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WO1993017102A1 - MODULATION DE FONCTIONS CELLULAIRES AVEC DES PROTEINES HnRNP - Google Patents

MODULATION DE FONCTIONS CELLULAIRES AVEC DES PROTEINES HnRNP Download PDF

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Publication number
WO1993017102A1
WO1993017102A1 PCT/US1993/001543 US9301543W WO9317102A1 WO 1993017102 A1 WO1993017102 A1 WO 1993017102A1 US 9301543 W US9301543 W US 9301543W WO 9317102 A1 WO9317102 A1 WO 9317102A1
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cell
hnrnp
protein
nucleus
proteins
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PCT/US1993/001543
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English (en)
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Gideon Dreyfuss
Serafín PIÑOL-ROMA
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The Trustees Of The University Of Pennsylvania
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Publication of WO1993017102A1 publication Critical patent/WO1993017102A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is directed to the modulation of cellular function by hnRNP proteins.
  • transgenic animals can be produced by the transport of foreign DNA into the nucleus of a cell.
  • antisense therapies are effected by the targeting of oligonucleotides to nucleic acids in the nucleus.
  • research, diagnostic and therapeutic strategies can involve the targeting of antibodies and other proteins to nuclear material.
  • One method of introducing a foreign molecule into the nucleus of a cell is by microinjection.
  • this procedure is time consuming and requires a high degree of skill and special equipment. As a result, this procedure cannot practically be applied to a large number of cells.
  • Another method of introducing a foreign molecule, in particular DNA, into the nucleus of a cell is by calcium-phosphate or DEAE- dextran mediated transfection. This procedure can be applied to a large number of cells but results in limited uptake rates, on the order of 20%. Further, some cell lines are resistant to transfection by this method.
  • Protoplast fusion a method limited to DNA manipulation, involves the fusion of protoplasts from bacteria carrying the plasmid DNA of interest with cultured cells. Although this procedure has high efficiency, the manipulations are time-consuming.
  • Some methods of introducing molecules into the nucleus of cells involve passing the molecules through the cytoplasm, across the nuclear membrane, and into the nucleus.
  • the nuclear membrane is selective and, in the case of large or charged molecules, generally impermeable to such moLecules in the absence of an active transport mechanism.
  • Active transport mechanisms are generally specific to particular molecules or types of molecules and often act slowly.
  • Synthetic active transport mechanisms for molecules are therefore desirable whereby molecules may be shuttled from the cytoplasm, across the nuclear membrane, and into the nucleus.
  • Such synthetic active transport mechanisms would augment natural active transport mechanisms by providing import of molecules to the nucleus which, under natural conditions, would be excluded from the nucleus due to the lack of an appropriate active transport mechanism. Further, such synthetic active transport mechanisms would also augment the rate by which desired molecules pass through the nuclear membrane, thus increasing the amount of molecule which can be directed to the nucleus, and consequently, the efficacy of molecules acting within the nucleus.
  • Synthetic active transport systems modeled after a naturally occurring mechanism such can be a be particularly valuable tool because it is less likely to be toxic to cells and less likely to interfere with natural cellular mechanisms.
  • Synthetic active transport methods useful in diagnostic, research and therapeutic practices are provided. Such methods allow for the transport of molecules which are foreign to the cell, and are useful to augment the transport of molecules which have naturally-occurring, but rate limiting active transport mechanisms.
  • methods of transporting a molecule into the nucleus of a cell comprise appending a molecule to at least a portion of a hnRNP protein to form a complex and introducing the complex into the cell.
  • the invention is also directed to complexes comprising at least a portion of a hnRNP protein appended to at least a portion of a gene.
  • Complexes comprising at least a portion of a hnRNP protein appended to an oligonucleotide are also provided by the present invention. Further, complexes comprising at least a portion of a hnRNP protein appended to a polypeptide having activity within the nucleus of a cell are provided. Methods of transporting genetic material, nucleic acids and polypeptides into the nucleus of a cell are also provided whereby a complex comprising hnRNP protein and said genetic material, nucleic acids, or polypeptides are introduced into a cell.
