RESUMEN
TRIT1 is a highly conserved tRNA isopentenyl transferase that modifies a subset of tRNAs in human cells and is a candidate tumor suppressor in lung cancer in certain ethnic populations. The yeast homologue, Mod5, has similar tRNA-modifying functions in the cytoplasm and is required for the transcriptional silencing activity of RNA polymerase II promoters near tRNA genes in the nucleus, a phenomenon termed tRNA gene mediated (tgm) silencing. Furthermore, Mod5 can fold into amyloid fibers in vitro and in vivo, which confers resistance to certain fungicides in yeast. Since TRIT1 complements both tRNA modifying and tgm-silencing activities in yeast where the Mod5 gene has been deleted, it seemed possible that TRIT1 might also have amyloid-forming capabilities. Here we show that TRIT1, like Mod5, directly binds to tRNAs that are both substrate and non-substrates for modification with similar affinity, and to an unstructured, non-tRNA. Binding appears to involve distinct protein-RNA multimers which decrease in electrophoretic mobility as the protein to RNA ratio increases. Furthermore, we characterize TRIT1 as a novel human amyloid fiber forming protein. We discuss these data in light of TRIT1's functional roles and possible implications for disease.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Amiloide/biosíntesis , ARN de Transferencia/metabolismo , Transferasas Alquil y Aril/química , Secuencia de Aminoácidos , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Mod5 is the yeast tRNA isopentenyl transferase, an enzyme that is conserved from bacteria to humans. Mod5 is primarily cytoplasmic where it modifies the A37 position of a few tRNAs, and the yeast enzyme has been shown capable of forming heritable, amyloid-like aggregates that confer a selective advantage in the presence of specific antifungal agents. A subpopulation of Mod5 is also found associated with nuclear tRNA genes, where it contributes tRNA-gene mediated (tgm) silencing of local transcription by RNA polymerase II. The tgm-silencing function of Mod5 has been observed in yeast and a Mod5-deletion in yeast can be complemented by the plant and human tRNA isopentenyl transferases, but not the bacterial enzymes, possibly due to the lack of an extended C-terminal domain found in eukaryotes. In light of this additional nuclear role for Mod5 we discuss the proposed role of the human homologue of Mod5, TRIT1, as a tumor suppressor protein.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Núcleo Celular/enzimología , Citoplasma/enzimología , Neoplasias/genética , Neoplasias/metabolismo , Transferasas Alquil y Aril/genética , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Genes Supresores de Tumor , Humanos , Datos de Secuencia Molecular , Pliegue de Proteína , Homología de Secuencia de AminoácidoAsunto(s)
Núcleo Celular/enzimología , Endorribonucleasas/metabolismo , ARN Catalítico/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Bases , Endorribonucleasas/química , Endorribonucleasas/genética , Conformación de Ácido Nucleico , Filogenia , ARN Catalítico/química , ARN Catalítico/genética , ARN de Hongos/química , ARN de Hongos/genética , Ribonucleasa P , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the three-hybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme.
Asunto(s)
Endorribonucleasas/metabolismo , ARN Catalítico/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Endorribonucleasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Precursores del ARN/metabolismo , ARN Catalítico/química , ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa P , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidad de la EspecieRESUMEN
RNA affinity tags would be very useful for the study of RNAs and ribonucleoproteins (RNPs) as a means for rapid detection, immobilization, and purification. To develop a new affinity tag, streptavidin-binding RNA ligands, termed "aptamers," were identified from a random RNA library using in vitro selection. Individual aptamers were classified into two groups based on common sequences, and representative members of the groups had sufficiently low dissociation constants to suggest they would be useful affinity tools. Binding of the aptamers to streptavidin was blocked by presaturation of the streptavidin with biotin, and biotin could be used to dissociate RNA/streptavidin complexes. To investigate the practicality of using the aptamer as an affinity tag, one of the higher affinity aptamers was inserted into RPR1 RNA, the large RNA subunit of RNase P. The aptamer-tagged RNase P could be specifically isolated using commercially available streptavidin-agarose and recovered in a catalytically active form when biotin was used as an eluting agent under mild conditions. The aptamer tag was also used to demonstrate that RNase P exists in a monomeric form, and is not tightly associated with RNase MRP, a closely related ribonucleoprotein enzyme. These results show that the streptavidin aptamers are potentially powerful tools for the study of RNAs or RNPs.
