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PubMed2002

Genomic organization and differential expression of Kalirin isoforms.

Multidomain guanine nucleotide (GDP/GTP) exchange factor (GEF) proteins coordinate diverse inputs that signal the actin cytoskeleton. Mammals have two such proteins (Kalirin, Trio), while Drosophila has one, which plays essential roles within and outside the nervous system. For Kalirin, numerous isoforms containing different combinations of functional domains are generated through alternative splicing and use of alternative transcriptional start sites. These different isoforms potentially allow a wide variety of proteins to interact with Kalirin, thereby affecting the activity of the functional domains. Humans, like rats, express a large set of Kalirin isoform mRNAs, and we identified a novel Kalirin isoform, containing only the second GEF domain. Kalirin isoforms are predominantly expressed in the brain, while Trio is expressed in a wider variety of tissues. Alternative splicing and transcription of Kalirin are differentially regulated during development in rats and humans, resulting in expression of isoforms of Kalirin containing different functional domains at different times and locations. The prevalence of Kalirin in the cortex throughout life suggests roles in axonal development and the mature brain.

Alternative Splicing↗
PubMed1994

Subcellular distribution of human immunodeficiency virus type 1 Rev and colocalization of Rev with RNA splicing factors in a speckled pattern in the nucleoplasm.

The human immunodeficiency virus type 1 (HIV-1) Rev (regulator of virion protein expression) protein exemplifies a new type of posttranscriptional regulation. One main function of Rev is to increase the cytoplasmic expression of unspliced and incompletely spliced retroviral mRNAs from which viral structural proteins are made. In that way, Rev is essential in order to complete the retroviral life cycle. The biology of Rev in the host cell has remained elusive. In this study, a complex distribution of Rev in single cells was found. Rev was found in the cytoplasm, in a perinuclear zone, in the nucleoplasm, and in the nucleoli. In the nucleoplasm, Rev colocalized in a speckled pattern with host cell factors known to assemble on nascent transcripts. Those factors are involved in the processing of heterogeneous RNA to spliced mRNA in the nucleoplasm of all cells. The distribution of Rev was dependent only on Rev and host cell interactions, since neither the Rev target RNA nor other HIV proteins were expressed in the cells. Rev was found in the same subcellular compartments of cells treated for extended periods with cycloheximide, an inhibitor of protein synthesis. This finding implies that Rev shuttles continuously between cytoplasmic and nucleoplasmic compartments. The results suggest a potential role for Rev both in the RNA-splicing process and in the nucleocytoplasmic transport of Rev-dependent HIV mRNA.

Amino Acid Sequence↗
PubMed2001

From gene to protein in higher plant mitochondria.

Higher plant mitochondria contain a genetic system with a genome, transcription and translation processes, which have to be logistically integrated with the two other genomes in the nucleus and the plastid. In plant mitochondria, after transcripts have been synthesised, at least in some cases by a phage-type RNA polymerase, they have to go through a complex processing apparatus, which depends on protein factors imported from the cytosol. Processing involves cis- and trans-splicing, internal RNA editing and maturation at the transcript termini, these steps often occurring in parallel. Transcript life is terminated by RNA degradation mechanisms, one of which involves polyadenylation. RNA metabolism seems to be a key element of the regulation of gene expression in higher plant mitochondria.

Genome, Plant↗
PubMed2005

Coevolution of tRNA intron motifs and tRNA endonuclease architecture in Archaea.

Members of the three kingdoms of life contain tRNA genes with introns. The introns in pre-tRNAs of Bacteria are self-splicing, whereas introns in archaeal and eukaryal pre-tRNAs are removed by splicing endonucleases. We have studied the structures of the endonucleases of Archaea and the architecture of the sites recognized in their pre-tRNA substrates. Three endonuclease structures are known in the Archaea: a homotetramer in some Euryarchaea, a homodimer in other Euryarchaea, and a heterotetramer in the Crenarchaeota. The homotetramer cleaves only the canonical bulge-helix-bulge structure in its substrates. Variants of the substrate structure, termed bulge-helix-loops, appear in the pre-tRNAs of the Crenarcheota and Nanoarcheota. These variant structures can be cleaved only by the homodimer or heterotetramer forms of the endonucleases. Thus, the structures of the endonucleases and their substrates appear to have evolved together.

