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PubMed1989

Mutation at the Chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II.

The nuclear NAC2 locus of C. reinhardtii encodes a trans-acting factor that confers stability on the chloroplast message for the D2 protein of photosystem II (PS II). A mutant that carries a lesion in the NAC2 gene is unable to accumulate the psbD transcript encoding D2, but synthesizes all other PS II proteins normally. However, the PS II complex is completely unstable in the nac2-26 mutant, and all major PS II polypeptides, including the three oxygen-evolving enhancing proteins, are absent or greatly reduced in this strain because of posttranslational degradation. In C. reinhardtii the second exon of the trans-spliced psaA mRNA is cotranscribed with psbD (Choquet et al., 1988), but the nac2-26 mutation normally has no effect on psaA message maturation or stability. However, in double mutants carrying the nac2-26 mutation, as well as a mutation that prevents psaA splicing, splicing intermediates carrying psbD sequences are degraded. These results suggest that the NAC2 gene product acts in a very specific manner to control the half-life of psbD transcripts.

Chlamydomonas↗
PubMed2006

Testican-1 is dispensable for mouse development.

Testicans are proteoglycans belonging to the BM-40/SPARC/osteonectin family of extracellular calcium-binding proteins. Testican-1 is strongly expressed in the brain and has been reported to modulate neuronal attachment and matrix metalloproteinase activation. Characterization of the mouse testican-1 gene (Ticn1), consisting of 12 exons out of which exon 3 is alternatively spliced, allowed the construction of a gene targeting construct. Mice deficient in testican-1 showed no obvious morphological or behavioral abnormalities, were fertile, and had normal life spans. Despite the fact that neither of the testican-1 homologues expressed in the brain, testican-2, testican-3 and SC1/hevin, showed an increased expression in Ticn1 null mice, these results, together with those from other gene targetings, indicate extensive functional redundancy among brain proteoglycans.

Animals↗
PubMed1999

Regulated transcription of the histone H2B genes of Trypanosoma brucei.

In Trypanosoma brucei, the genes encoding histone H2B are organized in a cluster of about 10-15 tandemly linked copies per haploid genome. The H2B transcripts are processed by trans-splicing and polyadenylation, and encode a polypeptide of 111 residues with a molecular mass of 12.5 kDa. H2B mRNAs are differentially expressed during the parasite life-cycle and are present at higher levels in dividing procyclic and bloodstream slender forms than in the nondividing bloodstream stumpy forms. Analysis of H2B mRNA levels during the synchronous differentiation from stumpy to procyclics forms revealed that the abundance of these transcripts is regulated through the cell-cycle, reaching maximum levels during S-phase. Addition of hydroxyurea to procyclic forms in culture specifically decreased H2B mRNA levels by about twofold, an effect not linked to its 3' untranslated region. Inhibition of protein synthesis prevented this decrease.

Amino Acid Sequence↗
PubMed1997

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea.

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5' upstream region of the grisea ORF. One mutation is a G --> A transition at the 5' splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Ascomycota↗
PubMed2004

Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and identify restrictive dermopathy as a lethal neonatal laminopathy.

