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PubMed2004

Characterization of Ceap-11 and Ceap-16, two novel splicing-variant-proteins, associated with centrosome, microtubule aggregation and cell proliferation.

A novel human gene, encoding two polypeptide-isoforms, has been identified from human fetal liver cDNA library. These two alternatively spliced polypeptide-variants are associated with centrosomes, and are designated Ceap-11 and Ceap-16, respectively, according to the acronym Ceap for centrosomal-associated protein and the approximate relative molecular mass. The high degree of sequence similarity between Ceap proteins of divergent species indicates that the Ceap homologous genes are significantly conserved in evolution and constitute a new gene family without any functional information until now. Human Ceap gene is mapped on 10q24.2. These two Ceap cDNA isoforms are generated by RNA alternative splicing on the 5' terminus of the Ceap gene, and are composed of four and five exons, respectively. Ceap-11 and Ceap-16 are co-immunoprecipitated and co-located with gamma-tubulin; ectopic overexpression of these two proteins in NIH3T3 cells induces microtubule aggregation and cell proliferation; the protein level of Ceap in certain tumors is significantly higher than that in corresponding normal tissues. Taken together, our data provide the first evidence for the function of Ceap-11 and Ceap-16, the two novel human proteins, namely, association with centrosome, microtubule aggregation and cell proliferation.

Alternative Splicing↗
PubMed1999

The Mycobacterium tuberculosis recA intein can be used in an ORFTRAP to select for open reading frames.

The DNA repair protein RecA of Mycobacterium tuberculosis contains an intein, a self-splicing protein element. We have employed this Mtu recA intein to create a selection system for successful intein splicing by inserting it into a kanamycin-resistance gene so that functional antibiotic resistance can only be restored upon protein splicing. We then proceeded to develop an ORFTRAP, i.e., a selection system for the cloning of open reading frames (ORFs). The ORFTRAP exploits the self-splicing properties of inteins (which depend on full-length in-frame translation of a precursor protein) by allowing protein splicing to occur when DNA fragments encoding ORFs are inserted into the Mtu recA intein, whereas DNA fragments containing non-ORFs are selected against. Regions of the Mtu recA intein that tolerate the insertion of additional amino acids were identified by Bgl II linker scanning mutagenesis, and a respective construct was chosen as the ORFTRAP. To test the maximum insert size that could be cloned into ORFTRAP, DNA fragments of increasing length from the Listeria monocytogenes hly gene as well as a genomic library of Haemophilus influenzae were inserted and it was found that the longest permissive inserts were 425 bp and 251 bp, respectively. The H. influenzae ORFTRAP library also demonstrated the strength (strong selection power) and weakness (insertion of very small fragments) of the system. Further modifications should make the ORFTRAP useful for protein expression, epitope mapping, and antigen screening.

Amino Acid Sequence↗
PubMed1983

A method for mapping RNA initiation, termination, splice, and protein binding sites. Ribosome binding sites on beta-globin messenger RNA.

A new method for mapping RNA initiation, termination, and splice sites was developed. The method involves: 1) hybridization of RNA to end-labeled single-stranded DNA; 2) mild digestion of the hybrid with a single strand specific nuclease; 3) high resolution gel electrophoresis and autoradiography. The regions of the labeled probe which are resistant to nuclease digestion are mapped by measuring the distance from the labeled end. The sequence can be determined by running the end-labeled probe sequenced according to the protocol of Maxam and Gilbert (Maxam, A.M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) at the same time. The feasibility of this method was tested with adult chicken beta-globin mRNA, and we found that the transcription initiation, termination, and RNA splice sites can be determined to within a few bases of the known sites. Using this method we also found that ribosomes bind to beta-globin mRNA from 22 bases upstream from the translation start codon (AUG) to 15 bases past the stop codon (UAA).

Animals↗
PubMed2004

Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel).