Archaeal Proteins↗
PubMedDate not supplied

Impediments to the development of additional vaccines: vaccines against important diseases that will not be available in the next decade.

The availability of new biotechnologies has led to the prediction that new or improved vaccines can be developed for 27 diseases within the next decade. The reasons why such optimism cannot be extended to the availability of vaccines for many other infectious diseases are considered by reviewing the steps in vaccine development, from identification of the etiologic agent to construction of attenuated or inactivated vaccines. Impediments to development may exist or arise at any point in this pathway (e.g., multiplicity of serotypes, inability to cultivate the pathogen, multistage life cycles with multiple antigens, unpredictability of epidemics, inadequate knowledge of pathogenesis and immunity, fear of gene splicing, need for an adjuvant, and lack of profitability). Diseases for which vaccines are not likely to be available in the next decade include trachoma, onchocerciasis, pneumonia due to Legionella and to mycoplasmas, amebiasis and giardiasis, schistosomiasis, syphilis, chlamydial urethritis, trypanosomiasis, leishmaniasis, and filariasis, and non-A, non-B hepatitis.

Communicable Disease Control↗
PubMed1990

A novel human immunodeficiency virus type 1 protein, tev, shares sequences with tat, env, and rev proteins.

We have characterized a novel 28-kilodalton protein, p28tev, detected in human immunodeficiency virus type 1-infected cells. tev is recognized by both tat and rev monospecific antibodies. tev is initiated at the tat AUG and contains the first exon of tat at its amino terminus, a small portion of env in the middle, and the second exon of rev at its carboxy terminus. A cDNA clone producing tev was cloned and expressed in human cells. Sequence analysis revealed that the tev mRNA is generated by splicing to a novel exon located in the env region. This identifies a fourth class of multiply spliced human immunodeficiency virus mRNAs, produced in infected and transfected cells. tev is regulated during the virus life cycle similarly to the other regulatory proteins, tat, rev, and nef, and displays both tat and rev activities in functional assays. Since tev contains important functional domains of tat and rev and is produced very early after transfection, it may be an important regulator in the initial phase of virus expression. Another rev-related protein, p18(6)Drev, containing env and rev sequences, was characterized and was found not to have detectable rev activity.

Amino Acid Sequence↗
PubMed2006

Two cases of severe neonatal hypertrophic cardiomyopathy caused by compound heterozygous mutations in the MYBPC3 gene.

BACKGROUND: Idiopathic (primary) hypertrophic cardiomyopathy (HCM) is mainly caused by mutations in genes encoding sarcomeric proteins. One of the most commonly mutated HCM genes is the myosin binding protein C (MYBPC3) gene. Mutations in this gene lead mainly to truncation of the protein which gives rise to a relatively mild phenotype. Pure HCM in neonates is rare and most of the time childhood HCM occurs in association with another underlying condition. OBJECTIVE: To study the presence of mutations in the MYBPC3 gene in idiopathic childhood HCM. METHODS: MYBPC3 coding region and splice junction variation were analysed by denaturing high performance liquid chromatography (DHPLC) and sequencing in DNA isolated from two neonates with severe unexplained HCM, who died within the first weeks of life. RESULTS: Truncating mutations were found in both alleles of the MYBPC3 gene in both patients, suggesting there was no functional copy of the MYBPC3 protein. Patient 1 carried the maternally inherited c.2373_2374insG mutation and the paternally inherited splice-donor site mutation c.1624+1G-->A. Patient 2 carried the maternally inherited frameshift mutation c.3288delA (p.Glu1096fsX92) and the paternally inherited non-sense mutation c.2827C-->T (p.Arg943X). CONCLUSIONS: The findings indicate the need for mutation analysis of genes encoding sarcomeric proteins in childhood HCM and the possibility of compound heterozygosity.

Cardiomyopathy, Hypertrophic↗
PubMed2005

Mild Pelizaeus-Merzbacher disease caused by a point mutation affecting correct splicing of PLP1 mRNA.

