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PubMed2003

In vivo association of the stability control protein alphaCP with actively translating mRNAs.

Posttranscriptional controls play a major role in eucaryotic gene expression. These controls are mediated by sequence-specific interactions of cis-acting determinants in target mRNAs with one or more protein factors. The positioning of a subset of these mRNA-protein (RNP) complexes within the 3' untranslated region (3' UTR) may allow them to remain associated with the mRNA during active translation. Robust expression of human alpha-globin mRNA during erythroid differentiation has been linked to formation of a binary complex between a KH-domain protein, alphaCP, and a 3' UTR C-rich motif. Detection of this "alpha-complex" has been limited to in vitro studies, and the functional state of the alpha-globin mRNA targeted by alphaCP has not been defined. In the present study we demonstrate that a significant fraction of alphaCP is associated with polysomal mRNA. Targeted analysis of the polysomal RNP complexes revealed that alphaCP is specifically bound to actively translating alpha-globin mRNA. The bound alphaCP is restricted to the poly(C)-rich 3' UTR motif and is dislodged when ribosomes are allowed to enter this region. These data validate the general importance of the 3' UTR as a sheltered site for RNP complexes and support a specific model in which the stabilizing function of alphaCP is mediated on actively translating target mRNAs.

3' Untranslated Regions↗

PubMed1980

Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes.

Antibodies were raised in chickens against heterogeneous nuclear RNA (hnRNA)-binding proteins from 30S ribonucleoprotein (RNP) complexes of mouse Taper hepatoma ascites cell nuclei. The antibody preparations were characterized for immunological specificity and purity by double-diffusion gels, binding to specific bands in SDS polyacrylamide gels, and crossed immunoelectrophoresis. Antibodies raised against either whole 30S RNP complexes or purified RNP core proteins had a strong selective affinity for the four 34,000- to 40,000-dalton polypeptides which comprise the major structural proteins of hnRNP. The intracellular distribution of 30S RNP antigens in mouse ascites cells was determined by indirect immunofluorescence microsacopy. In interphase cells immunofluorescent sites were restricted to the nucleus, and nucleoli were free of fluorescence. The chicken anti-mouse-RNP antibodies were also able to react with cells from many different vertebrate species, showing a similar nucleus-restricted localization of the reacting sites. The antibodies also bound chick 30S RNP-proteins and reacted with the nuclei of chick cells. An exception to this was the failure of the antibody to bind to adult chick erythrocytes, suggesting that these major hnRNA binding proteins may be found only in nuclei capable of RNA synthesis.

Animals↗

PubMed1995

Ribonucleoprotein complex formation by the human hepatitis B virus polymerase.

Human hepatitis B virus (HBV) polymerase (pol or RT), when expressed in Xenopus oocytes upon injection of synthetic minimal pol RNA (RT RNA), assembles into a higher molecular weight complex with the characteristics of a ribonuclear protein (RNP) complex. In vitro RNA competition binding data suggest that RT RNA is preferentially packaged into this complex even though it lacks the authentic viral encapsidation signal, epsilon, and viral capsid protein sequences. Consistent with this finding, the in vitro polymerase reaction performed in pol-expressing oocyte extracts generates primarily HBV-specific DNAs even when the pol template is challenged with a coinjected non-HBV competitor RNA. These results suggest that interaction between pol and its cognate RNA can be mediated by sequences other than the known packaging elements. We speculate that HBV RNP complexes containing at least polymerase and viral RNA may play a role in viral nucleocapsid assembly and may help to segregate HBV reverse transcription from the cellular milieu in vivo.

Animals↗

PubMed1997

Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon.