The LEM motif is a sequence of 40-50 amino acids that has been identified in a number of non-related proteins of the inner nuclear membrane including the lamina-associated polypeptides 2 (LAP2), emerin, MAN1 and the Drosophila protein otefin. This evolutionary conserved sequence motif can mediate via the interaction with the small protein BAF the binding of LEM-domain proteins to DNA. Taking advantage of its sequenced genome we analyzed whether Drosophila possesses beside otefin additional genes coding for proteins with a LEM motif. A putative candidate gene was the annotated gene CG9424 which we named Bocksbeutel. Of all putative Drosophila LEM-domain proteins, otefin and Bocksbeutel exhibited the highest similarity in the LEM motif (53% identical amino acids). The Bocksbeutel gene can code for two isoforms of 399 and 351 amino acids that are produced by alternative splicing. In the alpha-isoform a transmembrane domain is localized close to the carboxyterminus. This segment is absent in the shorter beta-isoform. By RT-PCR we could show that in the embryo the mRNA coding for the alpha-isoform and in significantly lower amounts the mRNA coding for the beta-isoform are expressed. When expressed in transfected cells as GFP fusion proteins, the beta-isoform is localized predominantly in the nucleoplasm and the alpha-isoform is targeted to the nuclear envelope, indicating that Bocksbeutel-alpha is localized in the inner nuclear membrane. Bocksbeutel-alpha is the predominant isoform expressed in cells, larvae, and flies. Indirect immunofluorescence with Bocksbeutel-specific antibodies on tissues and cultured cells revealed that Bocksbeutel proteins are localized in the nuclear envelope and in the cytoplasm. By RNA interference we have down-regulated the expression of Bocksbeutel, BAF, otefin, and lamin DmO in Drosophila Kc167 cells. The down-regulation of Bocksbeutel and otefin had no influence on the viability of Kc167 cells and the intracellular localization of all other nuclear and nuclear envelope proteins analyzed. In contrast, when lamin DmO was reduced by RNAi the distribution of Bocksbeutel and otefin in the nuclear envelope of Kc167 cells was significantly altered. We conclude that the two LEM-domain proteins Bocksbeutel and otefin are no limiting components for the maintenance of the nuclear architecture in cultured Drosophila cells at interphase.

Alternative Splicing↗
PubMed2005

Identification of three novel mutations in the dihydropyrimidine dehydrogenase gene associated with altered pre-mRNA splicing or protein function.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil and thymine, as well as of the widely used chemotherapeutic drug 5-fluorouracil (5FU). Analysis of the DPD gene ( DPYD ) in two patients presenting with complete DPD deficiency and the parents of an affected child showed the presence of three novel mutations, including one splice site mutation IVS11 + 1G-->T and the missense mutations 731A-->C (E244V) and 1651G-->A (A551T). The G-->T mutation in the invariant GT splice donor site flanking exon 11 (IVS11 + 1G-->T) created a cryptic splice site within exon 11. As a consequence, a 141-bp fragment encoding the aminoacid residues 400-446 of the primary sequence of the DPD protein was missing in the mature DPD mRNA. Analysis of the crystal structure of pig DPD suggested that the E244V mutation might interfere with the electron flow between NADPH and the pyrimidine binding site of DPD. The A551T point mutation might prevent binding of the prosthetic group FMN and affect folding of the DPD protein. The identification of these novel mutations in DPYD will allow the identification of patients with an increased risk of developing severe 5FU-associated toxicity.

Adolescent↗
PubMed2004

Prp8 intein in fungal pathogens: target for potential antifungal drugs.

Inteins are self-splicing intervening sequences in proteins, and inteins of pathogenic organisms can be attractive drug targets. Here, we report an intein in important fungal pathogens including Aspergillus fumigatus, Aspergillus nidulans, Histoplasma capsulatum, and different serotypes of Cryptococcus neoformans. This intein is inside the extremely conserved and functionally essential Prp8 protein, and it varies in size from 170 aa in C. neoformans to 819 aa in A. fumigatus, which is caused by the presence or absence of an endonuclease domain and a putative tongs subdomain in the intein. Prp8 inteins of these organisms were demonstrated to do protein splicing in a recombinant protein in Escherichia coli. These findings revealed Prp8 inteins as attractive targets for potential antifungal drugs to be identified using existing selection and screening methods.

Amino Acid Sequence↗
PubMed1999

A genetic system yields self-cleaving inteins for bioseparations.

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

Amino Acid Sequence↗
PubMed2000

Modulation of pre-mRNA splicing and protein production of fibronectin by TGF-beta2 in porcine trabecular cells.

