RESUMO
Every RNA added to an in vitro polyadenylation extract became stably associated with both the heterogeneous nuclear ribonucleoprotein (hnRNP) A and C proteins, as assayed by immunoprecipitation analysis using specific monoclonal antibodies. UV-cross-linking analysis, however, which assays the specific spatial relationship of certain amino acids and RNA bases, indicated that the hnRNP C proteins, but not the A proteins, were associated with downstream sequences of the simian virus 40 late polyadenylation signal in a sequence-mediated manner. A tract of five consecutive uridylate residues was required for this interaction. The insertion of a five-base U tract into a pGEM4 polylinker-derived transcript was sufficient to direct sequence-specific cross-linking of the C proteins to RNA. Finally, the five-base uridylate tract restored efficient in vitro processing to several independent poly(A) signals in which it substituted for downstream element sequences. The role of the downstream element in polyadenylation efficiency, therefore, may be mediated by sequence-directed alignment or phasing of an hnRNP complex.
Assuntos
Poli A/genética , RNA Nuclear Heterogêneo/metabolismo , RNA/genética , Ribonucleoproteínas/metabolismo , Uracila , Uridina Trifosfato/metabolismo , Sequência de Bases , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Células HeLa/metabolismo , Células HeLa/efeitos da radiação , Ribonucleoproteínas Nucleares Heterogêneas Grupo C , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Ligação Proteica , RNA Mensageiro , Mapeamento por Restrição , Transcrição Gênica , Raios UltravioletaRESUMO
We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.
Assuntos
Poli A/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico , Vírus 40 dos Símios/genética , Sequência de Bases , Sítios de Ligação/genética , Dados de Sequência Molecular , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/isolamento & purificaçãoRESUMO
The heterogeneous nuclear ribonucleoprotein C1 and C2 proteins were preferentially cross-linked by treatment with UV light in nuclear extracts to RNAs containing six different polyadenylation signals. The domain required for the interaction was located downstream of the poly(A) cleavage site, since deletion of this segment from several polyadenylation substrate RNAs greatly reduced cross-linking efficiency. In addition, RNAs containing only downstream sequences were efficiently cross-linked to C proteins, while fully processed, polyadenylated RNAs were not. Analysis of mutated variants of the simian virus 40 late polyadenylation signal showed that uridylate-rich sequences located in the region between 30 and 55 nucleotides downstream of the cleavage site were required for efficient cross-linking of C proteins. This downstream domain of the simian virus 40 late poly(A) addition signal has been shown to influence the efficiency of the polyadenylation reaction. However, there was not a strict correlation between cross-linking of C proteins and the efficiency of polyadenylation.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo C , Fosfoproteínas/metabolismo , Poli A/metabolismo , RNA Nuclear Heterogêneo/fisiologia , Ribonucleoproteínas/fisiologia , Sequência de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas , Ribonucleoproteínas Nucleares Heterogêneas , Técnicas In Vitro , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Vírus 40 dos Símios/genéticaRESUMO
The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked to the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.
Assuntos
Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/biossíntese , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenoviridae/metabolismo , Sequência de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas , Modelos Genéticos , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Vírus 40 dos Símios/metabolismo , Relação Estrutura-Atividade , Raios Ultravioleta , Fatores de Poliadenilação e Clivagem de mRNARESUMO
A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.
Assuntos
Proteínas Nucleares/fisiologia , Poli A/metabolismo , Processamento Pós-Transcricional do RNA , Ribonucleoproteínas/fisiologia , Sequência de Bases , Sistema Livre de Células , Células HeLa , Humanos , Técnicas In Vitro , Peso Molecular , Ribonucleoproteínas/efeitos da radiação , Ribonucleoproteínas/ultraestrutura , Raios UltravioletaRESUMO
We have developed an in vitro system which faithfully reproduces several aspects of general mRNA stability. Poly(A)- RNAs were rapidly and efficiently degraded in this system with no detectable intermediates by a highly processive 3'-to-5' exonuclease activity. The addition of a poly(A) tail of at least 30 bases, or a 3' histone stem-loop element, specifically stabilized these transcripts. Stabilization by poly(A) required the interaction of proteins with the poly(A) tail but did not apparently require a 3' OH or interaction with the 5' cap structure. Finally, movement of the poly(A) tract internal to the 3' end caused a loss of its ability to stabilize transcripts incubated in the system but did not affect its ability to interact with poly(A) binding proteins. The requirement for the poly(A) tail to be proximal to the 3' end indicates that it mediates RNA stability by blocking the assembly, but not the action, of an exonuclease involved in RNA degradation in vitro.