Previous experiments using human teratocarcinoma cells indicated that p40, the protein encoded by the first open reading frame (ORF) of the human LINE-1 (L1Hs) retrotransposon, occurs in a large cytoplasmic ribonucleoprotein complex in direct association with L1Hs RNA(s), the p40 RNP complex. We have now investigated the interaction between partially purified p40 and L1Hs RNA in vitro using an RNA binding assay dependent on co-immunoprecipitation of p40 and bound RNA. These experiments identified two p40 binding sites on the full-length sense strand L1Hs RNA. Both sites are in the second ORF of the 6000 nt RNA: site A between residues 1999 and 2039 and site B between residues 4839 and 4875. The two RNA segments share homologous regions. Experiments involving UV cross-linking followed by immunoprecipitation indicate that p40 in the in vitro complex is directly associated with L1Hs RNA, as it is in the p40 RNP complex found in teratocarcinoma cells. Binding and competition experiments demonstrate that p40 binds to single-stranded RNA containing a p40 binding site, but not to single-stranded or double-stranded DNA, double-stranded RNA or a DNA-RNA hybrid containing a binding site sequence. Thus, p40 appears to be a sequence-specific, single-strand RNA binding protein.

Amino Acid Sequence↗

PubMed1996

Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP.

Animals↗

PubMed1997

Identification of AUF1 (heterogeneous nuclear ribonucleoprotein D) as a component of the alpha-globin mRNA stability complex.

mRNA turnover is an important regulatory component of gene expression and is significantly influenced by ribonucleoprotein (RNP) complexes which form on the mRNA. Studies of human alpha-globin mRNA stability have identified a specific RNP complex (alpha-complex) which forms on the 3' untranslated region (3'UTR) of the mRNA and appears to regulate the erythrocyte-specific accumulation of alpha-globin mRNA. One of the protein activities in this multiprotein complex is a poly(C)-binding activity which consists of two proteins, alphaCP1 and alphaCP2. Neither of these proteins, individually or as a pair, can bind the alpha-globin 3'UTR unless they are complexed with the remaining non-poly(C) binding proteins of the alpha-complex. With the yeast two-hybrid screen, a second alpha-complex protein was identified. This protein is a member of the previously identified A+U-rich (ARE) binding/degradation factor (AUF1) family of proteins, which are also known as the heterogeneous nuclear RNP (hnRNP) D proteins. We refer to these proteins as AUF1/hnRNP-D. Thus, a protein implicated in ARE-mediated mRNA decay is also an integral component of the mRNA stabilizing alpha-complex. The interaction of AUF1/hnRNP-D is more efficient with alphaCP1 relative to alphaCP2 both in vitro and in vivo, suggesting that the alpha-complex might be dynamic rather than a fixed complex. AUF1/hnRNP-D could, therefore, be a general mRNA turnover factor involved in both stabilization and decay of mRNA.

DNA-Binding Proteins↗

PubMed1997

Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids.

Assembly of hepadnaviruses depends on the formation of a ribonucleoprotein (RNP) complex comprising the viral polymerase polypeptide and an RNA segment, epsilon, present on pregenomic RNA. This interaction, in turn, activates the reverse transcription reaction, which is primed by a tyrosine residue on the polymerase. We have shown recently that the formation of this RNP complex in an avian hepadnavirus, the duck hepatitis B virus, depends on cellular factors that include the heat shock protein 90 (Hsp90). We now report that RNP formation also requires ATP hydrolysis and the function of p23, a recently identified chaperone partner for Hsp90. Furthermore, we also provide evidence that the chaperone complex is incorporated into the viral nucleocapsids in a polymerase-dependent reaction. Based on these findings, we propose a model for hepadnavirus assembly and priming of viral DNA synthesis where a dynamic, energy-driven process, mediated by a multi-component chaperone complex consisting of Hsp90, p23 and, potentially, additional factors, maintains the reverse transcriptase in a specific conformation that is competent for RNA packaging and protein priming of viral DNA synthesis.

Adenosine Triphosphate↗

PubMed2001

Annealing of RNA editing substrates facilitated by guide RNA-binding protein gBP21.