PURPOSE: To determine the effect of transforming growth factor (TGF)-beta2 on the pre-mRNA splicing pattern of fibronectin, as well as on the synthesis and secretion of this glycoprotein by porcine trabecular cells. METHODS: First-passage porcine trabecular cells were rendered quiescent and incubated in culture medium containing 15% newborn calf serum, in serum-free culture medium containing either activated TGF-beta2 (concentration range: 0.2-2.7 ng/ml) or activated TGF-beta1 (1 ng/ml), or in serum-free medium alone (untreated control samples). For investigation of alternative splicing, total RNA was extracted, and reverse transcription-polymerase chain reaction (RT-PCR) was performed with primer pairs located in exons flanking the exon (extra domain [ED]A, or EDB) that undergoes alternative splicing. The polymerase chain reaction (PCR) products were verified by Southern hybridization and quantified by using laser densitometry. The percentage of EDA-positive (+) isoforms was compared with that of the EDB+ isoforms among the groups. To study the effect of TGF-beta2 on the synthesis and secretion of fibronectin, total protein was extracted from both cultured cells and conditioned medium, Western blot analysis was performed using an anti-fibronectin antibody, and the products were quantified by laser densitometry. Immunocytochemical analysis was also performed on cultured trabecular cells to detect fibronectin. RESULTS: Fibronectin mRNA that was detected in untreated serum-starved control cells was EDA and EDB negative. Incubation of trabecular cells in medium containing 1 ng/ml TGF-beta2, 1 ng/ml TGF-beta1, or 15% newborn calf serum induced the expression of EDA+ and EDB+ mRNA to varying degrees. At concentrations of 0.2, 0.5, 1.5, and 2.7 ng/ml, TGF-beta2 increased the concentration of fibronectin by 2-, 3-, 3.8-, and 5-fold in the conditioned medium, and by 3-, 3.7-, 4-, and 4.3-fold in the cell extracts, respectively. The trabecular cells treated with TGF-beta2 exhibited strong immunoreaction for fibronectin, whereas the cells incubated in serum-free medium showed only minimal immunoreactivity. CONCLUSIONS: Our results demonstrate that TGF-beta2 and TGF-beta1 modified the alternative splicing pattern of fibronectin pre-mRNA and enhanced the synthesis and secretion of this extracellular matrix molecule by trabecular cells in a dose-dependent fashion. These findings indicate a mechanism whereby TGF-beta2, the concentration of which is elevated in aqueous humor of patients with primary open-angle glaucoma, contributes to the increased deposition of extracellular matrix molecules in the outflow pathway.

Alternative Splicing↗
PubMed2002

Alternative splicing during chondrogenesis: modulation of fibronectin exon EIIIA splicing by SR proteins.

The alternative exon EIIIA of the fibronectin gene is included in mRNAs produced in undifferentiated mesenchymal cells but excluded from differentiated chondrocytes. As members of the SR protein family of splicing factors have been demonstrated to be involved in the alternative splicing of other mRNAs, the role of SR proteins in chondrogenesis-associated EIIIA splicing was investigated. SR proteins interacted with chick exon EIIIA sequences that are required for exon inclusion in a gel mobility shift assay. Addition of SR proteins to in vitro splicing reactions increased the rate and extent of exon EIIIA inclusion. Co-transfection studies employing cDNAs encoding individual SR proteins revealed that SRp20 decreased mRNA accumulation in HeLa cells, which make A+ mRNA, apparently by interfering with pre-mRNA splicing. Co-transfection studies also demonstrated that SRp40 increased exon EIIIA inclusion in chondrocytes, but not in HeLa cells, suggesting the importance of cellular context for SR protein activity. Immunoblot analysis did not reveal a relative depletion of SRp40 in chondrocytic cells. Possible mechanisms for regulation of EIIIA splicing in particular, and chondrogenesis associated splicing in general, are discussed.

Alternative Splicing↗
PubMed2005

Overexpression of SR proteins and splice variants modulates chondrogenesis.

Fibronectin alternative exon EIIIA is largely included in undifferentiated mesenchymal cells of the developing limb bud, whereas the exon is excluded in differentiated chondrocytes. Inclusion of exon EIIIA in chondrocytic cells is increased by overexpression of SRp40, and, to a lesser extent, SRp75, but not SRp55. RT-PCR analysis using real-time PCR revealed that the levels of the mRNAs for these three proteins did not vary significantly in chick chondrocytes versus mesenchymal cells of the developing limb bud. However, a variant spliced form of SRp40, termed, SRp40LF, is detected preferentially in chondrocytes and in chondrifying mesenchymal cells. Forced overexpression of SRp40 or SRp75, but not SRp55, enhanced chondrogenic differentiation of chick limb mesenchymal cells in a high-density micromass assay. Overexpression of SRp40LF, which produces a truncated form of SRp40, also was strongly pro-chondrogenic. In a HeLa cell-based assay, SRp40LF fails to substitute for SRp40 in mediating an increase in exon EIIIA inclusion, suggesting that the latter event is not essential for the pro-chondrogenic effect. These results demonstrate the ability of these highly conserved splicing factors to modulate chondrogenesis and are consistent with earlier results that implicated exon EIIIA-containing isoforms of fibronectin in formation of chondrogenic condensations.