Restrictive dermopathy (RD), also called tight skin contracture syndrome (OMIM 275210), is a rare disorder mainly characterized by intrauterine growth retardation, tight and rigid skin with erosions, prominent superficial vasculature and epidermal hyperkeratosis, facial features (small mouth, small pinched nose and micrognathia), sparse/absent eyelashes and eyebrows, mineralization defects of the skull, thin dysplastic clavicles, pulmonary hypoplasia, multiple joint contractures and an early neonatal lethal course. Liveborn children usually die within the first week of life. The overall prevalence of consanguineous cases suggested an autosomal recessive inheritance. We explored nine fetuses/newborns children with RD. Two were found to have an heterozygous splicing mutation in the LMNA gene, leading to the complete or partial loss of exon 11 in mRNAs encoding Lamin A and resulting in a truncated Prelamin A protein. Lamins are major constituents of the nuclear lamina, a filamentous meshwork underlying the inner nuclear envelope. In the other seven patients, a unique heterozygous insertion leading to the creation of a premature termination codon was identified in the gene ZMPSTE24, also known as FACE-1 in human. This gene encodes a metalloproteinase specifically involved in the post-translational processing of Lamin A precursor. In all patients carrying a ZMPSTE24 mutation, loss of expression of Lamin A as well as abnormal patterns of nuclear sizes and shapes and mislocalization of Lamin-associated proteins was evidenced. Our results indicate that a common pathogenetic pathway, involving defects of the nuclear lamina and matrix, is involved in all RD cases. RD is thus one of the most deleterious laminopathies identified so far in humans caused by (primary or secondary) A-type Lamin defects and nuclear structural and functional alterations.

Bone and Bones↗
PubMed2000

Expression and splicing of FGF receptor mRNAs during APRE-19 cell differentiation in vitro.

PURPOSE: The expression and alternative splicing of the four FGF receptor (FGFR) mRNAs are regulated in a developmental- and tissue-specific fashion. Capability of differentiation in vitro of the retinal pigment epithelial cell line ARPE-19 has been previously demonstrated. In this study, the hypothesis that FGF receptor gene expression and the alternative splicing of the FGFR1 mRNA is regulated as a function of ARPE-19 differentiation in vitro was tested. METHODS: ARPE-19 cells were plated at sparse or confluent densities and maintained in culture up to 14 months. The expression of FGF receptors and the ratio of the FGFR1beta to FGFR1alpha splice variants of the FGFR1 transcript were quantified by a published PCR technique. Two in vivo samples of human RPE served as controls. RESULTS: Sparse cultures of ARPE-19 cells predominantly express FGFR1. When these cultures are allowed to differentiate, FGFR2 is also expressed. Samples of mRNA from RPE cells in vivo exhibit FGFR1 and FGFR2 expression as well as FGFR3 expression, a form that is minimally apparent in vitro. The ratio of the FGFR1beta to FGFR1alpha splice variant decreases as a function of cell differentiation in vitro and approaches the ratio observed in human RPE cells in vivo. Stimulation of cultures in vitro with FGF2 as a prototypical differentiation agent does not regulate the ratio of the FGFR1beta to FGFR1alpha splice variant. CONCLUSIONS: Differentiation of the ARPE-19 cell line in vitro recapitulates many but not all the in vivo patterns of FGFR expression and splicing. This in vitro system may be useful for selected studies on how cellular differentiation regulates FGF receptor gene expression and splicing.

Alternative Splicing↗
PubMed1993

Dominant retinitis pigmentosa associated with two rhodopsin gene mutations. Leu-40-Arg and an insertion disrupting the 5'-splice junction of exon 5.

OBJECTIVE: To determine the phenotypes of two families in which retinitis pigmentosa cosegregates with a rhodopsin (RHO) gene mutation: a leucine-to-arginine change at codon 40 (Leu-40-Arg) in one family, and a 150-base pair insertion that disrupts the RHO 5'-splice junction of exon 5 in another. PATIENTS: Three affected members of each family. RESULTS: The Leu-40-Arg mutation was associated with the onset of night blindness in the first decade of life. By the fourth decade, severe retinal functional loss was evident on dark-adapted static threshold perimetry, and electroretinographic responses were absent or barely detectable. In contrast, the RHO 150-base pair insertion was associated with the later onset of mild night vision difficulties; in two individuals, mild night vision difficulties were first noticed in the second decade while a third, a 25-year-old woman, was asymptomatic. Dark-adapted static threshold perimetry of this latter individual revealed a "regional" or class 2 pattern of retinal functional loss associated with equal loss of rod and cone electroretinographic responses. CONCLUSION: The RHO Leu-40-Arg mutation causes symptomatic retinal dysfunction by the end of the first decade while the insertion disrupting the 5'-splice junction of RHO exon 5 causes later onset "regional" or class 2 retinal dysfunction.