  • methods of altering the function or activity of cells are provided in which a complex comprising hnRNP and a polypeptide, nucleic acid, a portion of a gene, or an oligonucleotide is introduced into a cell.
  • Methods of modulating the production of protein by a cell are also provided by the present invention whereby a molecule which binds competitively to the mRNA binding site of a hnRNP protein is introduced into a cell.
  • Chemotherapeutic methods are also provided in which carcinoma cells are contacted by molecules which bind competitively to the mRNA binding site of a hnRNP protein.
  • Methods of transporting a molecule into the nucleus of a cell are provided in certain embodiments of the present invention. These methods comprise appending a molecule to at least a portion of a hnRNP protein to form a complex and introducing the complex into the cell.
  • the molecules may include a broad range of molecules such as genetic material, oligonucleotides and polypeptides. For example, at least a portion of a gene for a therapeutic agent can be appended to a hnRNP protein.
  • the genes may code for platelet derived growth factor, fibroblast growth factor, epidermal growth factor, transforming growth factor alpha, angiogenic factors, insulin, interferon, immunoglobulins, tissue plasminogen activator, LDL receptor or ⁇ -antitrypsin.
  • This list is not exhaustive as any gene for a therapeutic, diagnostic or research reagent can be appended to hnRNP molecules to facilitate transformation of cells and expression of the gene.
  • oligonucleotides can similarly be transported to the nucleus such as oligonucleotides "antisense" to human immunodeficiency virus (HIV), influenza viruses, or other viruses.
  • proteins can be appended to a hnRNP protein for transport- to the nucleus.
  • proteins can be transported to the nucleus expeditiously.
  • antibodies directed to nuclear structures can be transported via the synthetic active transport mechanism of the present invention.
  • oligonucleotide or polypeptide molecule may be used in some embodiments of the present invention, a portion of the same may be sufficient to achieve objects of the present invention.
  • the portion should represent, for example, coding regions or control regions of the gene.
  • the active portion of a polypeptide may be sufficient in certain embodiments of the present invention, for example, the portion of an antibody polypeptide which represents an epitope may be sufficient for recognition of a nuclear structure by said antibody and the recognition site of a gene regulatory protein may be useful in the present invention.
  • RNA polymerase II transcripts the Balbaiani ring mRNAs in Chironomus tentan ⁇ salivary glands, have been visualized in transit through the nuclear pores as ribonucleoprotein particles, Stevens and Swift, J. Cell Biol . 3_1: 55-78 (1966); Skoglund, et al., Cell 34.: 847-855 (1983); Mehlin, et al., E ⁇ p. Cell Res . 191- 72-77 (1991), although the identity of the protein components of these particles is unknown.
  • HnRNP proteins may also serve a shuttling function and thus may be excellent targets for modulation of the expression of proteins by a cell.
  • HnRNP complexes in vertebrate cells are composed of at least 20 main proteins. Choi, Y.D. , and G. Dreyfuss, Proc. Natl . Acad. Sci . USA 81: 7471-7475 (1984); Pi ⁇ ol-Roma, et al., Genes Develop. 2 : 215-227 (1988). It is believed that heterogenous nuclear RNAs (hnRNAs) , associate in the nucleus with the specific proteins that bind pre messenger RNA (hnRNP proteins). Dreyfuss, G. , Ann . Rev.
  • snRNPs small nuclear ribonucleoprotein particles
  • HnRNP proteins have been localized in the nucleoplasm. Martin, et al., Immunocytochemistry of nuclear hnRNP complexes. In The Cell Nucleus, ed. H. Busch, 119-144, Vol. 9 (Academic Press, NY 1981); Leser, et al. , J. Biol . Chem. 259: 1827-1833 (1984); Dreyfuss, et al. , Mol . Cell . Biol . 4.: 1104-1114 (1984b); Choi, Y.D. and G. Dreyfuss,) J. Cell Biol . 99.: 1997-2004 (1984b); Pi ⁇ ol-Roma, et al. , J.
