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G Dreyfuss

Publications and source records attributed to G Dreyfuss.

At least 145 records · Page 8Linked to original sources
PubMed1994

The determinants of RNA-binding specificity of the heterogeneous nuclear ribonucleoprotein C proteins.

The hnRNP C proteins (C1/C2) are tenacious nuclear pre-mRNA-binding proteins that belong to the large RNP motif family of RNA-binding proteins. This motif identifies an RNA-binding domain (RBD) that consists of a four-stranded antiparallel beta-sheet packed against two alpha-helices. Despite considerable information on the structure of the hnRNP C RBD, little is known about its RNA-binding properties. To address this we used in vitro selection/amplification from pools of random sequence RNA to determine the RNA-binding specificity of hnRNP C1. After 8 rounds of selection/amplification nearly all RNAs contained contiguous stretches of at least 5 U residues, and filter-binding assays demonstrated that this sequence constitutes a high-affinity (Kd = 170 nM) binding site for hnRNP C1. The highest affinity we measured for hnRNP C1 was for r(U)14 (Kd = 14 nM). An RBD-containing peptide fragment of hnRNP C1 (amino acids 2-94) bound oligoribonucleotides containing an hnRNP C1 high-affinity binding site with nearly equal affinity to that of hnRNP C1. Unlike hnRNP C1, however, this peptide also bound oligoribonucleotides that do not contain high-affinity hnRNP C1-binding sites. We identified a region of 10 amino acids, immediately COOH-terminal to the RNP motif (amino acids 95-104), that prevents the minimal RBD from binding nonspecific RNA ligands. We propose that the highly conserved beta alpha beta beta alpha beta core structure of the RNP motif RBD confers a general RNA binding activity to RNP motif RBDs and that the determinants of RNA-binding specificity reside in the most variable regions, the loops connecting the beta-strands and/or the contiguous NH2 and COOH termini of the RBD.

Amino Acid Sequence↗
PubMed1994

Conserved structures and diversity of functions of RNA-binding proteins.

In eukaryotic cells, a multitude of RNA-binding proteins play key roles in the posttranscriptional regulation of gene expression. Characterization of these proteins has led to the identification of several RNA-binding motifs, and recent experiments have begun to illustrate how several of them bind RNA. The significance of these interactions is reflected in the recent discoveries that several human and other vertebrate genetic disorders are caused by aberrant expression of RNA-binding proteins. The major RNA-binding motifs are described and examples of how they may function are given.

Amino Acid Sequence↗
PubMed1994

Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome.

The KH domain is an evolutionarily conserved sequence motif present in many RNA-binding proteins, including the pre-mRNA-binding (hnRNP) K protein and the fragile X mental retardation gene product (FMR1). We assessed the role of KH domains in RNA binding by mutagenesis of KH domains in hnRNP K and FMR1. Conserved residues of all three hnRNP K KH domains are required for its wild-type RNA binding. Interestingly, while fragile X syndrome is usually caused by lack of FMR1 expression, a previously reported mutation in a highly conserved residue of one of its two KH domains (Ile-304-->Asn) also results in mental retardation. We found that the binding of this mutant protein to RNA is severely impaired. These results demonstrate an essential role for KH domains in RNA binding. Furthermore, they strengthen the connection between fragile X syndrome and loss of the RNA binding activity of FMR1.

Amino Acid Sequence↗
PubMed1994

Essential role for a heterogeneous nuclear ribonucleoprotein (hnRNP) in oogenesis: hrp40 is absent from the germ line in the dorsoventral mutant squid.

The Drosophila melanogaster hrp40 proteins are abundant nuclear pre-mRNA-binding proteins that are similar to the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins of vertebrates. Recently, hrp40 has been shown to be encoded by the squid gene, which is required for dorsoventral axis formation during oogenesis. Eggs and embryos from homozygous squid mothers are severely dorsalized, and complete deletion of the squid gene results in lethality. Here we have examined the expression and localization of hrp40 in wild-type and squid mutant ovaries. Using a monoclonal antibody specific for hrp40, the same isoforms of hrp40 are detected in both wild-type and squid ovaries, but the amount of hrp40 is reduced in squid ovaries. Furthermore, immunolocalization of hrp40 in wild-type egg chambers shows that hrp40 is present in the nurse cells, oocyte, and follicle cells. In contrast, in squid mutant egg chambers, hrp40 is absent from the germ-line-derived nurse cells and oocyte, but it is detected in the somatic follicle cells. The absence of hrp40 from the germ-line-derived cells of developing egg chambers is likely to lead to the striking dorsalized phenotype of squid eggs. In addition, dramatic stage-specific changes in the cellular localization of hrp40 are seen; the protein found in the nurse cell nuclei during early stages of oogenesis migrates to the cytoplasm at later stages. These findings reveal dynamic patterns of expression and localization of hnRNP proteins during development and provide evidence for an essential role for hnRNP proteins.