RNA editing within the mitochondria of African trypanosomes is characterized by the insertion and deletion of uridylate residues into otherwise incomplete primary transcripts. The reaction takes place in a high molecular mass ribonucleoprotein (RNP) complex of uncertain composition. Furthermore, factors that interact with the RNP complex during the reaction are by and large unknown. Here we present evidence for an editing-related biochemical activity of the gRNA-binding protein gBP21. Using recombinant gBP21 preparations, we show that the protein stimulates the annealing of gRNAs to cognate pre-mRNAs in vitro. This represents the presumed first step of the editing reaction. Kinetic data establish an enhancement of the second order rate constant for the gRNA- pre-mRNA interaction. gBP21-mediated annealing is not exclusive for RNA editing substrates since complementary RNAs, unrelated to the editing process, can also be hybridized. The gBP21-dependent RNA annealing activity was identified in mitochondrial extracts of trypanosomes and can be inhibited by immunoprecipitation of the polypeptide. The data suggest a factor-like contribution of gBP21 to the RNA editing process by accelerating the rate of gRNA-pre-mRNA anchor formation.

Animals↗

PubMed1998

The specificity of rabies virus RNA encapsidation by nucleoprotein.

Rabies virus nucleoprotein (N) encapsidates negative-strand genomic RNA in vivo, and this RNA-N complex, together with the nominal viral phosphoprotein (P) and RNA polymerase (L), forms the active cytoplasmic ribonucleoprotein (RNP) complex in virus-infected cells and the RNP core in virus particles. The RNP complex is capable of initiating viral RNA transcription and replication in vivo and in vitro. To obtain insight into the events leading to the formation of the RNA-N complex, we have investigated the interaction between rabies virus N and the positive-strand leader RNA transcript. Binding studies revealed that recombinant N binds preferentially to rabies virus leader RNA and that N binding to leader RNA was 5 to 10 times stronger than to nonleader RNA. Encapsidation of leader RNA by N could be competetively inhibited by unlabeled leader RNA but not by nonleader RNA. Furthermore, N protein encapsidation of nonleader RNA but not the leader RNA was inhibited when P was simultaneously added into the encapsidation reaction, indicating that P helps confer the specificity of leader RNA encapsidation by N. The initiation signal for leader RNA encapsidation by N has been mapped to nucleotides 20-30 of the RNA sequence which is A rich. Studies with N-deletion mutants indicate that the intact N is required to encapsidate RNA, since deletion of amino acid residues from either the N- or the C-terminus of N abolishes the ability of N to encapsidate leader RNA.

Animals↗

PubMed1988

Interaction between anti-influenza viral polymerase antibodies and RNP particles using the in vitro transcription process and an immunogold labelling technique.

Immunogold labelling and in vitro transcription of influenza virus vRNA have been used to analyse the interaction of anti-influenza polymerase antibodies with influenza-ribonucleoprotein (RNP) complexes. The polymerase proteins (P proteins) were localized exclusively at one end of the RNP segments. In the course of transcription the amount of P protein decreased significantly. The in vitro transcriptase activity y of influenza A virus RNP complexes in the presence of anti-polymerase antibodies to the strain A/PR/8/34 was inhibited by 60%. In contrast, RNP transcriptase activity of influenza B virus was not inhibited by these antibodies.

Animals↗

PubMed1991

2'-O-alkyloligoribonucleotides, synthesis and applications in molecular biology.

Oligo(2'-O-alkylribonucleotides) have been synthesized in which alkyl is methyl, allyl and butyl. The various phosphoramidite monomers of 2'-O-alkyl uridine, cytidine, adenosine, guanosine, inosine and 2,6-diaminopurine riboside have been synthesized from a minimum of key intermediates. Extra protection of the lactam function in uracil and hypoxanthine proves useful. The high stability of the oligomers combined with incorporation of non-radioactive reporter groups such as fluorophores, biotin and 2,4-dinitrophenylamino (DNP) moities renders them as excellent antisense tools for studying RNA processing, for locating and visualising RNA and RNP complexes in cells, for examining splicing complexes by electron microscopy and for the affinity chromatography of RNA or RNP complexes.

