Ribonucleoprotein particles in cellular processes.
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Heterogeneous-Nuclear Ribonucleoproteins↗
Biomedical subjects
G Dreyfuss
Publications and source records attributed to G Dreyfuss.
Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.
Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) particles can be efficiently purified by a specific, rapid, and mild procedure using monoclonal antibodies to hnRNP proteins. We report here on the detailed analysis of the protein composition of immunopurified hnRNP particles from human HeLa cells. By two-dimensional gel electrophoresis, immunopurified hnRNP particles contain at least 24 polypeptides in the range of 34,000-120,000 daltons. The abundant 30,000-40,000 dalton proteins, A, B, and C, described previously, are a subset of these polypeptides. The protein compositions of hnRNP particles found in the nucleoplasm fraction and in the chromatin-nucleolar fraction are very similar. Upon addition of the polyanion heparin, most of the major proteins remain associated in heparin-resistant particles, and only several, mostly minor, proteins dissociate. This provides an aid in the classification of the proteins and an additional criterion for the definition of hnRNP particle components. Chromatography on single-stranded DNA (ssDNA)-agarose in a heparin- and moderate or high salt (higher than 300 mM NaCl)-resistant manner suggests that most, if not all, of these proteins are single-stranded nucleic acid-binding proteins. We describe a general method for the large-scale purification of hnRNP proteins by affinity chromatography on ssDNA columns and its use for the production of new monoclonal antibodies to hnRNP proteins.
Carrier Proteins↗
Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.
Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.
HeLa Cells↗
[A new series of antiparasitic organic arsenicals: the spiroarsoranes. Experimental trypanocidal activity].
Arsenical compounds are the main therapeutic way, effective in the neurological phase of trypanosomiasis. Unfortunately, their important toxicity prevents their easy administration. Structural cyclization of arsonic acid derivatives has led to a class of non-toxic compounds: spiroarsoranes. A 47 compound primary screening on an in vivo murine model of Trypanosoma brucei brucei resulted in the isolation of a very effective derivative after a single subcutaneous injection of 30 mg.kg-1 body weight (Chemotherapeutic Index = 21). In this study, trypanocidal activity of this compound was evaluated on an in vivo sheep model of trypanosomiasis by T. brucei brucei, according to experimental model of the Institute of Neurological Epidemiology and Tropical Neurology of Limoges (France). A single subcutaneous injection of 30 and 100 mg.kg-1 b.w., and 30 and 60 mg.kg-1 b.w. was given respectively during first and second phase of trypanosomiasis. Ovine pharmacokinetics of this compound will be evaluated in a further study.
Animals↗
[Antiparasitic activity of Cis-Pt (II) pentamidine in experimental trypanosomiasis of sheep caused by Trypanosoma brucei brucei].
The failure of trypanocidal therapeutics have made it a duty to use other classes of molecules which show a comparable trypanocidal efficacy with well-known medicines. In this study, the authors have evaluated the trypanocidal activity of a Cis-Platinum (II) salt, DNA-intercalating medicine used in antineoplastic therapeutics, associated with pentamidine: Cis-Pt (II) pentamidine. After primary screenings on a murine model of T. brucei brucei, Cis-Pt (II) pentamidine was evaluated on an experimental sheep trypanosomiasis model, standardized by the Institute of Neurological Epidemiology and Tropical Neurology of Limoges (France). Cis-Pt (II) pentamidine was able to cure infected mice, without relapse, after a single subcutaneous injection of 12.5 mg.kg-1 body weight. In sheep trypanosomiasis, the same treatment was used successfully during the first stage of infection. Neurological stage susceptibility and pharmacokinetics in sheep will be evaluated in a further study.
Amidines↗
Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells.