Asunto(s)
Marcadores de Afinidad , Endorribonucleasas/aislamiento & purificación , ARN Catalítico/aislamiento & purificación , ARN/metabolismo , Ribonucleoproteínas/metabolismo , Estreptavidina/metabolismo , Sitios de Unión , Evolución Molecular Dirigida , Ligandos , Modelos Moleculares , Conformación de Ácido Nucleico , ARN/aislamiento & purificación , Ribonucleasa P , Ribonucleoproteínas/aislamiento & purificaciónRESUMEN
Ribonuclease P is an ancient enzyme that cleaves pre-tRNAs to generate mature 5' ends. It contains an essential RNA subunit in Bacteria, Archaea, and Eukarya, but the degree to which the RNA subunit relies on proteins to supplement catalysis is highly variable. The eukaryotic nuclear holoenzyme has recently been found to contain almost twenty times the protein content of the bacterial enzymes, in addition to having split into at least two related enzymes with distinct substrate specificity. In this review, recent progress in understanding the molecular architecture and functions of nuclear forms of RNase P will be considered.
Asunto(s)
Endorribonucleasas/química , Endorribonucleasas/fisiología , Procesamiento Postranscripcional del ARN , ARN Catalítico/química , ARN Catalítico/fisiología , ARN de Transferencia/metabolismo , Animales , Evolución Biológica , Núcleo Celular/enzimología , Núcleo Celular/genética , Sustancias Macromoleculares , Conformación de Ácido Nucleico , Precursores del ARN/metabolismo , Ribonucleasa P , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Sephadex-binding RNA ligands (aptamers) were obtained through in vitro selection. They could be classified into two groups based on their consensus sequences and the aptamers from both groups showed strong binding to Sephadex G-100. One of the highest affinity aptamers, D8, was chosen for further characterization. Aptamer D8 bound to dextran B512, the soluble base material of Sephadex, but not to isomaltose, isomaltotriose and isomaltotetraose, suggesting that its optimal binding site might consist of more than four glucose residues linked via alpha-1,6 linkages. The aptamer was very specific to the Sephadex matrix and did not bind appreciably to other supporting matrices, such as Sepharose, Sephacryl, cellulose or pustulan. Using Sephadex G-100, the aptamer could be purified from a complex mixture of cellular RNA, giving an enrichment of at least 60 000-fold, compared with a non-specific control RNA. These RNA aptamers can be used as affinity tags for RNAs or RNA subunits of ribonucleoproteins to allow rapid purification from complex mixtures of RNA using only Sephadex.
Asunto(s)
Dextranos/química , ARN/química , ARN/aislamiento & purificación , Sefarosa/análogos & derivados , Resinas Acrílicas/química , Unión Competitiva , Secuencia de Carbohidratos , Celulosa/química , Células HeLa , Humanos , Ligandos , Sustancias Macromoleculares , Oligorribonucleótidos/química , Oligorribonucleótidos/aislamiento & purificación , Oligosacáridos/química , Polisacáridos/química , Sefarosa/química , Células Tumorales CultivadasRESUMEN
This unit provides thorough coverage of the most useful chemical and enzyme probes that can be used to examine RNA secondary and tertiary structure. Footprinting methods are presented using dimethyl sulfate, diethyl pyrocarbonate, ethylnitrosourea, kethoxal, CMCT, and nucleases. For chemical probes, both strand scission and primer extension detection protocols are included.