Antisense Elements (Genetics)↗

PubMed1995

[The small Alu-like RNA from the A-431 cell line specifically regulates the activity of the RNA polymerase III from human placental nuclei].

The influence of small Alu-like RNA, isolated from specific RNP complexes (alpha-RNP), on the activity of RNA polymerase III in cell-free system has been studied. The RNAs transcribed in vitro from Alu-DNA template (BLUR, 8) were isolated and subjected to polyacrylamide gel electrophoresis. A specific stimulation of RNA polymerase III activity by alpha-RNA was demonstrated.

Amantadine↗

PubMed2000

The 72/74-kDa polypeptides of the 70-110 S large heterogeneous nuclear ribonucleoprotein complex (LH-nRNP) represent a discrete subset of the hnRNP M protein family.

Pre-mRNA processing in eukaryotes is thought to take place on a multitude of nuclear ribonucleoprotein (RNP) complexes, the most abundant of them being the heterogeneous nuclear (hn) RNP complexes. The identification in mammalian nuclear extracts of a novel, less-abundant 70-110 S heterogeneous RNP, named large heterogeneous nuclear RNP (LH-nRNP), has previously been reported by Aidinis, Sekeris and Guialis (1995) Nucleic Acids Res. 23, 2742-2753. The structural composition of the LH-nRNP complex has been determined following the production of polyclonal antibodies against the major protein constituents of the complex, the pair of the 72/74-kDa polypeptides. In the present study evidence is shown to prove that the 72/74-kDa proteins are members of the hnRNP M protein family, hereafter referred to as 72/74(M) polypeptides. The extensive application of two-dimensional gel electrophoresis, combined with specific immunoprecipitation and immunoblotting assays, has allowed the assignment of the 72/74(M) proteins to a subset of the hnRNP M family, characteristic of the presence of the LH-nRNP complex and distinct from the hnRNP-associated M1-M4 components. Moreover, the immunoselection of the LH-nRNP complex from [(32)P]orthophosphate-labelled HeLa cells, with the parallel application of UV irradiation, has permitted the identification of the 72/74(M) polypeptides as the sole protein constituents of the complex in direct contact with the RNA. It is proposed that LH-nRNP constitutes a discrete subset of hnRNP complexes, having a possible role in establishing specific interactions between hnRNP and nuclear-matrix protein components.

Amino Acid Sequence↗

PubMed1987

Assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein.

The influenza A virus nucleoprotein previously expressed in Escherichia coli after fusion to 32 heterologous amino acids has now been purified and tested for its ability to form complexes with RNA in vitro. By using a simple filter binding assay, we show that ribonucleoprotein (RNP) complexes form readily with single-stranded RNA of viral or nonviral origin but not with double-stranded RNA. The RNP complexes formed were similar to authentic influenza virus RNPs in appearance under the electron microscope, in buoyant density in gradients of cesium chloride, and in sensitivities to pancreatic ribonuclease, to chaotropic reagents, and to high salt. We conclude that nucleoprotein synthesized in E. coli has all the properties required for correct assembly into ribonucleoprotein.

Binding, Competitive↗

PubMed1997

Poly (rC) binding protein 2 forms a ternary complex with the 5'-terminal sequences of poliovirus RNA and the viral 3CD proteinase.

