RESUMEN

Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes and maintains telomeric DNA. Studies of telomeres and telomerase are facilitated by the large number of linear DNA molecules found in ciliated protozoa, such as Tetrahymena thermophila. To examine the expression of telomerase, we investigated the transcription of the RNA polymerase III-directed gene encoding the RNA subunit (TER1) of this enzyme. A chimeric gene containing the Glaucoma chattoni TER1 transcribed region flanked by 5' and 3' Tetrahymena regions was used to identify promoter elements following transformation of Tetrahymena cells. Disruption of a conserved proximal sequence element (PSE) located at -55 in the Tetrahymena TER1 5' flanking region eliminated expression of the chimeric gene. In addition, mutation of an A/T-rich element at -25 decreased expression markedly. A gel mobility shift assay and protein-DNA cross-linking identified a PSE-binding polypeptide of 50-60 kDa in Tetrahymena extracts. Gel filtration analysis revealed a native molecular mass of approximately 160 kDa for this binding activity. Our results point to a similar architecture between ciliate telomerase RNA and metazoan U6 small nuclear RNA promoters.

Asunto(s)

Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , ARN Protozoario/genética , Telomerasa/genética , Tetrahymena thermophila/enzimología , Tetrahymena thermophila/genética , Animales , Secuencia de Bases , Línea Celular , Secuencia Conservada/genética , ADN Protozoario/química , ADN Protozoario/genética , ADN Protozoario/metabolismo , ADN Recombinante/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dosificación de Gen , Genes Protozoarios/genética , Peso Molecular , Mutación/genética , ARN Polimerasa III/metabolismo , ARN Protozoario/análisis , ARN Nuclear Pequeño/genética , Elementos de Respuesta/genética , Telomerasa/metabolismo , Moldes Genéticos , Tetrahymena thermophila/citología , Tetrahymenina/enzimología , Tetrahymenina/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética/genética

RESUMEN

Many vertebrate small nuclear RNA gene promoters contain an SPH motif in their distal control regions that can confer transcriptional stimulation by RNA polymerase II or RNA polymerase III. Using the human U6 gene SPH motif as a probe, we isolated a cDNA encoding human SPH-binding factor (hSBF) from a HeLa cell expression library. The coding region of hSBF is almost identical to ZNF143, a 626 amino acid, seven zinc finger protein of previously unknown function. Furthermore, the predicted amino acid sequence of hSBF is highly homologous to Xenopus laevis and mouse Staf proteins, that bind to SPH motifs and stimulate transcription of selenocysteine tRNA gene promoters. Recombinant hSBF expressed in vitro or from Escherichia coli bound specifically to the human U6 gene SPH motif as shown by DNase I footprinting and electrophoretic mobility shift assays using various mutant SPH sites as competitors. Antibodies prepared against recombinant hSBF inhibited assembly of native SBF-DNA complexes. Immunodepleted HeLa S100 transcription extract no longer supported elevated levels of transcription by RNA polymerase III from a U6 promoter containing an SPH motif, whereas addition of recombinant hSBF protein to the immunodepleted extract reconstituted stimulated transcription.

Asunto(s)

ADN Complementario/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , ARN Nuclear Pequeño/genética , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Anticuerpos , Sitios de Unión/genética , Clonación Molecular , Secuencia Conservada , ADN/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Escherichia coli/genética , Proteínas Fúngicas/inmunología , Células HeLa , Humanos , Técnicas In Vitro , Ratones , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/inmunología , Xenopus laevis

RESUMEN

The distal control region of a human U6 small nuclear RNA (snRNA) gene promoter contains two separable elements, octamer (OCT) and SPH, found in many vertebrate snRNA genes. Complete distal regions generally account for a 4- to 100-fold stimulation of snRNA gene promoters. We examined the mechanism of transcriptional stimulation by each element when linked to the proximal U6 promoter. Multimers of either OCT or SPH did not increase transcriptional levels above that with a single copy, either in transfected human cells or after in vitro transcription in a HeLa S100 extract. The orientation of a single SPH element differentially stimulated transcription in transfected cells, whereas the orientation of an octamer element was not important. Using Sarkosyl to limit transcription to a single-round, we concluded that promoters containing either OCT or SPH elements supported an increased number of preinitiation complexes in vitro. Furthermore, the rate of formation of U6 promoter preinitiation complexes resistant to low (0.015%) concentrations of Sarkosyl was accelerated on templates containing either OCT or SPH. However, neither element had a significant effect on the number of rounds of reinitiation in the S100 extract.