Amino Acid Sequence↗
PubMed1994

The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization.

More than 20 different heterogeneous nuclear ribonucleoproteins (hnRNPs) are associated with pre-mRNAs in the nucleus of mammalian cells and these proteins appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. The arrangement of hnRNP proteins on pre-mRNAs is likely to be unique for each RNA and may be determined by the different RNA-binding preferences of each of these proteins. hnRNP F (M(r) = 53 kD, pI = 6.1) and hnRNP H (M(r) = 56 kD, pI = 6.7-7.1) are abundant components of immunopurified hnRNP complexes and they have distinct nucleic acid binding properties. Unlike other hnRNP proteins which display a varying range of affinities for different ribonucleotidehomopolymers and ssDNA, hnRNP F and hnRNP H bind only to poly(rG) in vitro. hnRNP F and hnRNP H were purified from HeLa cells by poly(rG) affinity chromatography and oligonucleotides derived from peptide sequences were used to isolate a cDNA encoding hnRNP F. The predicted amino acid sequence of hnRNP F revealed a novel protein with three repeated domains related to the RNP consensus sequence RNA-binding domain. Monoclonal antibodies produced against bacterially expressed hnRNP F were specific for both hnRNP F and hnRNP H and recognized related proteins in divergent organisms, including in the yeast Saccharomyces cerevisiae. hnRNP F and hnRNP H are thus highly related immunologically and they share identical peptides. Interestingly, immunofluorescence microscopy revealed that hnRNP F and hnRNP H are concentrated in discrete regions of the nucleoplasm, in contrast to the general nucleoplasmic distribution of previously characterized hnRNP proteins. The unique RNA-binding properties, amino acid sequence and distinct intranuclear localization of hnRNP F and hnRNP H make them novel hnRNP proteins that are likely to be important for the processing of RNAs containing guanosine-rich sequences.

Amino Acid Sequence↗
PubMed1994

RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing.

Pre-mRNA is processed as a large complex of pre-mRNA, snRNPs and pre-mRNA binding proteins (hnRNP proteins). The significance of hnRNP proteins in mRNA biogenesis is likely to be reflected in their RNA binding properties. We have determined the RNA binding specificity of hnRNP A1 and of each of its two RNA binding domains (RBDs), by selection/amplification from pools of random sequence RNA. Unique RNA molecules were selected by hnRNP A1 and each individual RBD, suggesting that the RNA binding specificity of hnRNP A1 is the result of both RBDs acting as a single RNA binding composite. Interestingly, the consensus high-affinity hnRNP A1 binding site, UAGGGA/U, resembles the consensus sequences of vertebrate 5' and 3' splice sites. The highest affinity 'winner' sequence for hnRNP A1 contained a duplication of this sequence separated by two nucleotides, and was bound by hnRNP A1 with an apparent dissociation constant of 1 x 10(-9) M. hnRNP A1 also bound other RNA sequences, including pre-mRNA splice sites and an intron-derived sequence, but with reduced affinities, demonstrating that hnRNP A1 binds different RNA sequences with a > 100-fold range of affinities. These experiments demonstrate that hnRNP A1 is a sequence-specific RNA binding protein. UV light-induced protein-RNA crosslinking in nuclear extracts demonstrated that an oligoribonucleotide containing the A1 winner sequence can be used as a specific affinity reagent for hnRNP A1 and an unidentified 50 kDa protein. We also show that this oligoribonucleotide, as well as two others containing 5' and 3' pre-mRNA splice sites, are potent inhibitors of in vitro pre-mRNA splicing.

Animals↗
PubMed1994

RNA annealing activities in HeLa nuclei.

RNA-RNA base pairing plays a critical role in the interactions between pre-mRNAs and trans-acting factors during the processing of pre-mRNAs (hnRNAs) into mRNAs, and it is likely that specific factors are required to promote the annealing of RNAs. To identify particular nuclear components that have such activity, we fractionated HeLa nucleoplasm and assayed for activity which promoted the hybridization of a pre-mRNA with an antisense RNA probe complementary to 60 nucleotides (nt) encompassing the 3' splice site. At least nine major RNA annealing activities were identified and, surprisingly, eight of these copurified partially or to homogeneity with known hnRNP proteins. The activities of three of these proteins, hnRNP A1, C1 and U, were confirmed using purified recombinant proteins. Moreover, we found that the RNA binding domain alone of hnRNP C1/C2 had significant activity, indicating that this RNA annealing may result, at least partly, from chaperone activity: a direct modulation of RNA conformation by hnRNP proteins. The finding that hnRNP proteins have strong RNA annealing activity indicates that they can profoundly affect the interactions of pre-mRNAs with trans-acting factors and suggests this to be an important function of hnRNP proteins in the processing of pre-mRNAs.