Assuntos
Exorribonucleases/metabolismo , Poli A/metabolismo , RNA Mensageiro/metabolismo , Genes myc/genética , Humanos , Proteínas de Ligação a Poli(A) , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Vírus 40 dos Símios/genéticaRESUMO
Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H' was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H', and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.
Assuntos
Linfócitos B/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Primers do DNA/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Memória Imunológica , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Peso Molecular , Mutação , Plasmócitos/imunologia , Plasmócitos/metabolismo , Subunidades Proteicas , Precursores de RNA/genética , Ribonucleoproteínas/genética , Transfecção , Fatores de Poliadenilação e Clivagem de mRNARESUMO
The examination of posttranscriptional regulation of mRNA in mammalian cells is critical to discovering the role that mRNA plays in the initiation and maintenance of cellular processes. The complexity of the system defies a holistic approach and, therefore, we have devised an in vitro mRNA turnover assay that enables us to elucidate the factors involved in mRNA deadenylation and degradation. Our system, using an S100 HeLa extract and in vitro transcribed RNAs, accurately mimics the end products of mRNA turnover, which have been previously described using in vivo studies and, in addition, allows for the detailed study of factors that may play a role in regulated deadenylation and degradation. Another important aspect of our system is the ease with which it can be manipulated. We can provide any synthetic RNA molecule to the assay to test for specific sequence activity. Furthermore, the results are clear and accurately interpretable. We have demonstrated that our in vitro system accurately deadenylates and decays a capped and polyadenylated RNA molecule in a processive manner without nonspecific nuclease activity. Finally, we have demonstrated regulated instability in vitro using the AU-rich elements (AREs) from tumor necrosis factor-alpha (TNF-alpha) and granulocyte macrophage colony stimulating factor (GM-CSF) embedded within the RNA molecule. The presence of the AREs increased the deadenylation and the decay rates seen in vivo. We feel that this system can be expanded and adapted to examine a variety of mRNA regulatory events in mammalian cells.
Assuntos
Estabilidade de RNA , RNA Mensageiro/química , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Processamento Pós-Transcricional do RNA , Análise de Sequência de RNARESUMO
A 64 kd protein was shown to bind to RNAs that contain functional polyadenylation signals by a UV cross-linking procedure in which label was transferred from RNA substrate to protein in cell-free polyadenylation extracts. The 64 kd nuclear protein bound specifically to three different substrates (adenovirus type 5 L3, SV40 early, and SV40 late polyadenylation domains), as determined by competition experiments and partial protease analysis. Deleted derivatives of the SV40 late substrate that retained the sequence 5'-CUGCAAUAAACAAGUU-3' were able to bind the 64 kd polypeptide. This sequence contains the canonical AAUAAA element that has been shown to be indispensable for polyadenylation. A single nucleotide change, converting AAUAAA to AAGAAA, prevented binding of the 64 kd moiety. The 64 kd protein was shown to be distinct from poly(A) polymerase by biochemical fractionation.
Assuntos
Adenoviridae/genética , Proteínas Nucleares/metabolismo , Poli A/metabolismo , RNA Viral/metabolismo , Vírus 40 dos Símios/genética , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Mutação , Fotoquímica , Polinucleotídeo Adenililtransferase/metabolismo , Moldes GenéticosRESUMO
Successful cloning with cDNA expression libraries involves interaction of ligand molecules (probes) with expressed fusion proteins of interest. So far those ligands leading to successful results fall into three classes: (i) antibodies, (ii) protein ligands that interact with the protein of interest, and (iii) DNA sequences recognized by transcription factors. We have previously identified a 50-kDa protein (called DSEF-1) which interacts with a functionally important 14-base G-rich RNA sequence located downstream of the simian virus 40 late polyadenylation signal. By using small RNAs containing the DSEF-1 binding site as probes, a cDNA clone was isolated whose gene product interacted in a sequence-specific fashion with the DSEF-1 binding site. This RNA binding protein contains three potential RNA recognition motifs. We present here a procedure to obtain cDNA clones of RNA binding proteins using recognition site probes.