Alternative Splicing↗
PubMed2001

Herpes simplex virus type 1 2-kilobase latency-associated transcript intron associates with ribosomal proteins and splicing factors.

During latency of herpes simplex virus type 1 in sensory neurons, the transcription of viral genes is restricted to the latency-associated transcripts (LATs). The stable 2-kb LAT intron has been characterized previously and has been shown to accumulate to high levels in the nuclei of infected neurons. However, in productively infected tissue culture cells, this unique intron is also found in the cytoplasm. Although deletion mutant analysis has suggested that the region of the gene from which the intron is spliced plays a role in maintenance of latency or in reactivation from latency, no well-defined function has been ascribed specifically to the 2-kb LAT intron. Nevertheless, previous work has shown that it associates with 50S particles in the cytoplasm of acutely infected cells. Our studies tested the ability of the 2-kb LAT to dissociate from cytoplasmic protein complexes under various salt conditions. Results indicated that this association, which had been speculated to be mRNA-like, is actually more similar to the affinity of rRNAs for translational complexes. Furthermore, by immunoprecipitation analysis, we demonstrate that the 2-kb LAT associates with ribosomal as well as with splicing complexes in infected cells. Our results suggest that the 2-kb LAT is processed similarly to mRNAs in the nuclei of infected cells. However, in the cytoplasm, the 2-kb LAT may play a structural role in the ribosomal complex, similar to that of the cellular rRNAs, and therefore affect the functioning of the translational machinery.

Biological Transport↗
PubMed1996

Isolation, differential splicing and protein expression of a DNase on the human X chromosome.

A systematic search for genes differentially expressed in human tissues resulted in the isolation of a gene encoding a protein with high homology to DNase I. In addition to the recently described cDNA sequence (Parrish et al., 1995) we have isolated a transcript, alternatively spliced in the 5' noncoding region. The gene is located between the QM and the XAP-2 gene in Xq28 and encodes a 302 amino acid protein with 39% identity to human DNase I. Besides a high homology at the nucleotide and amino acid level, most exon-intron boundaries of DNase I and DNase X are identical, indicating that both genes may have evolved from a common ancestor. The predicted function was verified by expression of a recombinant protein in an inducible bacterial system and detection of DNase activity. In contrast to DNase I a 18 kdal amino terminal fragment of the full length 35 kdal protein exhibited DNase activity.

Journal Article↗
PubMed1996

Enhanced early developmental expression of the metabotropic glutamate receptor mGluR5 in rat brain: protein, mRNA splice variants, and regional distribution.

Glutamate stimulates phosphatidyl inositol hydrolysis and mobilizes intracellular calcium through the mediation of metabotropic glutamate receptors (mGluRs), in particular the "Group I" receptors mGluRs, mGluR1, and mGluR5. This activity is markedly enhanced in developing brain relative to the adult. To determine whether this may be due to an increased amount of mGluR5 present in the developing brain, we examined mGluR5 expression using western blotting to measure mGluR5 protein reverse transcription polymerase chain reaction (RT-PCR) to measure mGluR5 mRNA, and immunocytochemistry to assess the regional distribution of mGluR5 morphologically. Western blotting revealed that in all brain regions examined there is more mGluR5 protein present in developing brain than in the adult. In most regions, the developmental decrease was over two-fold. Total mGluR5 mRNA also decreased with development in most regions, but to a much lesser extent than the protein, suggesting that there is considerable post-transcriptional regulation of the expression of this receptor. RT-PCR analysis also demonstrated that in most regions the mGluR5a splice variant is most abundant in the young animals but mGluR5b predominates in the adult. Light microscopic immunocytochemistry indicated that expression is widespread in developing brain, and that the developmental decrease in receptor concentration is due to both an increased growth of receptor-poor tissue regions and decreased expression within receptor-rich regions.

Animals↗
PubMed2003

A genetic approach to identifying mitochondrial proteins.

The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.

Amino Acid Sequence↗
PubMed2003

Split dnaE genes encoding multiple novel inteins in Trichodesmium erythraeum.

Three inteins were found when analyzing a pair of split dnaE genes encoding the catalytic subunit of DNA polymerase III in the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. The three inteins (DnaE-1, DnaE-2, and DnaE-3) were clustered in a 70-amino acid (aa) region of the predicted DnaE protein. The DnaE-1 intein is 1258 aa long and three times as large as a typical intein, due to the presence of large tandem repeats in which a 57-aa sequence is repeated 17 times. The DnaE-2 intein has a more typical size of 428 aa with putative protein splicing and endonuclease domains. The DnaE-3 intein is a split intein consisting of a 102-aa N-terminal part and a 36-aa C-terminal part encoded on the first and second split dnaE genes, respectively. Synthesis of a mature DnaE protein is predicted to involve expression of two split dnaE genes followed by two protein cis-splicing reactions and one protein trans-splicing reaction. Tandem repeats in the DnaE-1 intein inhibited the protein splicing activity of this intein when tested in Escherichia coli cells and may potentially regulate DnaE synthesis in vivo.