Base Composition↗
PubMed1994

The mRNA poly(A)-binding protein: localization, abundance, and RNA-binding specificity.

The poly(A)-binding protein (PABP) binds to the messenger (mRNA) 3'-poly(A) tail found on most eukaryotic mRNAs and together with the poly(A) tail has been implicated in governing the stability and the translation of mRNA. In order to further understand the role of the PABP in these processes, we have undertaken a detailed analysis of the cellular localization, the abundance, and the RNA-binding properties of the human PABP (hPABP). We raised monoclonal antibodies against the 70-kDa hPABP and confocal immunofluorescence microscopy with these antibodies reveals that it is localized exclusively to the cytoplasm. The hPABP exhibits a very low turnover rate in these cells and quantitative immunoblotting experiments demonstrated that growing HeLa cells contain a surprisingly high number of approximately 8 x 10(6) PABP molecules per cell, which corresponds to an intracellular concentration of about 4 microM. In an in vitro selection/amplification assay from random sequence oligonucleotide pools the hPABP selects oligo(rA)-rich sequences and it binds oligo(rA)25 with an apparent Kd of 7 nM. The hPABP binds to unrelated RNA sequences with an about 100-fold lower affinity (Kd > or = 0.5 microM). The abundance of the hPABP indicates that there is an approximately three-fold excess of the protein over binding sites on cytoplasmic poly(A). This excess and the high concentration of the hPABP, which is three orders of magnitude above its Kd for oligo(rA)25, suggest that the hPABP may bind to additional, lower affinity binding sites in vivo.

Animals↗
PubMed1994

Comparison of the migrations of Fasciola hepatica sporocysts in Lymnaea truncatula and other related snail families.

The migration and definitive localization of sporocysts in the snail body were studied in four species (Aplexa hypnorum, Lymnaea glabra, L. truncatula, Physa acuta), each of which was exposed to a single miracidium and killed during a period ranging from the 1st to the 5th day postexposure. When penetration occurred through the mantle, tunnels were found mainly in the mantle or kidney in Lymnaea adults and also extended to the foot in many physidid adults and in newborns of the four species. The sporocysts preferentially localized in the zone surrounding the kidney and heart beginning on the 4th day in Lymnaea spp. and disappeared in Aplexa and Physa snails on the 3rd day. When penetration occurred through the foot and extended inconstantly to the mantle according to the snail species involved. Sporocysts were encountered in the foot as well as the mantle or the zone surrounding the kidney and heart. The significance of these results is discussed in relation to the snail host and the entry site used by the Fasciola hepatica miracidium.

Animals↗
PubMed1994

Field observations concerning infection of Lymnaea palustris by Fasciola hepatica.

Fasciola hepatica miracidia were experimentally introduced into five sites colonized by Lymnaea palustris over a period of 4 or 6 years. In the first four ponds, a progressive increase in the prevalence of the spring-generation juvenile snails was observed (from 0.4 to 18.1%), with a corresponding increase in the shell height of infected snails (from 3.6 to 7.8 mm). In the fifth habitat, the pond dried in 1990 causing the prevalence to drop as compared with the initial values and to subsequently increase in 1991-1992.

Analysis of Variance↗
PubMed1994

Several observations concerning cercarial sheddings of Fasciola gigantica from Lymnaea natalensis.

The shedding of cercariae from Lymnaea natalensis and their transformation into metacercariae occurred mainly at night, and 70.7% of the cysts were counted between 7 p.m. and 1 a.m. There is no infradian rhythm in the numerical distribution of the daily mean values as related to experiment duration or patent period duration. One quarter of the snails shed their cercariae in a single wave and the others in 2 to 13 waves. Floating cysts represent 35% of the total number of metacercariae and their number decreased during the two first waves of shedding. Aberrant metacercarial localizations were noted on the inner surface of the shell when the snail died.

Animals↗
PubMed1994

Fasciola hepatica: a study of the shedding of cercariae from Lymnaea truncatula raised under constant conditions of temperature and photoperiod.