Asunto(s)
Bioquímica/métodos , Endonucleasas/metabolismo , Conformación de Ácido Nucleico/efectos de los fármacos , ARN/química , ARN/metabolismo , Aldehídos/química , Bacteriófago T4/enzimología , Emparejamiento Base , Butanonas , CME-Carbodiimida/análogos & derivados , CME-Carbodiimida/química , Dietil Pirocarbonato/química , Electroforesis , Etilnitrosourea/química , Indicadores y Reactivos , Radioisótopos de Fósforo , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Estándares de Referencia , Ésteres del Ácido Sulfúrico/químicaRESUMEN
In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.
Asunto(s)
Nucléolo Celular/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Silenciador del Gen , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación Puntual , ARN de Transferencia/biosíntesis , ARN de Transferencia/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transcripción Genética , Clonación Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidroliasas/genética , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Precursores del ARN/biosíntesis , Precursores del ARN/genética , ARN de Hongos/biosíntesis , ARN de Hongos/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P. Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined. Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives. However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results. Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme. Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor. Further studies showed that the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure. The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer. Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition.
Asunto(s)
Endorribonucleasas/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Catalítico/metabolismo , ARN de Transferencia de Tirosina/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Emparejamiento Base , Secuencia de Bases , Núcleo Celular/enzimología , Endorribonucleasas/antagonistas & inhibidores , Modelos Químicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Precursores del ARN/química , ARN Catalítico/antagonistas & inhibidores , ARN Bicatenario/metabolismo , ARN Ribosómico 5S/farmacología , ARN de Transferencia/farmacología , Ribonucleasa P , Saccharomyces cerevisiae/enzimología , Especificidad por SustratoRESUMEN
The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.
Asunto(s)
Endorribonucleasas/metabolismo , Células Eucariotas/enzimología , Organotiofosfatos/metabolismo , Precursores del ARN/metabolismo , ARN Catalítico/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Cadmio/farmacología , Catálisis/efectos de los fármacos , Núcleo Celular/enzimología , Núcleo Celular/genética , Dictyostelium/citología , Dictyostelium/enzimología , Dictyostelium/genética , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/genética , Células Eucariotas/citología , Células Eucariotas/metabolismo , Ingeniería Genética , Humanos , Hidrólisis/efectos de los fármacos , Magnesio/farmacología , Manganeso/farmacología , Modelos Químicos , Precursores del ARN/química , Precursores del ARN/genética , ARN Catalítico/antagonistas & inhibidores , ARN Catalítico/genética , ARN de Transferencia/genética , Ribonucleasa P , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Ribonuclease P is the enzyme responsible for removing the 5'-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is not known, however, which subunits participate directly in phosphodiester-bond hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation that in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphodiester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-Rp nonbridging oxygen of the scissile bond is replaced by sulfur. In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not involve Mg(II) coordinated by the pro-Rp oxygen. To determine whether the mechanism of nuclear RNase P resembles more closely an RNA- or a protein-catalyzed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear RNase P to cleave pre-tRNA containing a sulfur substitution of the pro-Rp oxygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends on the presence of a thio-sensitive ligand to the pro-Rp oxygen of the scissile bond, and is consistent with a common, RNA-based mechanism for the bacterial and eukaryal enzymes.