We describe a 28-year-old male patient with a mild course of Pelizaeus-Merzbacher disease (PMD) who presented with developmental delay in his second year of life and was able to walk until 12 years of age. Several computed tomography scans in infancy and youth were normal, the diagnosis of PMD was eventually suggested by magnetic resonance imaging at the age of 24 years. Analysis of the proteolipid protein gene (PLP1) revealed a nucleotide exchange (c.762G>T) at the 3' border of exon 6, which did not entail an amino acid exchange but adversely affected splicing. PCR analysis of fibroblast cDNA showed that c.762G>T resulted in partial skipping of exon 6 in the PLP1 mRNA. Exclusion of exon 6 does not alter the reading frame but leads to absence of amino acids 232-253 that constitute a main part of the fourth transmembrane helix of the PLP protein. Remarkably, residual wild-type splicing was also detected in the patient's cultured fibroblasts. This might explain the mild phenotype in this case, as exon 6 skipping mutations resulted in a severe course of disease in other patients.

Adult↗
PubMed2003

A new retroelement constituted by a natural alternatively spliced RNA of murine replication-competent retroviruses.

Replication of simple retroviruses depends on the recruitment of a single large primary transcript toward splicing, transport/packaging and translation regulations. In this respect, we studied the novel SD' 4.4 kb RNA of murine leukemia retroviruses (MLV) which results from alternative splicing of the primary transcript. We showed that SD' RNA was required for optimal replication since expression of a pre-spliced SD' RNA trans-complemented the impaired infectivity of a SD'-defective mutant. We monitored the fate of this novel transcript throughout early and late events of the viral life cycle. SD' RNA was specifically incorporated into virions demonstrating that the unspliced RNA was not the unique viral RNA present in virions. Furthermore, SD' RNA was reverse transcribed and its DNA copy integrated into the host genome, thus constituting a new splice donor-associated retroelement (SDARE) in infected cells. Finally, we showed that SD' mRNA encoded a 50 kDa polyprotein, and to a lower extent an additional 60 kDa polyprotein, which harbored Gag and integrase domains.

Alternative Splicing↗
PubMed1992

Functional LH receptor appears in the neonatal rat ovary after changes in the alternative splicing pattern of the LH receptor mRNA.

The LH receptor (R) function, as assessed by ligand binding and stimulation of cAMP production, is initiated in the neonatal rat ovary around day 7 of life. To correlate this with the onset of expression of the LHR gene, we assessed the presence of the cognate mRNA in the developing rat ovary from day 17 of fetal life onwards using PCR multiplication of reverse transcribed LHR mRNA, followed by southern hybridization and sequencing of the PCR products. Our findings indicate that only truncated versions of LHR mRNA are present in the developing rat ovary as early as day 17 fetal life and up to day 7 post partum. Concomitant with the appearance of a functional LH receptor (after day 7 post partum), two larger LHR mRNA species (2.6 and 6.7 kb) appear, which are also present in the adult ovary. These findings indicate that the LHR gene is probably constitutively expressed in the developing rat ovary, and that the onset of translation of functional LH receptor occurs through a change in the alternative splicing pattern of LHR mRNA.

Animals↗
PubMed1996

Coding potential of the X region of human T-cell leukemia/lymphotropic virus type II.

Human T-cell leukemia/lymphotropic viruses type I and type II (HTLV-I and HTLV-II) are complex human retroviruses showing a similar genetic organization but substantially different biologic and pathogenic properties. As in other complex retroviruses, the 3' portion of the HTLV genome contains the peculiar "X region" comprising several partially overlapping open reading frames (ORFs). To search for a possible basis for the pathogenic differences between the two viruses, a number of studies have been carried out to analyze the coding potential of the X region of HTLV-I and, more recently, of HTLV-II. This review focuses on the coding potential of the HTLV-II X region and presents a comparison with that of HTLV-I. Expression of different ORFs present in the X region may be accessed through two expression strategies: alternative splicing and translation of more than one protein from the same mRNA. Initial analyses of the X region proteins indicate that some differ significantly from their HTLV-I homologues, thus providing possible clues to the understanding of the complex life cycle and pathogenicity of the two viruses.

Alternative Splicing↗
PubMed1999

Antisense oligonucleotides targeting malarial aldolase inhibit the asexual erythrocytic stages of Plasmodium falciparum.