Investigations on the shedding of cercariae of Fasciola hepatica were carried out in Lymnaea truncatula in order to verify the existence of a low-frequency periodicity in the numerical distribution of metacercariae per snail and per day when the snails are raised under controlled conditions. Preadult L. truncatula were thus collected in the field, individually exposed to two miracidia, and subsequently raised until their death under constant temperature (20 degrees C) and photoperiod (12 h/12 h diurnal rhythm). The 102 snails shedding parasites produced 24,325 metacercariae of which 5% were floating cysts. The daily production of cercariae was maximal during the first 30 days of the patent period, subsequently decreased until day 114, and ceased on day 124. No infradian-type rhythm was noted in the daily distribution of mean values. The snails shed their cercariae in one to 14 waves; 20.6% and 15.7% of the snails produced their parasites in four and five periods respectively. The authors suggest that the seven-day periodicity found by Audousset et al. (1989) in the daily distribution of cercariae produced by three colonies of L. truncatula raised in seminatural conditions must be attributed only to the influence of environmental factors.

Animals↗
PubMed1994

Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.

3T3 Cells↗
PubMed1993

hnRNP G: sequence and characterization of a glycosylated RNA-binding protein.

The autoantigen p43 is a nuclear protein initially identified with autoantibodies from dogs with a lupus-like syndrome. Here we show that p43 is an RNA-binding protein, and identify it as hnRNP G, a previously described component of heterogeneous nuclear ribonucleoprotein complexes. We demonstrate that p43/hnRNP G is glycosylated, and identify the modification as O-linked N-acetylglucosamine. A full-length cDNA clone for hnRNP G has been isolated and sequenced, and the predicted amino acid sequence for hnRNP G shows that it contains one RNP-consensus RNA binding domain (RBD) at the amino terminus and a carboxyl domain rich in serines, arginines and glycines. The RBD of human hnRNP G shows striking similarities with the RBDs of several plant RNA-binding proteins.

Acetylglucosamine↗
PubMed1993

Specific binding of heterogeneous ribonucleoprotein particle protein K to the human c-myc promoter, in vitro.

A homopurine/homopyrimidine-like sequence is found 100-150 base pairs upstream of the human c-myc promoter P1. This element, termed the CT-element, has been shown to augment expression from P1, and it serves as a positive transcriptional element when coupled to a heterologous promoter in vivo and in vitro. Synthetic oligonucleotides comprising this element were used to form DNA-protein complexes in electrophoretic mobility shift assays. By using conventional and affinity methods, 61- and 34-kDa proteins were shown to be associated with these complexes. Amino acid sequence analysis and immunological methods have identified these proteins as heterogeneous ribonucleoprotein particle (hnRNP) proteins K and A1. Surprisingly, hnRNP protein K binds to the pyrimidine-rich strand of the CT-element in a sequence-specific manner as well as to the double-stranded molecule. Cotransfection of vectors encoding hnRNP protein K in the sense or anti-sense orientations with reporter plasmids driven by wild-type or mutant CT-elements demonstrates that hnRNP protein K augments gene expression in a cis-element-dependent manner. Taken together, these results suggest that hnRNP protein K may play a role in the transcriptional regulation of the human c-myc gene.

Amino Acid Sequence↗
PubMed1993

The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein.

Fragile X syndrome is one of the most common human genetic diseases and the most common cause of hereditary mental retardation. The gene that causes fragile X syndrome, FMR1, was recently identified and sequenced and found to encode a putative protein of unknown function. Here we report that FMR1 contains two types of sequence motifs recently found in RNA-binding proteins: an RGG box and two heterogeneous nuclear RNP K homology domains. We also demonstrate that FMR1 binds RNA in vitro. Using antibodies to FMR1, we detect its expression in divergent organisms and in cells of unaffected humans, but fragile X-affected patients express little or no FMR1. These findings demonstrate that FMR1 expression is directly correlated with the fragile X syndrome and suggest that anti-FMR1 antibodies will be important for diagnosis of fragile X syndrome. Furthermore, the RNA binding activity of FMR1 opens the way to understanding the function of FMR1.

Amino Acid Sequence↗
PubMed1993

The pre-mRNA binding K protein contains a novel evolutionarily conserved motif.

The K protein is among the major pre-mRNA-binding proteins (hnRNPs) in vertebrate cell nuclei. It binds tenaciously to cytidine-rich sequences and is the major oligo(rC/dC)-binding protein in vertebrate cells. We have cloned a cDNA of the Xenopus laevis hnRNP K and determined its sequence. The X.laevis hnRNP K is a 47 kD protein that is remarkably similar to its human 66 kD counterpart except for two large internal deletions. The sequence of hnRNP K contains a 45 amino acid repeated motif which is almost completely conserved between the X.laevis and human proteins. We found that this repeated motif, the KH motif (for K homology), shows significant homology to several proteins some of which are known nucleic acids binding proteins. The homology is particularly strong with the archeabacterial ribosomal protein S3 and with the saccharomyces cerevisiae protein MER1 which is required for meiosis-specific splicing of the MER 2 transcript. As several of the proteins that contain the KH motif are known to bind RNA, this domain may be involved in RNA binding.

Amino Acid Sequence↗