Asunto(s)

Regiones Promotoras Genéticas , ARN Nuclear Pequeño/genética , Secuencia de Bases , Cartilla de ADN/genética , Detergentes , Elementos de Facilitación Genéticos , Células HeLa , Humanos , Plásmidos/genética , Sarcosina/análogos & derivados , Transcripción Genética , Activación Transcripcional , Transfección

RESUMEN

Vertebrate small nuclear RNA (snRNA) gene promoters contain a distal, enhancer-like region that is composed of an octamer motif adjacent to at least one other element. Here we show that a human U6 snRNA distal region contains a SPH motif previously found in several chicken snRNA gene enhancers and the 5'-flanking region of vertebrate selenocysteine tRNA genes. SPH binding factor (SBF) was detected in either chicken or HeLa cell extracts that could bind SPH elements in a species-independent manner. Both human and chicken SBF required divalent cation to bind effectively to DNA. DNase I footprinting experiments indicated that human SBF specifically protected the human U6 SPH element. Furthermore, a SBF polypeptide of approximately 85 kDa was detected in both HeLa and chicken extracts following ultraviolet light-mediated cross-linking to human U6 or chicken U4 SPH elements. A part of the human U6 SPH element was quite sensitive to mutation, as demonstrated by both specific protein binding and transcription assays. From these data it is apparent that the distal regions of some RNA polymerase III- and RNA polymerase II-transcribed small RNA promoters are virtually identical in composition, and their mechanisms of transcriptional activation are possibly quite similar.

Asunto(s)

Regiones Promotoras Genéticas , ARN Nuclear/genética , Proteínas de Unión al ARN/genética , Animales , Pollos , Células HeLa , Humanos , Unión Proteica , ARN Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética

RESUMEN

The promoter of vertebrate U6 small nuclear RNA genes consists of a TATA box and a snRNA proximal sequence element (PSE), and the combination of these two elements directs RNA polymerase III transcription. We detected RNA polymerase II transcription as well as pol III transcription from the human U6 promoter in a HeLa nuclear extract. The pol II-specific transcription was independent of the PSE and dependent on the presence of the TATA box. Both pol III- and pol II-specific transcription were stimulated by addition of recombinant TATA-binding protein (TBP). We conclude that both pol III and pol II preinitiation complexes can assemble on the U6 promoter in vitro and could compete during the bona fide process in the cell.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/biosíntesis , TATA Box , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/biosíntesis , Células HeLa , Humanos , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteína de Unión a TATA-Box , Factores de Transcripción/biosíntesis , Vertebrados

RESUMEN

The proximal promoter of a human U6 small nuclear RNA (snRNA)-encoding gene contains two separate elements, the proximal sequence element (PSE) and the TATA box. We investigated the interaction of the PSE- and TATA-binding proteins (PBP and TBP) with normal and mutant U6 proximal promoters using an electrophoretic mobility shift assay. We detected a complex containing both PBP and TBP bound to the wild-type U6 promoter. Efficient formation of the triple complex was dependent on the presence of the PSE and the TATA box on the template DNA. Mutant U6 promoters containing an increased spacing between the PSE and TATA box of 5 or 10 bp were impaired in the ability to form a complex that includes TBP. We infer from these results that PBP and TBP interact when their binding sites are properly positioned in a U6 gene promoter.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , ARN Nuclear Pequeño/genética , TATA Box , Factores de Transcripción/metabolismo , Secuencia de Bases , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Mutación , Proteína de Unión a TATA-Box , Transcripción Genética

RESUMEN

In order to explore the coupling of transcription with splicing in mammalian cells we have prepared hybrid genes in which either the human U2 promoter, recognized by RNA polymerase II, or the human U6 promoter, recognized by RNA polymerase III, was fused to an intron-containing gene segment. Neither human small nuclear RNA gene contains an intron although U6 genes from some species of yeast contain a short intervening sequence. Following transfection of human cells and analysis of specific RNAs by primer extension we found that the chimeric U2 promoter-derived transcript was efficiently spliced but the RNA polymerase III transcript driven by the U6 promoter remained unspliced. Hence, the splicing apparatus differentiates between transcripts produced from two closely related promoters that are distinguished by RNA polymerase selectivity.