Adult↗
PubMed2002

Identification of an alternatively spliced RNA for the Ras suppressor RSU-1 in human gliomas.

Previous studies demonstrated that the Ras suppressor, RSU-1, localizes to human chromosome 10p13, a region frequently deleted in high grade gliomas, and that RSU-1 expression inhibited the tumorigenesis of a glioblastoma cell line. We have now examined RNA from human glial tumors for RSU-1 expression by RT-PCR using primers for the 5' and 3' ends of the RSU-1 open reading frame. Analysis of the amplified RSU-1 cDNA demonstrated that in addition to the entire 858 bp RSU-1 open reading frame, a shorter 725 bp RSU-1 fragment was amplified as well. Sequencing of this product revealed that it encoded a RSU-1 cDNA product which was missing a single 133 bp internal exon. This exon-deleted product was found in 30% of the high grade gliomas studied and 2/3 oligodendrogliomas, but not in other CNS tumors, bladder or colon tumors or normal tissue. The exon-deleted RSU-1 product was infrequently detected in RNA from human tumor cell lines. Expression of an HA-tagged form of the deleted RSU-1 protein in transfected Cos 1 cells revealed that the protein was unstable, with a half life of less than 1 h, in contrast to the full length HA-tagged Rsu-1 protein which was stable for more than 4 h. These results suggest that the alternative splicing of the RSU-1 RNA to produce the exon-deleted form constitutes a mechanism for reduction or loss of functional Rsu-1. Because the expression of Rsu-1 can inhibit malignant growth of glioblastoma cells, the depletion of Rsu-1, via the production of the alternatively spliced form of RSU-1, may inhibit growth regulation in tumors.

Alternative Splicing↗
PubMed1990

Nobel lecture. Self-splicing and enzymatic activity of an intervening sequence RNA from Tetrahymena.

A living cell requires thousands of different chemical reactions to utilize energy, move, grow, respond to external stimuli and reproduce itself. While these reactions take place spontaneously, they rarely proceed at a rate fast enough for life. Enzymes, biological catalysts found in all cells, greatly accelerate the rates of these chemical reactions and impart on them extraordinary specificity. In 1926, James B. Summer crystallized the enzyme urease and found that it was a protein. Skeptics argued that the enzymatic activity might reside in a trace component of the preparation rather than in the protein (Haldane, 1930), and it took another decade for the generality of Summer's finding to be established. As more and more examples of protein enzymes were found, it began to appear that biological catalysis would be exclusively the realm of proteins. In 1981 and 1982, my research group and I found a case in which RNA, a form of genetic material, was able to cleave and rejoin its own nucleotide linkages. This self-splicing RNA provided the first example of a catalytic active site formed of ribonucleic acid. This lecture gives a personal view of the events that led to our realization of RNA self-splicing and the catalytic potential of RNA. It provides yet another illustration of the circuitous path by which scientific inquiry often proceeds. The decision to expand so many words describing the early experiments means that much of our current knowledge about the system will not be mentioned. For a more comprehensive view of the mechanism and structure of the Tetrahymena self-splicing RNA and RNA catalysis in general, the reader is directed to a number of recent reviews (Cech & Bass, 1986: Cech, 1987, 1988a, 1990; Burke, 1988; Altman, 1989). Possible medical and pharmaceutical implications of RNA catalysis have also been described recently (Cech, 1988b).

Animals↗
PubMed2002

Biochemical and cellular characteristics of the four splice variants of protein kinase CK1alpha from zebrafish (Danio rerio).