  • hnRNP proteins involved the exchange of hnRNP for mRNA-binding proteins accompanying transport of mRNA from the nucleus to the cytoplasm. Dreyfuss, G.A. , A. Rev. Cell . Biol . 2.: 459-498 (1986); Dreyfuss, et al., Mol . Cell . Biol . 4: 415-423 (1984).
  • hnRNP protein which disperse during mitosis reaccumulate in the newly formed daughter cell nuclei by two modes: transcription-independent (e.g. C proteins) and transcription-dependent (e.g. Al, A2 and E proteins).
  • C proteins transcription-independent proteins
  • Al transcription-dependent proteins
  • E transcription-dependent proteins
  • Pi ⁇ ol-Roma S. and G. Dreyfuss, Science 253:312- 314 (1991) .
  • HnRNP proteins may thus also have cytoplasmic functions.
  • Al is bound to mRNA and RNA polymerase II transcription is necessary before it can return to the nucleus.
  • cytoplasmic ribonucleoprotein complex of mRNA with hnRNP proteins is the substrate of nuclear-cytoplas ic transport of mRNA. Further, these transcription-dependent hnRNP proteins accumulate in the cytoplasm after treatment with polymerase II inhibitors, whereas several other hnRNP proteins such as U appear to be restricted to the nucleus. It is likely that export of transcription-dependent hnRNP proteins from the nucleus is by active transport.
  • hnRNP proteins have been isolated and identified as useful in the present invention. Transcription-dependent hnRNP proteins such as Al, A2 or E are useful in certain embodiments of the present invention. Naturally occurring hnRNP proteins may be useful in purified or semi-purified forms. In addition, sequences of these hnRNP proteins can be determined in accordance with methods provided by Pi ⁇ ol-Roma, et al.. Journal of Cell Biology 109: 2575-2587 (1989) and synthetic hnRNP proteins can be synthesized by standard protein synthesis techniques known to those skilled in the art. All or at least a portion of the hnRNP protein can be used.
  • a portion from about 20 to about 1000 amino acids in length is sufficient to transport a molecule across the nuclear membrane. In other embodiments of the present invention, a portion from about 100 to about 500 amino acids in length is sufficient. In still other embodiments of the present invention, a portion from about 100 to about 300 amino acids in length is sufficient to transport a molecule across the nuclear membrane.
  • any of a range of methods of joining two distinct moieties is encompassed.
  • crosslinker molecules such as disuccinimidyl suberate (DSS) , N-hydroxysuccinimidyl- (4-azidobenzoate) (HSAB) and m-maleimido-benzoyl-tf- hydroxysuccinimide ester (MBS) can be used to link molecules and hnRNP proteins.
  • DSS disuccinimidyl suberate
  • HSAB N-hydroxysuccinimidyl- (4-azidobenzoate)
  • MFS m-maleimido-benzoyl-tf- hydroxysuccinimide ester
  • covalent linkers useful in the present invention will be apparent to those skilled in the art.
  • the molecules and hnRNP proteins may also be appended to form a complex by direct covalent bonding between hnRNP proteins and molecules such as nucleic acid molecules.
  • the specific binding affinity of some hnRNP proteins towards a particular ribonucleotide can be harnessed to achieve specific binding, such as for example, the specific binding affinity of hnRNP E protein for polyG. Swanson and Dreyfuss, Molecular and Cellular Biology 8.(5) 2237-2241 (1988).
  • the complex is in the form of a fusion protein, expressed within the cell itself comprising a portion of a molecule and a portion of a hnRNP protein joined together, or "appended" to form a complex.
  • the resulting complex may be introduced into the cell by any of a range of methods known to those skilled in the art. Any of the known techniques such as those described in Sambrook, et al. , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, NY) , incorporated by reference herein in its entirety may be used.