The proteins that interact with cytoplasmic and nuclear polyadenylated RNA in adenovirus type 5 (Ad5) infection of HeLa cells were examined by UV-induced RNA-protein cross-linking in intact cells. The Ad5 100-kilodalton late nonvirion protein (100K protein) was cross-linked to both host and viral polyadenylated cytoplasmic RNA (mRNA). The cross-linking of the 100K protein to mRNA appears to correlate with productive infection, because the protein is not cross-linked to mRNA in abortive infection of wild-type Ad5 in monkey cells (CV-1) even though normal amounts of it are produced. However, when CV-1 cells are infected with Ad5 hr404, and Ad5 mutant which overcomes the host restriction to wild-type Ad5 infection in these cells, the 100K protein is cross-linked to mRNA. To identify and obtain antibodies to RNA-contacting proteins, a mouse was immunized with oligo(dT)-selected cross-linked RNA-protein complexes from Ad5-infected cells and the serum was used for immunoblotting experiments. It was found that in addition to the 100K protein, the Ad5 72K DNA-binding protein is also associated with RNA in the infected cells. The 72K DNA-binding protein is cross-linked to polyadenylated nuclear RNA sequences. These findings indicate that adenovirus proteins interact with RNAs in the infected cell and suggest possible mechanisms for the effects of the virus on mRNA metabolism.
Adenoviruses, Human↗
Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins.
In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins.
Amino Acid Sequence↗
[Antimalarial activity of various isoquinoline alkaloids in the mouse].
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Alkaloids↗
[Trypanocidal activity of isoquinoline alkaloids in mice].
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Alkaloids↗
Heterogeneous nuclear ribonucleoproteins: role in RNA splicing.
Splicing in vitro of a messenger RNA (mRNA) precursor (pre-mRNA) is inhibited by a monoclonal antibody to the C proteins (anti-C) of the heterogeneous nuclear RNA (hnRNA)-ribonucleoprotein (hnRNP) particles. This antibody, 4F4, inhibits an early step of the reaction: cleavage at the 3' end of the upstream exon and the formation of the intron lariat. In contrast, boiled 4F4, or a different monoclonal antibody (designated 2B12) to the C proteins, or antibodies to other hnRNP proteins (120 and 68 kilodaltons) and nonimmune mouse antibodies have no inhibitory effect. The 4F4 antibody does not prevent the adenosine triphosphate-dependent formation of a 60S splicing complex (spliceosome). Furthermore, the 60S splicing complex contains C proteins, and it can be immunoprecipitated with 4F4. Depletion of C proteins from the splicing extract by immunoadsorption with either of the two monoclonal antibodies to the C proteins (4F4 or 2B12) results in the loss of splicing activity, whereas mock-depletion with nonimmune mouse antibodies bodies has no effect. A 60S splicing complex does not form in a C protein-depleted nuclear extract. These results indicate an essential role for proteins of the hnRNP complex in the splicing of mRNA precursors.
Adenosine Triphosphate↗
Molecular cloning of cDNA for the nuclear ribonucleoprotein particle C proteins: a conserved gene family.
The C proteins, C1 and C2, are major constituents of the heterogeneous nuclear RNA (hnRNA) ribonucleoprotein (hnRNP) complex in vertebrates. C1 and C2 are antigenically related phosphoproteins that are in contact with hnRNA in intact cells and bind to RNA tightly in vitro. A cDNA clone for the C proteins was isolated by immunological screening of a human lambda gt11 expression vector cDNA library with monoclonal antibodies. The lacZ-cDNA fusion protein is recognized by two different anti-C protein monoclonal antibodies. HeLa cell mRNA that was hybrid-selected with the cDNA clone (1.1 kilobases long) was translated in vitro and yielded both the C1 and C2 proteins (41 and 43 kDa, respectively). RNA blot analysis showed strong hybridization to two polyadenylylated transcripts, of about 1.4 kb and 1.9 kb, in human cells. Genomic DNA blot analysis showed multiple hybridizing restriction fragments in human and mouse, and homologous DNA sequences are found across eukaryotes from human to yeast. These findings suggest that the sequences encoding the hnRNP C proteins are members of a conserved gene family and they open the way for detailed molecular and genetic studies of these proteins.