Assuntos
DNA Viral/genética , Biblioteca Gênica , Proteínas de Ligação a RNA/genética , Bacteriófago lambda/genética , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Colódio , DNA Viral/isolamento & purificação , Células HeLa , Humanos , Membranas Artificiais , Dados de Sequência Molecular , Sondas RNA , Proteínas de Ligação a RNA/metabolismo , Sensibilidade e EspecificidadeRESUMO
Computer predictions identified similarities to a 14-base G-rich element in numerous mRNAs at a variety of locations. A Northwestern screening strategy was used to obtain a cDNA clone from a HeLa cell library using the G-rich RNA element as a probe. A cellular protein (called GRSF-1), which was encoded by this cDNA, binds RNAs containing the G-rich element. GRSF-1 was distinct from DSEF-1, a nuclear protein we have previously identified that interacts with the G-rich element, based on differences in molecular weight and partial peptide maps, as well as the lack of cross-reactivity with GRSF-1 specific monoclonal antibodies. Using indirect immunofluorescence microscopy, we localized GRSF-1 to the cytoplasm. In vivo UV cross-linking further demonstrated that GRSF-1 was bound to poly(A)+ mRNA in living human cells. Western blot analysis revealed four cytoplasmic proteins which expressed GRSF-1 specific epitopes. GRSF-1 contains three potential RNA recognition motifs and two auxiliary domains. Curiously, the domain organization of GRSF-1 is similar to the RNA binding proteins PUB1, ELAV, HuD, Hel-N1, mcs94-1 and RBP9.
Assuntos
Sequência Conservada , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Clonagem Molecular , DNA , Guanina/análise , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas de Ligação a Poli(A) , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Homologia de Sequência de AminoácidosRESUMO
We have previously identified a G-rich sequence (GRS) as an auxiliary downstream element (AUX DSE) which influences the processing efficiency of the SV40 late polyadenylation signal. We have now determined that sequences downstream of the core U-rich element (URE) form a fundamental part of mammalian polyadenylation signals. These novel AUX DSEs all influenced the efficiency of 3'-end processing in vitro by stabilizing the assembly of CstF on the core downstream URE. Three possible mechanisms by which AUX DSEs mediate efficient in vitro 3'-end processing have been explored. First, AUX DSEs can promote processing efficiency by maintaining the core elements in an unstructured domain which allows the general polyadenylation factors to efficiently assemble on the RNA substrate. Second, AUX DSEs can enhance processing by forming a stable structure which helps focus binding of CstF to the core downstream URE. Finally, the GRS element, but not the binding site for the bacteriophage R17 coat protein, can substitute for the auxiliary downstream region of the adenovirus L3 polyadenylation signal. This suggests that AUX DSE binding proteins may play an active role in stimulating 3'-end processing by stabilizing the association of CstF with the RNA substrate. AUX DSEs, therefore, serve as a integral part of the polyadenylation signal and can affect signal strength and possibly regulation.
Assuntos
Proteínas do Capsídeo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Adenovírus Humanos/genética , Animais , Sítios de Ligação , Capsídeo/química , Extratos Celulares , Dependovirus/genética , Células HeLa , Humanos , Vírus do Tumor Mamário do Camundongo , Conformação de Ácido Nucleico , Precursores de RNA/genética , RNA Mensageiro/química , RNA Viral/química , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas Recombinantes de Fusão , Vírus 40 dos Símios/genética , Raios Ultravioleta , Fatores de Poliadenilação e Clivagem de mRNARESUMO
Autoantibodies reactive with specific nuclear and cytoplasmic small RNAs were identified by immunoprecipitation of HeLa cell RNA. Approximately 30% of antisera examined from patients with autoimmune disorders contained anti-RNA antibodies. Two previously undescribed specificities--anti U1 RNA and anti-initiator methionine tRNA--were identified. Anti-RNA antibodies were reactive with gel-purified species as well as with RNA synthesized in vitro using the SP-6 transcription system. Antigenic mapping using two sera specific for the human initiator methionine tRNA revealed separate epitopes, one of which is conserved in formyl-methionine initiator tRNA from Escherichia coli. RNA fragmentation studies further suggested that secondary or tertiary tRNA structure is required for antibody recognition. The mammalian U1 RNA specific antibodies did not precipitate small RNAs of yeast but were highly reactive with yeast ribosomal RNA, thus indicating a possible relationship between these RNA species. Results obtained with these antisera are discussed in terms of higher order structure of small RNA molecules as well as the role of nucleic acid antibodies in the autoimmune phenomenon.