Asunto(s)
Núcleo Celular/enzimología , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , ARN Catalítico/química , ARN Catalítico/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Bases , Catálisis , Cloruros/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/aislamiento & purificación , Escherichia coli/enzimología , Células Eucariotas/citología , Células Eucariotas/enzimología , Células Eucariotas/metabolismo , Magnesio/metabolismo , Compuestos de Manganeso/metabolismo , Mutación/genética , Conformación de Ácido Nucleico , Compuestos Organotiofosforados/metabolismo , Oxígeno/metabolismo , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Catalítico/genética , ARN Catalítico/aislamiento & purificación , ARN de Transferencia de Fenilalanina/química , ARN de Transferencia de Fenilalanina/genética , ARN de Transferencia de Fenilalanina/metabolismo , Ribonucleasa P , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Tat activation-response region (TAR) decoys have been developed for use in gene therapy for people infected with human immunodeficiency virus type 1 (HIV-1). When a TAR RNA decoy is overexpressed, it will bind Tat, thus leaving less of this crucial protein to bind to and activate the natural transcriptional promoter of HIV-1. Previous TAR decoy constructs have used HIV-1 TAR. However, recent epidemiological and biological data began to suggest that the TAR region from the human immunodeficiency virus type 2 (HIV-2) may suppress HIV-1 transcription and hence replication. We created a vector which overexpresses TAR-2 under the control of the human U6 small nuclear RNA gene promoter and here show that the U6-TAR-2 decoy construct potently inhibits both HIV-2 and HIV-1 gene expression. Further, this decoy construct is able to markedly suppress HIV-1 replication. Thus, we have directly proven that TAR-2 can suppress HIV-1 replication and suggest that the HIV-2 TAR decoy may prove useful for combating HIV-1 infection.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/terapia , Proteínas Bacterianas/fisiología , Proteínas de Escherichia coli , Productos del Gen tat/fisiología , Terapia Genética , VIH-1/genética , VIH-2/genética , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular , Elementos de Respuesta , Replicación Viral , Células Quimiorreceptoras , Expresión Génica , VIH-1/fisiología , Humanos , Regiones Promotoras Genéticas , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
There is little information as to the location of early tRNA biosynthesis. Using fluorescent in situ hybridization in the budding yeast, Saccharomyces cerevisiae, examples of nuclear pre-tRNAs are shown to reside primarily in the nucleoli. We also probed the RNA subunit of RNase P. The majority of the signal from RNase P probes was nucleolar, with less intense signals in the nucleoplasm. These results demonstrate that a major portion of the tRNA processing pathway is compartmentalized in nucleoli with rRNA synthesis and ribosomal assembly. The spatial juxtaposition suggests the possibility of direct coordination between tRNA and ribosome biosynthesis.
Asunto(s)
Nucléolo Celular/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Hongos/biosíntesis , ARN de Transferencia/biosíntesis , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Endorribonucleasas/metabolismo , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Precursores del ARN/metabolismo , Sondas ARN , ARN Catalítico/metabolismo , ARN Ribosómico/biosíntesis , ARN de Transferencia/metabolismo , Ribonucleasa P , Saccharomyces cerevisiae/genéticaRESUMEN
Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that cleaves precursor tRNA transcripts to give mature 5' ends. RNase P in eubacteria has a large, catalytic RNA subunit and a small protein subunit that are required for precursor tRNA cleavage in vivo. Although the eukaryotic holoenzymes have similar, large RNA subunits, previous work in a number of systems has suggested that the eukaryotic enzymes require a greater protein content. We have purified the Saccharomyces cerevisiae nuclear RNase P to apparent homogeneity, allowing the first comprehensive analysis of an unexpectedly complex subunit composition. Peptide sequencing by ion trap mass spectrometry identifies nine proteins that copurify with the nuclear RNase P RNA subunit, totaling 20-fold more protein than in the bacterial enzyme. All of these proteins are encoded by genes essential for RNase P activity and for cell viability. Previous genetic studies suggested that four proteins might be subunits of both RNase P and RNase MRP, the related rRNA processing enzyme. We demonstrate that all four of these proteins, Pop1p, Pop3p, Pop4p, and Rpp1p, are integral subunits of RNase P. In addition, four of the five newly identified protein subunits, Pop5p, Pop6p, Pop7p, and Pop8p, also appear to be shared between RNase P and RNase MRP. Only one polypeptide, Rpr2p, is unique to the RNase P holoenzyme by genetic depletion and immunoprecipitation studies. The large increase in the number of protein subunits over eubacterial RNase P is consistent with an increase in functional complexity in eukaryotes. The degree of structural similarity between nuclear RNase P and RNase MRP suggests that some aspects of their functions in pre-tRNA and pre-rRNA processing pathways might overlap or be coordinated.