A major obstacle in the global effort to control malaria is the paucity of anti-malarial drugs. This is compounded by the continuing emergence and spread of resistance to old and new anti-malarial drugs in the malarial parasites. Here we describe the anti-malarial effect of phosphorothioate antisense (AS) oligodeoxynucleotides (ODNs) targeting the aldolase enzyme of Plasmodium falciparum, using the asexual blood stages of the parasite grown in vitro. The blood stages of P. falciparum depend almost entirely on the energy produced by their own glycolysis. Aldolase, the fourth enzyme of the glycolytic pathway, is highly upregulated during the malarial 48-h life cycle. We found that the mRNA of this enzyme can be inhibited, in a sequence specific manner, using AS-ODN to the splice sites on the pre-mRNA of malarial aldolase. At the enzyme level, both specific AS-ODNs for the splice sites, as well as for the translation initiation site on mature mRNA, can inhibit aldolase enzyme activity within the trophozoites of P. falciparum. Furthermore, this downregulation of the malarial aldolase results in a reduction in the production of ATP within the parasite. Finally, the treatment reduces parasitemia. In summary, AS-ODNs targeting the aldolase gene of P. falciparum can interfere with the blood-stage life cycle of this parasite in vitro by inhibiting the expression of the enzyme aldolase which results in decreased malarial glycolysis and energy production. Thus, we conclude that blockade of the expression of malarial glycolytic enzymes using specific AS-ODNs has the potential of a new anti-malarial strategy.

Adenosine Triphosphate↗
PubMed1996

A G-1-to-A acceptor splice site LDLR mutant allele leads to reduced relative transcript levels in patients with heterozygous familial hypercholesterolemia.

A plethora of different mutations in the gene for the low density receptor (LDLR) are responsible for the autosomal dominant inherited disorder familial hypercholesterolemia (FH). However, only a few splice site mutations have been identified in this gene. We here report a defect presumably affecting the splicing of precursor mRNA, resulting from a novel mutation, a G to A transition at the terminal nucleotide of intron 12, of the LDLR gene detected in three unrelated families with heterozygous FH. This mutation markedly reduced the steady-state transcript level of the mutant LDLR allele as compared to the corresponding normal LDLR allele in heterozygous FH patients as measured by a fluorescence based, allele-specific quantitation technique. In the FH families, the acceptor splice site mutation cosegregates with hypercholesterolemia, and it is associated with onset of ischemic heart disease in the fifth and sixth decade of life.

Adult↗
PubMed2002

Intron retention and frameshift mutations result in severe pyruvate carboxylase deficiency in two male siblings.

This paper describes the molecular characterization of two male siblings displaying the complex (Type B) form of pyruvate carboxylase (PC) deficiency in which severe neonatal lactic acidosis and redox abnormalities results in death within the first few weeks of life. The two male siblings were found to be compound heterozygous for a TAGG deletion at the exon15/intron15 splice site (IVS15+2-5delTAGG) and a dinucleotide deletion in exon 16 (2491-2492delGT) of the PC gene. We also demonstrate through RT-PCR and sequencing of aberrant transcripts that the IVS15+2-5delTAGG results in the retention of intron 15 during pre-mRNA splicing. In addition, both deletions are predicted to result in a frameshift to generate a premature termination codon such that the encoded mRNA could be subject to nonsense mediated decay.

Alternative Splicing↗
PubMed2006

Altered DNA damage response in Caenorhabditis elegans with impaired poly(ADP-ribose) glycohydrolases genes expression.