  • a molecule occurring naturally in the cell is bonded to or otherwise appended to a hnRNP protein via a crosslinking molecule while in the cytoplasm.
  • an hnRNP protein may be modified by addition of a crosslinker, and introduced into the cytoplasm of a cell by methods described herein. Thereafter, said hnRNP-crosslinker can append to target molecules via ultraviolet activated or other crosslinking.
  • the hnRNP- molecule complex is introduced into the cell by expression as a fusion protein within the cell in accordance with known techniques such as those described in Sambrook, et al.
  • a complex or components of the complex may be microinjected into a cell. Transfection methods such as calcium-phosphate and DEAE-dextran mediated transfection may also be used to introduce a complex or its components into the cytoplasm of a cell.
  • a hnRNP-crosslinker moiety or hnRNP- molecule complexes linked with or without a crosslinker may be introduced by these methods.
  • fusion products may be introduced into the cell by means of cellular expression of a fusion protein comprising a hnRNP protein and a molecule such as a polypeptide.
  • a fusion protein comprising a hnRNP protein and a molecule such as a polypeptide.
  • the hnRNP protein will function as a shuttle to carry the molecule through the nuclear membrane via an active transport mechanism. It is further theorized that the hnRNP proteins function to shuttle molecules, such as mRNA out of the nucleus. HnRNP proteins appear to shuttle mRNA from the nucleus to the cytoplasm where the mRNA is translated into proteins. Methods of altering the function or activity of a cell by contacting the cell with a complex are also provided by the present invention.
  • a cell is transformed to express a gene such as platelet derived growth factor, fibroblast growth factor, epidermal growth factor, transforming growth factor alpha, angiogenic factors, insulin, interferon, immunoglobulins, tissue plasminogen activator, LDL receptor or ⁇ -antitrypsin genes.
  • a gene such as platelet derived growth factor, fibroblast growth factor, epidermal growth factor, transforming growth factor alpha, angiogenic factors, insulin, interferon, immunoglobulins, tissue plasminogen activator, LDL receptor or ⁇ -antitrypsin genes.
  • an oligonucleotide is transported into the nucleus of the cell to interfere with gene expression within a cell such as the expression of human immunodeficiency virus.
  • genes and oligonucleotides can be used to alter the function or activity of a cell, as long as the basic tenets of the present invention are adhered to.
  • Polypeptides may also be targeted to the nucleus in order to alter the function or activity of a cell such as for example by providing augmented transport of proteins naturally expressed in the cytoplasm of the cell.
  • gene regulatory proteins expressed in the cytoplasm can be expeditiously transported to the nucleus to enhance inhibition or activation of the expression of a protein which effects cellular function or activity.
  • Methods of modulating the expression of protein by a cell are also provided by the present invention whereby a molecule which binds competitively to an mRNA binding site of a hnRNP protein is introduced into a cell. While not wishing to be bound to any particular theory, it is believed that by competitively inhibiting binding of mRNA to a hnRNP protein transport of mRNA into the cytoplasm will be suppressed, and translation of the mRNA can be modulated.
  • an antibody which recognizes an mRNA binding site of a hnRNP protein may bind so as to prevent mRNA binding, i.e., the binding of a hnRNP protein and said antibody (or other molecule) is preferential to the binding of mRNA.
  • Oligonucleotides which hybridize to at least a portion of a gene coding for a hnRNP protein can also modulate the expression of protein by a cell by blocking the expression of the protein, thereby reducing or eliminating the number of hnRNP proteins available to transport mRNA into the cytoplasm.
  • This procedure may be particularly applicable, for example, to the modulation of the expression of proteins in hyperproliferative cells, especially carcinoma cells.
  • molecules which bind competitively to a mRNA binding site of a hnRNP protein prevent the expression of proteins by said cells, and inhibit or eliminate the proliferation of said cells.