Poly(rC) binding protein 2 (PCBP2) forms a specific ribonucleoprotein (RNP) complex with the 5'-terminal sequences of poliovirus genomic RNA, as determined by electrophoretic mobility shift assay. Mutational analysis showed that binding requires the wild-type nucleotide sequence at positions 20-25. This sequence is predicted to localize to a specific stem-loop within a cloverleaf-like secondary structure element at the 5'-terminus of the viral RNA. Addition of purified poliovirus 3CD to the PCBP2/RNA binding reaction results in the formation of a ternary complex, whose electrophoretic mobility is further retarded. These properties are consistent with those described for the unidentified cellular protein in the RNP complex described by Andino et al. (Andino R, Rieckhof GE, Achacoso PL, Baltimore D, 1993, EMBO J 12:3587-3598). Dicistronic RNAs containing mutations in the 5' cloverleaf-like structure of poliovirus that abate PCBP2 binding show a decrease in RNA replication and translation of gene products directed by the poliovirus 5' noncoding region in vitro, suggesting that the interaction of PCBP2 with these sequences performs a dual role in the virus life cycle by facilitating both viral protein synthesis and initiation of viral RNA synthesis.

3C Viral Proteases↗

PubMed2003

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components.

A stable 100-kD complex from mitochondria of Leishmania tarentolae containing two RNA-binding proteins, Ltp26 and Ltp28, was identified by cross-linking to unpaired 4-thiouridine nucleotides in a partially duplex RNA substrate. The genes were cloned and expressed and the complex was reconstituted from recombinant proteins in the absence of RNA or additional factors. The Ltp26 and Ltp28 proteins are homologs of gBP27 and gBP29 from Crithidia fasciculata and gBP25 and gBP21 from Trypanosoma brucei, respectively. The purified Ltp26/Ltp28 complex, the individual recombinant proteins, and the reconstituted complex are each capable of catalyzing the annealing of complementary RNAs, as was previously shown for gBP21 from T. brucei. A high-molecular-weight RNP complex consisting of the Ltp26/Ltp28 complex and several 55-60-kD proteins together with guide RNA could be purified from mitochondrial extract of L. tarentolae transfected with Ltp28-TAP. This complex also interacted in a less stable manner with the RNA ligase-containing L-complex and with the 3' TUTase. The Ltp26/Ltp28 RNP complex is a candidate for catalyzing the annealing of guide RNA and pre-edited mRNA in the initial step of RNA editing.

Amino Acid Sequence↗

PubMed1993

Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing.

A cis-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in control of the relative levels of spliced and unspliced viral mRNAs. Insertion of the NRS into the intron of an adenovirus pre-mRNA resulted in inhibition of splicing in vitro before the first cleavage step. Analyses of spliceosome assembly with this substrate showed that it formed large RNP complexes that did not migrate like mature spliceosomes on native gels. Affinity selection of the RNP complexes formed on NRS-containing pre-mRNAs showed an association with U11 and U12 snRNPs, as well as with the spliceosomal snRNPs. Immunoprecipitation with antisera specific for U1 and U2 snRNPS showed binding of both snRNPs to NRS RNA. A 7-nucleotide missense mutation in the NRS that prevented binding of U11 and U12 snRNPs impaired NRS activity in vivo, suggesting a functional role for U11 and U12 snRNPs in the inhibition of splicing mediated by the RSV NRS RNA.

Adenoviridae↗

PubMed1976

Detection of mRNA sequences in nuclear 30S ribonucleoprotein subcomplexes.

RNA from nuclear 30S ribonucleoprotein (RNP) complexes of mouse ascites cells has been shows to contain sequences homologous to poly(A) + mRNA by its ability to hybridize with complementary DNA prepared from poly(A) + mRNA template. Analysis of the hybridization kinetics of poly(A) + mRNA with its own complementary DNA revealed several abundancy classes. The total complexity of poly(A) + mRNA from ascites cells was estimated to be approximately 30,000 sequences of average molecular weight (6 X 10(5)). When the hybridization reaction of 30S RNP-RNA with mRNA-specific cDNA was compared to the homologous reaction the majority, and most probably all, of the poly(A) + mRNA sequences were found to be present in the RNA. The kinetics of hybridization suggest that 10-15% of the RNA in this RNP complex is homologous to poly(A) + mRNA. The 30S RNP subcomplexes therefore contain nuclear poly(A) + mRNA sequences as well as the bulk of heterogeneous RNA.

Base Sequence↗