Animals↗
Interaction of mRNA with proteins in vesicular stomatitis virus-infected cells.
The interaction of mRNA with proteins in vesicular stomatitis virus (VSV)-infected cells was studied by photochemical cross-linking in intact cells. The major [35S]methionine-labeled proteins which became cross-linked by UV light to mRNA in uninfected and in VSV-infected HeLa cells were similar and had apparent mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to 135, 93, 72, 68, 53, 50, 43, and 36 kilodaltons. The proteins which were cross-linked in vivo specifically to the five mRNAs of VSV were labeled through radioactive nucleotides incorporated only into VSV mRNAs under conditions (5 micrograms of actinomycin D per ml) in which only VSV mRNAs are labeled. The same major mRNP proteins that became cross-linked to host mRNAs also became cross-linked to VSV mRNAs, although several quantitative differences were detected. Photochemical cross-linking and immunoblotting of cross-linked mRNPs with VSV antiserum demonstrated that in addition to host proteins VSV mRNAs also became cross-linked to the VSV-encoded N protein. The poly(A) segment of both host and VSV mRNAs was associated in vivo selectively with the 72-kilodalton polypeptide. The major proteins of mRNA-ribonucleoprotein complexes are therefore ubiquitous and common to different mRNAs. Furthermore, since the major messenger ribonucleoproteins interact also with VSV mRNAs even though these mRNAs are transcribed in the cytoplasm, it appears that nuclear transcription and nucleocytoplasmic transport are not necessary for mRNA to interact with these proteins.
Cross-Linking Reagents↗
mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence.
We identified and produced antibodies to the major proteins that interact with poly(A)+ RNAs in the yeast Saccharomyces cerevisiae. The major proteins which were cross-linked by UV light to poly(A)+ RNA in intact yeast cells had apparent molecular weights of 72,000, 60,000, and 50,000. The poly(A) segment of the RNA was selectively cross-linked to the 72,000-molecular-weight protein (72K protein). Mice immunized with purified UV-cross-linked RNA-protein (RNP) complexes produced antibodies to the three major RNP proteins. A yeast genomic DNA library constructed in the lambda gt11 expression vector was screened with the anti-RNP serum, and recombinant bacteriophage clones were isolated. One recombinant phage, lambda YPA72.1, bearing a 2.5-kilobase insert, produced a large beta-galactosidase-RNP fusion protein. Affinity-selected antibodies from the anti-RNP serum on this fusion protein recognized a single 72K protein which was cross-linked to the poly(A) segment of RNA in the intact cell. Furthermore, the fusion protein of lambda YPA72.1 had specific poly(A)-binding activity. Therefore, lambda YPA72.1 encodes the 72K poly(A)-binding protein. Immunofluorescence microscopy showed that this protein was localized in the cytoplasm. Hybrid-selected mRNA translated in vitro produced the 72K poly(A)-binding protein, and mRNA blot analysis detected a single 2.1-kilobase mRNA. DNA blot analysis suggested a single gene for the poly(A)-binding protein. DNA sequence analysis of genomic clones spanning the entire gene revealed a long open reading frame encoding a 64,272-molecular-weight protein with several distinct domains and repeating structural elements. A sequence of 11 to 13 amino acids is repeated three times in this protein. Strikingly, this repeated sequence (RNP consensus sequence) is highly homologous to a sequence that is repeated twice in a major mammalian heterogeneous nuclear RNP protein, A1. The conservation of the repetitive RNP consensus sequence suggests an important function and a common evolutionary origin for messenger RNP and heterogeneous nuclear RNP proteins.
Amino Acid Sequence↗
Structure and function of nuclear and cytoplasmic ribonucleoprotein particles.
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Animals↗
Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): a unique supramolecular assembly.