Protein kinase CK1 (previously known as casein kinase I) conforms to a subgroup of the great protein kinase family found in eukaryotic organisms. The CK1 subgroup of vertebrates contains seven members known as alpha, beta, gamma1, gamma2, gamma3, delta, and epsilon. The CK1alpha gene can generate four variants (CK1alpha, CK1alphaS, CK1alphaL, and CK1alphaLS) through alternate splicing, characterized by the presence or absence of two additional coding sequences. Exon "L" encodes a 28-amino acid stretch that is inserted after lysine 152, in the center of the catalytic domain. The "S" insert encodes 12 amino acid residues and is located close to the carboxyl terminus of the protein. This work reports some biochemical and cellular properties of the four CK1alpha variants found to be expressed in zebrafish (Danio rerio). The results obtained indicate that the presence of the "L" insert affects several biochemical properties of CK1alpha: (a) it increases the apparent Km for ATP twofold, from approximately 30 to approximately 60 microM; (b) it decreases the sensitivity to the CKI-7 inhibitor, raising the I50 values from 113 to approximately 230 microM; (c) it greatly decreases the heat stability of the enzyme at 40 degrees C. In addition, the insertion of the "L" fragment exerts very important effects on some cellular properties of the enzyme. CK1alphaL concentrates in the cell nucleus, excluding nucleoli, while the CK1alpha variant is predominantly cytoplasmic, although some presence is observed in the nucleus. This finding supports the thesis that the basic-rich region found in the "L" insert acts as a nuclear localization signal. The "L" insert-containing variant was also found to be more rapidly degraded (half-life of 100 min) than the CK1alpha variant (half-life of 400 min) in transfected Cos-7 cells.

Alternative Splicing↗
PubMed2005

Characterization of a multisubunit transcription factor complex essential for spliced-leader RNA gene transcription in Trypanosoma brucei.

In the unicellular human parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., the spliced-leader (SL) RNA is a key molecule in gene expression donating its 5'-terminal region in SL addition trans splicing of nuclear pre-mRNA. While there is no evidence that this process exists in mammals, it is obligatory in mRNA maturation of trypanosomatid parasites. Hence, throughout their life cycle, these organisms crucially depend on high levels of SL RNA synthesis. As putative SL RNA gene transcription factors, a partially characterized small nuclear RNA-activating protein complex (SNAP(c)) and the TATA-binding protein related factor 4 (TRF4) have been identified thus far. Here, by tagging TRF4 with a novel epitope combination termed PTP, we tandem affinity purified from crude T. brucei extracts a stable and transcriptionally active complex of six proteins. Besides TRF4 these were identified as extremely divergent subunits of SNAP(c) and of transcription factor IIA (TFIIA). The latter finding was unexpected since genome databases of trypanosomatid parasites appeared to lack general class II transcription factors. As we demonstrate, the TRF4/SNAP(c)/TFIIA complex binds specifically to the SL RNA gene promoter upstream sequence element and is absolutely essential for SL RNA gene transcription in vitro.

5' Untranslated Regions↗
PubMedDate not supplied

Messenger RNA decay of macrophage colony-stimulating factor in human ovarian carcinomas in vitro.