Asunto(s)
Núcleo Celular/enzimología , Endorribonucleasas/aislamiento & purificación , ARN Catalítico/aislamiento & purificación , Secuencia de Aminoácidos , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Datos de Secuencia Molecular , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , Ribonucleasa P , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/enzimología , Alineación de SecuenciaRESUMEN
Eukaryotic precursor (pre)-tRNAs are processed at both ends prior to maturation. Pre-tRNAs and other nascent transcripts synthesized by RNA polymerase III are bound at their 3' ends at the sequence motif UUUOH [3' oligo(U)] by the La antigen, a conserved phosphoprotein whose role in RNA processing has been associated previously with 3'-end maturation only. We show that in addition to its role in tRNA 3'-end maturation, human La protein can also modulate 5' processing of pre-tRNAs. Both the La antigen's N-terminal RNA-binding domain and its C-terminal basic region are required for attenuation of pre-tRNA 5' processing. RNA binding and nuclease protection assays with a variety of pre-tRNA substrates and mutant La proteins indicate that 5' protection is a highly selective activity of La. This activity is dependent on 3' oligo(U) in the pre-tRNA for interaction with the N-terminal RNA binding domain of La and interaction of the C-terminal basic region of La with the 5' triphosphate end of nascent pre-tRNA. Phosphorylation of La is known to occur on serine 366, adjacent to the C-terminal basic region. We show that this modification interferes with the La antigen's ability to protect pre-tRNAiMet from 5' processing either by HeLa extract or purified RNase P but that it does not affect interaction with the 3' end of pre-tRNA. These findings provide the first evidence to indicate that tRNA 5'-end maturation may be regulated in eukaryotes. Implications of triphosphate recognition is discussed as is a role for La phosphoprotein in controlling transcriptional and posttranscriptional events in the biogenesis of polymerase III transcripts.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Autoantígenos/metabolismo , Precursores del ARN/metabolismo , Ribonucleoproteínas/metabolismo , Factores de Transcripción/metabolismo , Sistema Libre de Células , Endorribonucleasas/metabolismo , Células HeLa , Humanos , Fosforilación , Poli U/metabolismo , ARN Bacteriano/metabolismo , ARN Catalítico/metabolismo , ARN de Transferencia de Metionina/metabolismo , Ribonucleasa P , Antígeno SS-BRESUMEN
Cell surface oligosaccharides have been shown to play essential biological roles in such diverse biological phenomena as cellular adhesion, molecular recognition, and inflammatory response. The development of high-affinity ligands capable of selectively recognizing a variety of small motifs in different oligosaccharides would be of significant interest as experimental and diagnostic tools. As a step toward this goal we have developed DNA ligands that recognize the disaccharide cellobiose, whether in soluble form or as the repeating unit of the polymer, cellulose. These DNA "aptamers" bind with high selectivity to cellobiose with little or no affinity for the related disaccharides lactose, maltose, and gentiobiose. Thus, the DNA ligands can discriminate sugar epimers, anomers, and disaccharide linkages.