Amino Acid Sequence↗
PubMed2026

Prenatal BPA exposure perturbs RNA-binding protein-mediated splicing regulation and synaptogenesis in the developing cerebellum in a sex-dependent manner.

BACKGROUND: Autism spectrum disorder (ASD) is a pervasive neurodevelopmental condition characterized by social communication deficits, exhibiting a male bias in prevalence. Emerging evidence suggests that prenatal exposure to bisphenol A (BPA) may perturb neurodevelopmental trajectories relevant to ASD. While the cerebellum is increasingly recognized as a brain region implicated in ASD pathophysiology, the impact of gestational BPA exposure on its post-transcriptional alternative splicing machinery remains fundamentally undefined. METHODS: Here, we investigated sex-dependent effects of prenatal BPA exposure on the alternative splicing landscape of the neonatal rat cerebellum. We utilized RNA-seq to profile differential alternative splicing (DAS) events. Ingenuity Pathway Analysis (IPA) was used to predict biological functions and canonical pathways, and to construct the interactome network of DAS genes. To explore candidate upstream regulatory mechanisms, we performed in silico molecular docking and used high-resolution melting (HRM) qRT-PCR to validate selected splicing events. Furthermore, we assessed in vitro cellular phenotypes in primary cerebellar neurons by measuring MTS-based viability and Syn1/Psd95 puncta colocalization. RESULTS: Prenatal BPA exposure was associated with widespread DAS in genes enriched for ASD-relevant pathways in the neonatal rat cerebellum. To our knowledge, this study is the first to report molecular docking analyses predicting favorable interactions between BPA and several candidate RNA-binding proteins (RBPs), including CPEB1, RALYL, HNRNPDL, and ACO1. Our findings support a model in which BPA may perturb RBP-associated splicing regulation, including altered splicing of chromatin regulators such as Ccar1 in males. These molecular and cellular findings were accompanied by sex-stratified differences in neuronal viability and synaptic puncta measurements. BPA exposure was associated with an increased MTS viability signal in male primary cerebellar neurons, together with significant reductions in Psd95 and Syn1 puncta density, whereas female neurons showed significantly increased synaptic puncta colocalization together with reduced viability. CONCLUSIONS: In this study, we propose that prenatal BPA may be relevant to ASD-related neurodevelopmental pathways through sex-dependent changes in RBP-associated alternative splicing, including altered splicing of Ccar1 in males, together with distinct cellular outcomes. Together, these findings identify the developing cerebellum as a sensitive target of prenatal BPA exposure and highlight alternative splicing as a candidate pathway relevant to ASD biology.

Animals↗
PubMed1995

Regulation of alternative splicing of protein kinase C beta by insulin.

Insulin regulates a diverse array of cellular signaling processes involved in the control of growth, differentiation, and cellular metabolism. Insulin increases glucose transport via a protein kinase C (PKC)-dependent pathway in BC3H-1 myocytes, but the function of specific PKC isozymes in insulin action has not been elucidated. Two isoforms of PKC beta result via alternative splicing of precursor mRNA. As now shown, both isoforms are present in BC3H-1 myocytes, and insulin induces alternative splicing of the PKC beta mRNA thereby switching expression from PKC beta I to PKC beta II mRNA. This effect occurs rapidly (15 min after insulin treatment) and is dose-dependent. The switch in mRNA is reflected by increases in the protein levels of PKC beta II. High levels of 12-0-tetradecanoylphorbol-13-acetate, which are commonly used to deplete or down-regulate PKC in cells, also induce the switch to PKC beta II mRNA following overnight treatment, and protein levels of PKC beta II reflected mRNA increases. To investigate the functional importance of the shift in PKC beta isoform expression, stable transfectants of NIH-3T3 fibroblasts overexpressing PKC beta I and PKC beta II were established. The overexpression of PKC beta II but not PKC beta I in NIH-3T3 cells significantly enhanced insulin effects on glucose transport. This suggests that PKC beta II may be more selective than PKC beta I for enhancing the glucose transport effects of insulin in at least certain cells and, furthermore, that insulin can regulate the expression of PKC beta II by alternative mRNA splicing.

3T3 Cells↗