Assuntos
Autoanticorpos/imunologia , RNA Nuclear Pequeno/imunologia , Aminoacil-RNA de Transferência/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Eletroforese em Gel de Poliacrilamida , Epitopos/análise , Células HeLa/análise , Humanos , Conformação de Ácido NucleicoRESUMO
Hepatic hydroxysteroid sulfotransferase-a (HST-a) gene expression was examined in young male (age 22-26 days) and female rats (age 22-30 days), and in older male (age 42-45 days) and female (age 49-55 days) rats. Northern and slot blot analyses of poly(A)+RNA revealed that HST-a was differentially expressed with respect to both age and gender with female rats expressing higher levels of HST-a in both age groups. Hepatic HST-a mRNA levels were approximately 4 to 6-fold higher in females compared to males in both age groups examined. HST-a expression increased with age in both male and female rats. HST-a expression was approximately 8 to 10-fold higher in 42-45 day old males relative to 22-26 day old males. HST-a mRNA levels were approximately 3 to 7-fold higher in 49-55 day old females relative to females in the 22-30 day age group. These data suggest that HST-a gene expression is transcriptionally controlled and that HST-a regulation is subject to hormonal and developmental modulation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fígado/crescimento & desenvolvimento , Sulfotransferases , Sulfurtransferases/genética , Envelhecimento , Animais , Sequência de Bases , Northern Blotting , Feminino , Fígado/enzimologia , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Poli A/genética , Poli A/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Endogâmicos , Caracteres SexuaisRESUMO
The hydroxysteroid sulfotransferases (HSTs) are phase II drug metabolizing enzymes which play a role in xenobiotic detoxication, carcinogen activation, and intratissue hormone modulation. Rat liver contains three principal HST enzymes. Of these, HST-a is most abundant in female rat liver. The phase I drug metabolizing enzyme cytochrome P450 1A1 (CYP1A1) provides a major pathway for polycyclic aromatic hydrocarbon (PAH) carcinogen activation, whereas HST-a, through enzymatic sulfation, offers an alternative pathway for PAH carcinogen activation. Transcription of the rat hepatic CYP1A1 gene is induced in response to treatment with PAH carcinogen 3-methylcholanthrene (3-MC). In view of the potential importance of enzymatic sulfation to the complex process of PAH carcinogenesis, the effect of 3-MC on HST-a gene expression was investigated. Groups of prepubescent (aged approximately 22-30 days) and young adult female (aged approximately 69-84 days) Sprague-Dawley rats were injected ip with corn oil vehicle or with 3-MC (80 mg/kg). After fasting for 24 hr, rats were terminated and hepatic HST-a gene expression was analyzed using slot blot, Northern blot, and Western blot analyses. Initial Northern blot and slot blot analyses demonstrated a substantial suppression of rat hepatic HST-a mRNA in both prepubescent and young adult female rats. Subsequent Northern blot analyses on liver tissue isolated from female rats aged approximately 55 days confirmed CYP1A1 mRNA induction in 3-MC-treated animals and demonstrated a dose-dependent suppression of HST-a mRNA expression of approximately 25, 50, and 75% in rats treated with 10, 25, and 80 mg/kg 3-MC ip, respectively. In contrast, HST-a protein levels remained unaltered 24 hr after 3-MC treatment. A time-course study revealed that hepatic HST-a mRNA levels returned to control levels by 36 hr following 3-MC treatment. These results suggest that xenobiotics such as 3-MC modulate HST-a mRNA expression and that HST-a mRNA suppression after 3-MC treatment may occur at the level of transcription.
Assuntos
Expressão Gênica/efeitos dos fármacos , Fígado/enzimologia , Metilcolantreno/toxicidade , Sulfotransferases/genética , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sulfotransferases/biossínteseRESUMO
The pathways and machinery involved in the regulated turnover of mRNAs in mammalian cells are largely unknown. We have developed an in vitro system using HeLa cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that faithfully reproduces in vivo aspects of regulated mRNA turnover. RNA substrates for use in the system that contain a poly(A) tail precisely at their 3' end can be readily prepared using a ligation-polymerase chain reaction approach. The system also uses standard cytoplasmic S100 extracts that are activated through the sequestration of poly(A)-binding proteins by the addition of cold poly(A) RNA. On incubation in the system, the poly(A) tail is removed from RNA substrates by a sequence-specific deadenylase activity and the body of the transcript is ultimately degraded in the system with no apparent intermediates by an ATP-dependent ribonulceolytic activity. AU-rich destability elements can regulate the rates of both deadenylation and degradation in the system. This in vitro system, therefore, should allow the elucidation of pathways of mRNA turnover, identification of the cellular factors involved, and insights into the mechanisms that regulate the half-life of a mRNA.