Asunto(s)
Celobiosa/metabolismo , ADN/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Celulosa/metabolismo , Biblioteca de Genes , Cinética , Ligandos , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
The P10/11-P12 RNA domain of yeast RNase P contains several highly conserved nucleotides within a conserved secondary structure. This RNA domain is essential for enzyme function in vivo, where it has a demonstrated role in divalent cation utilization. To better understand the function of this domain, its structure and alterations in response to magnesium have been investigated in vitro. A secondary structure model of the P10/11-P12 RNA domain had been previously developed by phylogenetic analysis. Computer modeling and energy minimization were applied to the Saccharomyces cerevisiae P10/11-P12 domain to explore alternatives and additional interactions not predicted by the phylogenetic consensus. The working secondary structure models were challenged with data obtained from 1H NMR and in vitro chemical and enzymatic probing experiments. The solution structure of the isolated domain was found to conform to the phylogenetic prediction within the context of the holoenzyme. Structure probing data also discriminated among additional base contacts predicted by energy minimization. The withdrawal of magnesium does not appear to cause gross refolding or rearrangement of the RNA domain structure. Instead, subtle changes occur in the solution accessibility of specific nucleotide positions. Most of the conserved nucleotides reported to be involved in magnesium utilization in vivo also display magnesium-dependent changes in vitro.
Asunto(s)
Endorribonucleasas/química , Endorribonucleasas/genética , ARN Catalítico/química , ARN Catalítico/genética , ARN de Hongos/química , ARN de Hongos/genética , Secuencia de Bases , Simulación por Computador , Secuencia Conservada , Cartilla de ADN/genética , Endorribonucleasas/metabolismo , Magnesio/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Catalítico/metabolismo , ARN de Hongos/metabolismo , Ribonucleasa P , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , TermodinámicaRESUMEN
Nuclear pre-tRNA transcripts often contain an extension of the aminoacyl stem formed by base pairing between the 5'-leader and 3'-trailing sequences, but the -1 position preceding the mature 5' end is usually left unpaired. Considering recently proposed tertiary structural models for RNase P RNAs, we hypothesize that the -1 mismatch prevents a strong, coaxially extended aminoacyl stem, which might otherwise sterically interfere with substrate positioning in the RNase P active site. This hypothesis is tested by creating uninterrupted aminoacyl stem extensions in four nuclear tRNA precursors that normally have a mismatched nucleotide at position -1, and comparing their cleavage rates with those of the normal precursors. Determinations of Km and kcat values for a normal and an altered pre-tRNA(SUP53), which exhibits the most subtle structural alteration immediately upstream of the cleavage site, indicate that the mismatch at position -1 is an important structural requirement for both substrate affinity and efficient catalysis (and/or product release) by nuclear RNase P. This conclusion is further supported in vivo, where the pre-tRNA(SUP53) mutant precursor lacking the -1 mismatch is shown to accumulate.
Asunto(s)
Endorribonucleasas/metabolismo , Conformación de Ácido Nucleico , Precursores del ARN/química , ARN Catalítico/metabolismo , ARN de Hongos/química , ARN de Transferencia/química , Hidrólisis , Precursores del ARN/metabolismo , ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa P , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Effective intracellular expression of small RNA therapeutics depends on a number of factors. The RNA, whether antisense, ribozyme, or RNA aptamer, must be efficiently transcribed, stabilized against rapid degradation, folded correctly, and directed to the part of the cell where it can be most effective. To overcome a number of these problems we have been testing expression cassettes based on the human tRNA(met) and U6 snRNA promoters, in which transcripts encoding small RNA inserts are protected against attack from the 3' and Transient expression in cultured cells results in 10(9)-2 x 10(7) full-length transcripts per cell, depending partially on the promoter construct used but also on the nature of the insert RNA 5' gamma-Phosphate methylation (capping) depended, as expected, on the inclusion of specific U6 snRNA sequences from positions +19 to +27. In situ localization of the transcripts shows that both tRNA and U6 promoter transcripts give primarily punctate nuclear patterns, and that capping of transcripts is not required for nuclear retention. Several different insert RNAs directed against HIV-1 were tested by cotransfection with HIV-1 provirus and assay for subsequent viral reverse transcriptase production. These include antisense RNA, hairpin and hammerhead ribozymes, and RNA ligands (aptamers) for Tat and Rev RNA binding proteins. Results show that Rev-binding RNAs efficiently block HIV-1 gene expression, whereas other RNAs have little or no effected when expressed in these cassettes.