  • Administering oligonucleotides which hybridize to at least a portion of a gene coding for a hnRNP protein may also be an effective method to prevent the proliferation of carcinoma cells.
  • HeLa cells were grown at 37°C, in DME supplemented with 10% fetal calf serum and penicillin-streptomycin.
  • Xenopus laevis kidney epithelial cells were grown at 29°C in DME with 20% H 2 0 and 10% fetal calf serum, supplemented with penicillin and streptomycin.
  • Heterokaryons were grown at 29°C in DME supplemented with 10% fetal calf serum, 10% H 2 0, and 1% penicillin and streptomycin. Where indicated actinomycin D (Calbiochem) was added directly to the culture medium to a final concentration of 0.04 or 5jug/ml.
  • HeLa-X. laevis heterokaryons were produced by polyethylene glycol-mediated cell fusion, by a modification of the procedure described in Borer et al., Cell 56: 379-390 (1989) . Briefly, X. laevis cells were grown on glass coverslips to subconfluent density. HeLa cells were then trypsinized and plated onto the X. laevis cell-bearing coverslips, and allowed to attach for 2-4 hours in heterokaryon growth medium. Each coverslip was then rinsed in PBS, and placed inverted onto a drop of 50% PEG (Gibco BRL) pre-warmed to 29°C, for 2 minutes.
  • PEG Gibco BRL
  • the coverslip was then rinsed twice in PBS to remove the PEG, and placed into a 35mm culture plate in heterokaryon growth medium. Heterokaryons were incubated at 29°C, 5% C0 2 for 4 hours prior to fixation. Where indicated, cycloheximide (20 ⁇ g/ml) was added to the HeLa-X. laevis cell co-cultures 30 minutes prior to fusion, and incubation following the fusion step was also continued in the presence of cycloheximide.
  • Cells grown on sterile glass coverslips were rinsed with PBS, fixed with 2% formaldehyde in PBS for 20 minutes at room temperature, and permeabilized with acetone at -20°C for 5 minutes.
  • the fixed, permeabilized cells were immediately processed for immunofluorescence microscopy staining as follows.
  • primary antibody incubation was carried out with the indicated hybridoma conditioned culture medium for 45 minutes at room temperature in a humidified chamber.
  • the slides were then rinsed in PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Cappel) , used at a 1:500 dilution, for 30 minutes.
  • FITC fluorescein isothiocyanate
  • the monoclonal antibodies 4F4 (Choi, Y.D. and G. Dreyfuss, J. Cell . Biol . 99 . : 1997-2004 (1984b)) and 4B10 (Pi ⁇ ol-Roma, et al. , Genes Devel . 2y. 215-227 (1988), to the hnRNP C and Al proteins respectively, were purified from ascites fluid by HPLC on a Dynamax PEI column (Rainin) following the manufacturer's suggestions. 4F4 was then labeled with fluorescein isothiocyanate (FITC; Sigma Chemical Company, St.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RNA-protein Crosslinking in Intact Cells and Isolation of hnRNP RNA-protein Crosslinking in Intact Cells and Isolation of hnRNP
  • RNA-protein crosslinking by UV light irradiation of cells on culture dishes, and isolation and analysis of RNPs was carried out as previously described (Dreyfuss, et al., Mol . Cell . Biol . 4.: 415-423 (1984a); Adam et al., J. Virol . J52: 614-622 (1986)).
  • the cells were lysed in RSB-100 (containing 0.5% Triton X-100, 0.5% aprotinin, l ⁇ g/ml each leupeptin and pepstatin A, and lOmM vanadyl-adenosine by four passages through a 25 gauge syringe needle.