The packaging of heterogeneous nuclear RNA (hnRNA), the fate of hnRNA in the nucleus, and the conversion of hnRNA to mRNA are believed to occur as the hnRNA transcript is associated with specific proteins to form a ribonucleoprotein complex termed the hnRNP complex. The identity and organization of the protein constituents of the hnRNP have been a matter of considerable controversy. We report here the isolation of the hnRNP complex from vertebrate cell nuclei, employing immunoprecipitation with monoclonal antibodies against the major proteins that are in contact with hnRNA in HeLa cells. Rapid immunoprecipitation from HeLa nucleoplasm with two different monoclonal antibodies to the hnRNP C proteins (41 and 43 kDa) isolates a similar complex that contains proteins and hnRNA of up to approximately equal to 10 kilobases. The major steady-state [35S]methionine-labeled proteins of the isolated complex are of 34 kDa, 36 kDa (A1 and A2), 37 kDa, 38 kDa (B1 and B2), 41 kDa, 43 kDa (C1 and C2), and doublets at 68 kDa and at 120 kDa. Additional proteins from 45 kDa to very high molecular mass are also seen. The major proteins of the complex appear identical by NaDodSO4/polyacrylamide gel electrophoresis to genuine hnRNP proteins--those which become crosslinked by UV light to the hnRNA in vivo. Immunoprecipitation with a different, noncrossreacting monoclonal antibody to the 120-kDa protein isolates an apparently identical complex of proteins that are present at a similar relative stoichiometry. Similar hnRNP complexes are found in rodent and avian cells. Nuclease digestions indicate that RNA plays a role in maintaining the integrity of the structure and that intact RNA of approximately equal to 125 nucleotides is sufficient to hold the complex of proteins together. The coimmunoprecipitation of the hnRNA and of all of the proteins through antibodies against different genuine hnRNP proteins and from divergent species strongly suggests that the hnRNP complex is a unitary structure of consistent, defined, and conserved components.
Animals↗
Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells.
The C proteins (C1 and C2) are major constituents of the 40S subparticle of heterogeneous nuclear ribonucleoprotein complexes (hnRNPs) (Beyer, A.L., M.E. Christensen, B.W. Walker, and W.M. LeStourgeon, 1977, Cell, 11:127-138) and are two of the most prominent proteins that become cross-linked by ultraviolet light to heterogeneous nuclear RNA (hnRNA) in vivo. Studies are described here on the characterization of the C proteins in vertebrate cells using monoclonal and polyclonal antibodies. Monoclonal antibodies to genuine RNP proteins, including the C proteins, were obtained by immunizing mice with purified complexes of poly(A)+ hnRNA and poly(A)+ mRNA with their contacting proteins in vivo obtained by ultraviolet cross-linking the complexes in intact cells (Dreyfuss, G., Y.D. Choi, and S.A. Adam, 1984, Mol. Cell. Biol., 4:1104-1114). One of the monoclonal antibodies identified the C proteins in widely divergent species ranging from human to lizard. In all species examined, there were two C proteins in the molecular weight range of from 39,000 to 42,000 for C1, and from 40,000 to 45,000 for C2. The two C proteins were found to be highly related to each other; they were recognized by the same monoclonal antibodies and antibodies raised against purified C1 reacted also with C2. In avian, rodent, and human cells the C proteins were phosphorylated and were in contact with hnRNA in vivo. Immunofluorescence microscopy demonstrated that the C proteins are segregated to the nucleus. Within the nucleus the C proteins were not found in nucleoli and were not associated with chromatin as seen in cells in prophase. These findings demonstrate that C proteins with similar characteristics to those in humans are ubiquitous components of hnRNPs in vertebrates.
Animals↗
Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.
Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 (38K) become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared. Immunofluorescence microscopy shows mostly cytoplasmic and some nuclear staining. These observations demonstrate that commonly used inhibitors of transcription affect the physical state of messenger ribonucleoproteins in vivo.
Camptothecin↗
Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.
Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.
Animals↗