OBJECTIVE: Recently, the importance of the macrophage colony-stimulating factor (CSF-1) and its receptor (encoded by the c-fms proto-oncogene) in patients with epithelial ovarian cancer has been recognized. Overexpression of CSF-1 denotes poor prognosis. Macrophage colony-stimulating factor may be one of a group of inflammatory cytokines, whose 3' untranslated region (UTR) contains AU-rich stretches and whose expression may be largely dependent on mRNA decay. The purposes of this study were to investigate the effect of protein synthesis inhibition on CSF-1 transcript expression, to help determine whether there is a role for labile intermediary proteins in the regulation of CSF-1 expression, and to explore the transcriptional or post-transcriptional mechanisms that could underlie such overexpression of CSF-1 transcripts by protein synthesis inhibitors. Although regulation of CSF-1 gene expression has been investigated in hematopoietic cells, such studies have not been carried out in any epithelial cancer. METHODS: Northern blot analyses for CSF-1 expression were performed on total cellular RNA extracted from primary and established ovarian cancer cell lines in the absence or presence of proteins synthesis inhibitors with different modes of action. The probe for the AU-rich exon 10 of CSF-1 was prepared by amplification of the terminal 143 bp of the 3' UTR of human CSF-1 by polymerase chain reaction. Transcription rates were assessed in the presence or absence of cycloheximide (CH) in nuclei of ovarian cancer cells by run-off analyses. The effect of CH on CSF-1 mRNA half-life was measured by actinomycin D chase experiments in SKOV3 cells. RESULTS: We demonstrate that CSF-1 mRNA was expressed by all of a panel of primary and established ovarian cancer cell lines. There are at least three CSF-1 transcripts expressed by ovarian cancer cells; we demonstrate that the 4.2-kb CSF-1 transcript contains exon-6 sequences, which are spliced from the 3.4- and 1.9-kb transcripts, whereas both the 4.2- and 3.4-kb CSF-1 transcripts contain the 3' AU-rich exon 10. Treatment with several different protein synthesis inhibitors resulted in marked overexpression of CSF-1 transcript levels, suggesting a potential role for labile proteins in the regulation of CSF-1 expression. The predominant effect of CH is on the two CSF-1 transcripts that contain exon 10. Although CH does not change the rate of CSF-1 gene transcription, measurement of CSF-1 mRNA stability reveals a prolongation of CSF-1 transcript half-life by CH from 4.5 hours to significantly greater than 6 hours in ovarian cancer cells. CONCLUSIONS: Augmented CSF-1 transcript stability underlies the marked overexpression of CSF-1 seen with protein synthesis inhibitors. Our results suggest the involvement of a labile regulatory protein that contributes to CSF-1 mRNA decay in ovarian cancer cells. Our data suggest further that exon 10 (not the spliced exon-6 sequences) of the CSF-1 transcript may contain an instability determinant.

Cycloheximide↗
PubMed1997

Inhibition of Hepatitis Delta Virus Genomic Ribozyme Self-Cleavage by Aminoglycosides.

Subgenomic regions of hepatitis delta virus (HDV) RNA contains ribozyme whose activities are important to viral life cycles and depend on a unique pseudoknot structure. To explore the characters of HDV ribozyme, antibiotics of the aminoglycoside, which has been shown inhibiting self-splicing of group I intron and useful in elucidating its structure, were tested for their effect on HDV genomic ribozyme. Aminoglycosides, including tobramycin, netromycin, neomycin and gentamicin effectively inhibited HDV genomic ribozyme self-cleavage in vitro at a concentration comparable to that inhibiting group I intron self-splicing. The extent of inhibition depended upon the concentration of magnesium ion. Chemical modification mapping of HDV ribozyme RNA indicated that the susceptibility of nucleotide 703 to the modifying agent was enhanced in the presence of tobramycin, suggesting a conformational shift of HDV ribozyme, probably due to an interaction with the aminoglycoside. Finally, we examined the effect of aminoglycoside on HDV cleavage and replication in cell lines, however, none of the aminoglycoside effective in vitro exerted suppressive effects in vivo. Our results represented as an initial effort in utilizing aminoglycoside to probe the structure of HDV ribozyme and to compare its reaction mechanism with those of other related ribozymes.

Journal Article↗
PubMed1995

Ontogeny of membrane cofactor protein: phenotypic divergence in the fetal heart.