Poly(ADP-ribosyl)ation is one of the first cellular responses induced by DNA damage. Poly(ADP-ribose) is rapidly synthesized by nick-sensor poly(ADP-ribose) polymerases, which facilitate DNA repair enzymes to process DNA damage. ADP-ribose polymers are rapidly catabolized into free ADP-ribose units by poly(ADP-ribose) glycohydrolase (PARG). The metabolism of poly(ADP-ribose) is a well-defined biochemical process for which a physiological role in animals is just beginning to emerge. Two Caenorhabditis elegans PARGs, PME-3 and PME-4, have been cloned by our group. The pme-3 gene encodes an enzyme of 89kDa having less than 18% overall identity with human PARG but 42% identity with the PARG signature motif. The pme-4 gene codes for a PARG of 55kDa with approximately 22% overall identity with human PARG and 40% identity with the PARG signature motif. Two alternatively spliced forms of PME-3 were identified with an SL1 splice leader on both forms of the mRNA and were found to be expressed throughout the worm's life cycle. Similarly, pme-4 was shown to be expressed in all developmental stages of the worm. Recombinant enzymes that were expressed in bacteria displayed a PARG activity that may partly account for the PARG activity measured in the total worm extract. Reporter gene analysis of pme-3 and pme-4 using a GFP fusion construct showed that pme-3 and pme-4 are mainly expressed in nerve cells. PME-3 was shown to be nuclear while PME-4 localized to the cytoplasm. Worms with pme-3 and pme-4 expression knocked-down by RNAi showed a significant sensitivity toward ionizing radiations. Taken together, these data provide evidence for a physiological role for PARGs in DNA damage response and survival. It also shows that PARGs are evolutionarily conserved enzymes and that they are part of an ancient cellular response to DNA damage.

Amino Acid Sequence↗
PubMed2006

Cortisol resistance in conditions such as asthma and the involvement of 11beta-HSD-2: a hypothesis.

The two types of corticosteroid hormones, the mineralocorticoids and glucocorticoids, act in a complementary manner and are functionally closely linked. Aldosterone and glucocorticoids express their functions through the glucocorticoid receptors (GRs; GRalpha and GRbeta) and the mineralocorticoid receptors (MRs). Commercially available steroidal drugs used in conditions such as asthma may act on both GR and MR receptors; although glucocorticoid-receptor agonists play a fundamental role in the treatment of inflammatory conditions, prolonged exposure may have adverse effects such as the development of resistance. Glucocorticoid resistance in such conditions has been observed to be accompanied by a downregulation of GRalpha, a twofold decrease in GR protein half-life, downregulation of GRalpha mRNA expression, and enhanced expression of GRbeta. Other suggestions for glucocorticoid resistance include alternative splicing of the GR gene with subsequent expression of the GRbeta protein isoform, defective regulation of gene transcription of the GR gene or GR mutations, defective DNA binding and transactivating domains of the GR. In addition, we would like to suggest that dysregulation of the MR enzyme 11beta-HSD-2 may be one of the causes of resistance. When expressed in cells with MRs, this enzyme's major role is to prevent permanent occupancy of MR by glucocorticoid hormones, allowing concentration-dependent binding of aldosterone to MR. However, deficiency of the 11beta-HSD isoforms (particularly 11beta-HSD-2) leads to the activation of MRs by glucocorticoids rather than the glucocorticoids interacting with its "normal" receptors, the GRs. We will substantiate on support for our hypothesis in the dysregulation of this enzyme, which is typically associated with significantly higher levels of circulating plasma cortisol and elevated levels of cholesterol, little or no response to systemic glucocorticoids, and problems associated with homeostasis primarily in the distal nephron and distal colon. These are some of the symptoms typically noted in cortisol resistance.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2006

Mecp2 deficiency is associated with learning and cognitive deficits and altered gene activity in the hippocampal region of mice.

Rett syndrome (RTT) is a debilitating neurological condition associated with mutations in the X-linked MECP2 gene, where apparently normal development is seen prior to the onset of cognitive and motor deterioration at 6-18 months of life. A targeted deletion of the methyl-CpG-binding domain (MBD) coding region and disruption of mRNA splicing was introduced in the mouse, resulting in a complete loss of Mecp2 transcripts and protein. Postnatal comparison of XO and XY mutant Mecp2 allele-containing null mice revealed similar effects on mouse growth and viability, suggesting that phenotypic manifestations are not modulated by the Y-chromosome. Further assessment of Mecp2-null XY mice highlighted cerebellar and hippocampal/amygdala-based learning deficits in addition to reduced motor dexterity and decreased anxiety levels. Brain tissues containing the hippocampal formation of XY Mecp2-null mice also displayed significant changes in genetic activity, which are related to the severity of the mutant phenotype.

Animals↗