Assuntos
Citoplasma/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Sistema Livre de Células/metabolismo , Células HeLa , Humanos , Processamento Pós-Transcricional do RNARESUMO
The New Jersey serotype of vesicular stomatitis virus (VSV-NJ) was found to synthesize a minus strand leader RNA of 44-46 bases long and a plus strand leader RNA of 47-50 bases long in infected cells. The minus strand leader RNA of VSV-NJ was found associated with the host cell La protein in infected cells by immunoprecipitation with antisera from patients with systemic lupus erythematosus. These results differ from those reported previously (J. Wilusz , M. G. Kurilla , and J. D. Keene (1983). Proc. Natl. Acad. Sci. USA 80, 5827-5831) for the similarly sized species of minus strand leader RNA made by the Indiana serotype of VSV (VSV-IND). Despite sequence differences between the 3' ends of the plus strand leader RNAs of the two serotypes, the plus strand leader RNA of VSV-NJ was found to have a pattern of La protein accumulation similar to that reported previously for the plus strand leader RNA of VSV-IND. These results provide additional support for a role for La protein in VSV replication and help further delineate the sequence requirements for La protein binding to VSV leader RNAs.
Assuntos
Antígenos/metabolismo , RNA/metabolismo , Ribonucleoproteínas , Fatores de Transcrição/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Vesiculovirus , Animais , Autoantígenos , Sequência de Bases , Linhagem Celular , Cricetinae , Rim , Sorotipagem , Especificidade da Espécie , Vírus da Estomatite Vesicular Indiana/classificação , Antígeno SS-BRESUMO
We have previously shown that the presence of a poly(A) tail blocks the activity of a highly efficient 3'-->5' exonuclease in HeLa extracts. Similar activities have been implicated in RNA turnover in vivo. It is not clear, however, what protects poly(A)-non-mRNAs from the action of this enzyme. A stem-loop structure located at the 3'-end of U11 RNA was required to protect this transcript from the exonuclease in vitro. Similar 3' stem-loop structures, or extensive base pairinginvolving the 3'-end, are present on all mature small stable RNAs. The placement of artificial stem-loop structures at the 3'-end also protected RNA substrates, suggesting that RNA structure alone is sufficient to block the initiation of the exonuclease. The placement of RNA structures at internal positions of substrate trans-cripts did not affect the activity of the exonuclease or lead to the accumulation of degradation intermediates. Pol III precursor transcripts contain short poly(U) tracts rather than structure at their 3'-ends. Terminal poly(U) tracts protected RNA substrates from the 3'-->5' exonuclease in a protein-dependent fashion. Although La protein is found associated with the terminal U tracts of pol III precursor transcripts both in vivo and in vitro, La protein was not required for poly(U) to protect RNA substrates from the 3'-->5' exonuclease. In summary, these data reveal a variety of ways RNAs have evolved to protect themselves from this exonuclease.
Assuntos
Regiões 3' não Traduzidas/química , Exodesoxirribonucleases/metabolismo , Conformação de Ácido Nucleico , Poli U , RNA/química , RNA/metabolismo , Sequência de Bases , Exodesoxirribonuclease V , Células HeLa , Humanos , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
The 3'-terminal regions of the genomes of vesicular stomatitis virus obtained from two long-term, independently initiated persistent infections of L cells were found to contain several sequence mutations. In contrast to the hypermutability displayed in the 5'-terminal regions of the genomes of viruses obtained from persistent infections of baby hamster kidney (BHK) cells (P. J. O'Hara, F. M. Horodyski, S. T. Nichol, and J. J. Holland, J. Virol. 49, 793-798, 1984), no 5' mutations were detected in viruses from L-cell carrier lines. The absence of detectable defective interfering (DI) particles in the L-cell carrier cultures may account for this difference. Plus-strand leader RNA made by the viruses from persistently infected L cells failed to accumulate from 5 to 8 hr postinfection unlike the accumulation noted for the leader RNA generated by wild-type VSV. Minus-strand leader RNA, on the other hand, accumulated at a similar or increased rate compared to wild type. The relationship of these observations to the processes of host shutoff, viral transcription, and replication are discussed.