  • DRB 5,6-dichlororibofuranosyl benzimidazole
  • the cytoplasmic accumulation of Al is readily reversible upon restoration of transcription, by placing cells back int a medium without the inhibitor at 37°C. This shows that the cells retain their viability under these inhibition conditions, and that the cytoplasmic Al is not impaired in its ability to be imported back into the nucleus. No such reversal is observed after placing the DRB-treated cells into fresh DRB- free medium at 4°C. This could be explained by the failure of the cells to resume transcription due to the low temperature, and/or the requirement for an active transport mechanism for the return of the cytoplasmic Al to the nucleus. In all of these experiments the C proteins, in contrast to Al, are detected exclusively in the nucleus.
  • Al HnRNP when in the Cytoplasm is Bound to Poly(A) + RNA.
  • Al is an avid RNA-binding protein, and it was of interest to determine whether the Al protein is in free form or whether it is bound to mRNA when it is in the cytoplasm.
  • actinomycin D 5 ⁇ g/ml
  • RNA-crosslinked proteins were released from t he complexes by RNase digestion and analyzed by SDS-PAGE and immunoblotting.
  • HnRNP proteins are purified from HeLa nucleoplasm in accordance with procedures described in Swanson and Dreyfuss,
  • Oligonucleotides coding for the loop forming site of influenza virus encoding polymerase 3 of the influenza virus, TTGACGAAATT (SEQ ID NO: 1) are synthesized using standard procedures known in the art, incorporating a polyA tail at the 3' end of the oligonucleotide and a modified oligonucleotide linker at the 5' end of the oligonucleotide.
  • Oligonucleotides and purified hnRNP Al protein are incubated and resulting complexes are purified using an oligodT cellulose column.
  • Purified complex and unlinked oligonucleotides are each incubated with HeLa cells infected with influenza virus.
  • the effectiveness of inhibition of the influenza virus by the complex and unlinked oligonucleotide is determined by monitoring the number of viable cells remaining after a 48 hour incubation. Cells incubated with complex will be more viable than cells incubated with unlinked oligonucleotide due to the increased transport of hnRNP-oligonucleotides into the nucleus.
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne un procédé de transport actif synthétique permettant de transporter des molécules du cytoplasme, à travers la membrane nucléaire et dans le noyau. Des compositions utiles pour effectuer ce transport sont également décrites et comprennent des complexes hn-RNP-molécule. En appliquant les procédés de la présente invention, on peut effectuer la modulation de l'expression d'une protéine par une cellule. D'autres fonctions ou activités cellulaires peuvent également être modifiées selon les procédés de la présente invention.
PCT/US1993/001543 1992-02-19 1993-02-18 MODULATION DE FONCTIONS CELLULAIRES AVEC DES PROTEINES HnRNP WO1993017102A1 (fr)

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Cited By (6)

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WO1996017074A3 (fr) * 1994-12-02 1996-08-29 Univ Yale Peptides derives de l'arn polymerase ii et leurs procedes d'utilisation
US6090784A (en) * 1995-12-01 2000-07-18 Yale University RNA polymerase II peptides and methods of use
US8658608B2 (en) 2005-11-23 2014-02-25 Yale University Modified triple-helix forming oligonucleotides for targeted mutagenesis
US11136597B2 (en) 2016-02-16 2021-10-05 Yale University Compositions for enhancing targeted gene editing and methods of use thereof
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WO1994004696A1 (fr) * 1992-08-25 1994-03-03 Miles Inc. Apport nucleaire de macromolecules facilite par un signal de translocation
WO1996017074A3 (fr) * 1994-12-02 1996-08-29 Univ Yale Peptides derives de l'arn polymerase ii et leurs procedes d'utilisation
US6090784A (en) * 1995-12-01 2000-07-18 Yale University RNA polymerase II peptides and methods of use
US8658608B2 (en) 2005-11-23 2014-02-25 Yale University Modified triple-helix forming oligonucleotides for targeted mutagenesis
US11136597B2 (en) 2016-02-16 2021-10-05 Yale University Compositions for enhancing targeted gene editing and methods of use thereof
US12268774B2 (en) 2017-04-04 2025-04-08 Yale University Compositions and methods for in utero delivery

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