Human adult cells are protected from complement-induced damage in part by membrane cofactor protein (MCP, CD46). To examine fetal characteristics which might influence autoantibody-mediated diseases acquired in utero, such as heart block in neonatal lupus, the tissue expression of MCP was studied. Using a high ratio of acrylamide:bisacrylamide, immunoblots of tissues from six fetuses (aged 19-24 weeks) probed with rabbit anti-MCP antibodies revealed a band at 60 KD in addition to the known 65 KD and 55 KD isoforms which comprise the codominant allelic system of MCP. Five fetuses expressed the most common MCP polymorphism (predominance of the 65 KD isoform, upper band alpha-phenotype) in the kidney, spleen, liver and lung. In contrast, all hearts from these five fetuses demonstrated a different pattern in which there was a marked decrease in the intensity of the 65 KD band and accentuation of the lower molecular weight bands. In a sixth fetus, which expressed the second most common polymorphism (equal expression of the 65 KD and 55 KD MCP isoforms, alpha beta-phenotype), the heart was similar to the other tissues. These studies confirm the expression of MCP in early gestational life. Preferential expression of the MCP beta-isoform in the majority of fetal hearts irrespective of the phenotype of other organs, suggests tissue-specific RNA splicing or post-translational modification which may relate to autoantibody-mediated injury in diseases such as neonatal lupus.

Antigens, CD↗
PubMed2003

Neural dysfunction and neurodegeneration in Drosophila Na+/K+ ATPase alpha subunit mutants.

The Na+/K+ ATPase asymmetrically distributes sodium and potassium ions across the plasma membrane to generate and maintain the membrane potential in many cell types. Although these pumps have been hypothesized to be involved in various human neurological disorders, including seizures and neurodegeneration, direct genetic evidence has been lacking. Here, we describe novel mutations in the Drosophila gene encoding the alpha (catalytic) subunit of the Na+/K+ ATPase that lead to behavioral abnormalities, reduced life span, and severe neuronal hyperexcitability. These phenotypes parallel the occurrence of extensive, age-dependent neurodegeneration. We have also discovered that the ATPalpha transcripts undergo alternative splicing that substantially increases the diversity of potential proteins. Our data show that maintenance of neuronal viability is dependent on normal sodium pump activity and establish Drosophila as a useful model for investigating the role of the pump in human neurodegenerative and seizure disorders.

Action Potentials↗
PubMed1992

Expression and regulation of the evi-1 gene in the human factor-dependent leukemia cell line, UCSD/AML1.

The human factor-dependent leukemia cell line UCSD/AML1 contains the t(3;3) (q21;q26) characteristic of the syndrome of acute leukemia with high platelets. The human homologue of the murine leukemia oncogene evi-1 was recently localized to chromosome 3q24-3q28 and transcription of evi-1 is a frequent event in mouse-retrovirus-induced leukemias (17). To determine whether translocations near human 3q24 might induce similar genetic changes, we examined and compared evi-1 and c-myc expression and regulation in UCSD/AML1 cells. Steady-state evi-1 transcripts were detected in UCSD/AML1 and murine leukemia M1 cells, but were not present in HL60 or Namalwa human leukemia cells. Transcription assays showed the evi-1 gene was actively transcribed in UCSD/AML1, but not HL60 nuclei. Evi-1 transcript sizes and half-life were similar in UCSD/AML1 and human HEC-1B carcinoma cells which express evi-1 transcripts, but do not have abnormalities involving chromosome 3. An alternative splice site detected by polymerase chain reaction was present in transcripts from both cell lines. Regulation of evi-1 RNA in UCSD/AML1 cells was similar to that of actin transcripts in response to cycloheximide or phorbol-ester-induced macrophage differentiation. After withdrawal of granulocyte/macrophage colony-stimulating factor (GM-CSF), evi-1, actin, and histone H3 transcripts declined in concert with exit from the cell cycle. Minor differences in rates of recovery were noted for these three genes after GM-CSF restimulation. In contrast, c-myc was expressed at high levels in UCSD/AML1 cells and showed evidence for specific regulation in response to cycloheximide, phorbol ester, and GM-CSF withdrawal and restimulation. These studies suggest the 3q translocation in UCSD/AML1 cells is associated with evi-1 transcription and expression of a potential transforming gene. In contrast to c-myc, evi-1 expression is minimally altered by biologically active chemicals or growth factor stimulation.

Animals↗