splicing factor arginine/serine-rich 14). Both genes encode proteins containing a SURP motif; this domain is found in several splicing proteins including Drosophila alternative splicing regulator, suppressor-of-white-apricot (SWAP) and the yeast splicing factor, prp21p. In addition, SF4 and SFRS14 contain a G-patch domain at their C-termini, a motif present in a large number of eukaryotic RNA-binding proteins. SFRS14 also contains an N-terminal region that is rich in arginine/serine residues, suggesting SFRS14 is a novel member of the SR-related family of pre-mRNA processing factors. We have also identified the mouse orthologues of SF4 and SFRS14, based on conserved domain organization and high sequence similarity. Interestingly, SFRS14 undergoes alternative 3′-end processing events that are conserved between human and mouse, suggesting a functional significance.

Identification of semaphorin E gene expression in metastatic human lung adenocarcinoma cells by mRNA differential display.

Human lung adenocarcinoma cell lines HAL-8Luc and HAL-24Luc differ in their metastatic potential. HAL-8Luc cells metastasize to lungs when injected either intravenously or intramuscularly. in mice while HAL-24Luc cells do not. The differential display method is used to identify genes differentially expressed between the two cell lines and the findings are extensively discussed. Lung cancer is the leading form of cancer in most countries, and metastasis is the main cause of death in oncological patients. The metastatic phenotype of tumor cells is the result of genetic events altering the RNA and protein expression of normal cells. Our objective was to identify genes expressed differentially between metastatic and nonmetastatic human lung adenocarcinoma cells that might be used as a prognostic factor. The differential display technique was used to compare the RNA expression patterns distinguishing metastatic (HAL-8Luc) and nonmetastatic (HAL-24Luc) human lung adenocarcinoma cells, two genetically close cell lines. Differential expression of three cDNAs was confirmed by Northern blot analysis. Two sequences corresponding to a putative splicing factor and a proliferation-related factor cDNAs were underexpressed in the metastatic cells relative to the nonmetastatic ones. Interestingly, we found that human semaphorin E mRNA was several fold overexpressed in the metastatic cells. This recently identified gene encodes a protein whose expression has been related to several cell survival mechanisms as well as to immunosuppression. Our results point to the relevance of semaphorin E in metastatic spread of human lung adenocarcinoma cells.

Identification of semaphorin E gene expression in metastatic human lung adenocarcinoma cells by mRNA differential display

Synopsis Human lung adenocarcinoma cell lines HAL-8Luc and HAL-24Luc differ in their metastatic potential. HAL-8Luc cells metastasize to lungs when injected either intravenously or intramuscularly. in mice while HAL-24Luc cells do not. The differential display method is used to identify genes differentially expressed between the two cell lines and the findings are extensively discussed. Background Lung cancer is the leading form of cancer in most countries, and metastasis is the main cause of death in oncological patients. The metastatic phenotype of tumor cells is the result of genetic events altering the RNA and protein expression of normal cells. Our objective was to identify genes expressed differentially between metastatic and nonmetastatic human lung adenocarcinoma cells that might be used as a prognostic factor. Methods The differential display technique was used to compare the RNA expression patterns distinguishing metastatic (HAL-8Luc) and nonmetastatic (HAL-24Luc) human lung adenocarcinoma cells, two genetically close cell lines. Results Differential expression of three cDNAs was confirmed by Northern blot analysis. Two sequences corresponding to a putative splicing factor and a proliferation-related factor cDNAs were underexpressed in the metastatic cells relative to the nonmetastatic ones. Interestingly, we found that human semaphorin E mRNA was several fold overexpressed in the metastatic cells. This recently identified gene encodes a protein whose expression has been related to several cell survival mechanisms as well as to immunosuppression. Conclusion Our results point to the relevance of semaphorin E in metastatic spread of human lung adenocarcinoma cells. J. Surg. Oncol. 1999;72:18–23. © 1999 Wiley-Liss, Inc.

Repeat-Induced Point Mutations inPad-1,a Putative RNA Splicing Factor fromNeurospora crassa,Confer Dominant Lethal Effects on Ascus Development

We describe the characterization of a gene,Pad-1,fromNeurospora crassawhich displays sequence characteristics of the RS class of hnRNA-binding proteins (hnRNP) and mRNA splicing factors. This is the first report of the isolation of a putative hnRNP gene fromN. crassa.PAD-1 showed 30% identity and 57% similarity to a protein, HCC1, which was isolated using autoantibodies from patients suffering from hepatocellular carcinoma. Both HCC1 and PAD-1 show amino acid sequence similarities to the human splicing factor, U2AF65. Mutations induced inPad-1by repeat-induced point (RIP) mutation show dominant effects on ascus and ascospore formation, a novel phenotypic class of RIP mutants. A mutant isolated from thePad-1RIP cross displayed a severe vegetative growth defect and dominant effects on ascus development, indicating thatPad-1is essential for both asexual and sexual development.

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.