Asunto(s)

Intrones/genética , Regiones Promotoras Genéticas/genética , Precursores del ARN/metabolismo , Empalme del ARN , ARN Nuclear Pequeño/genética , Secuencia de Bases , Células Cultivadas , ADN Recombinante , Humanos , Datos de Secuencia Molecular , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Transfección , Tubulina (Proteína)/biosíntesis , Tubulina (Proteína)/genética

RESUMEN

The promoters of vertebrate U6 small nuclear RNA genes contain a distal control region whose presence results in at least an eightfold level of transcriptional activation in vivo. Previous transfection experiments have demonstrated that most of the distal control region of a human U6 gene resides in a restriction fragment located from -244 to -149 relative to the transcriptional start site. Three octamer-related motifs that bind recombinant Oct-1 transcription factor in vitro exist in this segment of DNA. However, transfection of human 293 cells with various plasmid templates in which these Oct-1 binding sites had been disrupted individually or in combination showed that only the consensus octamer motif located between positions -221 to -214 was functional. Even so, the consensus octamer motif mutant template was expressed at only a moderately reduced level relative to the wild-type promoter. When another octamer-related sequence located nearby, one that did not bind Oct-1 in vitro, was disrupted along with the perfect octamer site, expression was reduced fivefold in transfected cells. A factor that binds this functional, nonconsensus octamer site (NONOCT) was detected in crude cellular extracts. However, the NONOCT sequence was not essential for activation, since its disruption caused only a 40% reduction in U6 gene expression, and mutagenesis to convert the NONOCT sequence to a consensus octamer motif restored wild-type expression. Furthermore, in vitro transcription of a human U6 proximal promoter joined to a single copy of the octamer motif was stimulated by the addition of recombinant Oct-1 protein.

Asunto(s)

Regiones Promotoras Genéticas , ARN Nuclear Pequeño/genética , Secuencia de Bases , Línea Celular , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Genes , Factor C1 de la Célula Huésped , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Factor 1 de Transcripción de Unión a Octámeros , Oligodesoxirribonucleótidos/química , Mutación Puntual , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética , Transcripción Genética

RESUMEN

Transcription of vertebrate U6 snRNA genes by RNA polymerase III requires two sequence elements in the proximal promoter region: the PSE (proximal sequence element, found in snRNA promoters transcribed by RNA polymerase II) and the TATA element (found in many mRNA promoters). The locations of the PSE and the TATA box are important determinants for transcriptional start site selection in their respective RNA polymerase II promoters. In vertebrate U6 genes the PSE and the TATA elements are located in approximately the same positions as in the polymerase II transcribed genes, but their respective roles in initiation site selection are unknown. We have analyzed the effects of spacing changes between the PSE and the TATA element, and between the two elements and the normal U6 start site on human U6 gene transcription. The spacing requirement between the two elements is highly stringent, implying a possible interaction between the factors that bind them. Our results discount the possibility that the location of either the PSE or the TATA element, by itself, dictates efficient selection of a transcriptional start site. Instead, we suggest that the two elements form a compound promoter element whose location dictates the start site of transcription from the human U6 gene promoter.

Asunto(s)

Regiones Promotoras Genéticas , ARN Nuclear Pequeño/genética , TATA Box , Transcripción Genética , Secuencia de Bases , Línea Celular , Expresión Génica , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , ARN Polimerasa II/metabolismo , Eliminación de Secuencia , Transfección

RESUMEN

Vertebrate U6 small nuclear RNA (snRNA) loci exemplify a novel class of polymerase III-transcribed genes that lack an intragenic control region (ICR). Instead important transcriptional control elements are located in the 5'-flanking region and resemble those found in promoters and enhancers of polymerase II-transcribed genes. These include a proximal sequence element (PSE), a TATA element, and a distal region containing, at least, an octamer motif. We have used Sarkosyl to characterize steps in U6 promoter transcription in vitro in an unfractionated S100 extract and find very similar properties to those of the adenovirus VA1 gene that contains an ICR. Preformed preinitiation complexes are stable to 0.015% Sarkosyl and can undergo multiple rounds of initiation upon addition of nucleoside triphosphates. A higher concentration (0.075%) prevents reinitiation. In addition, we have investigated the formation of transcription complexes on this promoter in a S100 extract using a template competition assay. No stable complexes are detected with plasmid templates that contain clustered point mutations in the PSE nor with DNAs lacking the U6 5'-flanking region. A plasmid template containing mutations in the TATA element is partially deficient in competitive ability. Furthermore, the distal region upstream of position--148 is necessary for efficient stable complex formation. Within this region, the consensus octamer motif is one component needed to form complexes that withstand competition by a wild-type U6 promoter. The human U2 gene enhancer ligated to the U6 proximal region supports formation of a complex that competes at an intermediate level.

Asunto(s)

Elementos de Facilitación Genéticos , Genes Reguladores , Regiones Promotoras Genéticas , ARN Nuclear Pequeño/genética , TATA Box , Transcripción Genética , Adenovirus Humanos/genética , Secuencia de Bases , Unión Competitiva , Línea Celular , Deleción Cromosómica , Genes Virales/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Plásmidos , Mapeo Restrictivo , Sarcosina/análogos & derivados , Sarcosina/farmacología , Moldes Genéticos , Transcripción Genética/efectos de los fármacos

RESUMEN

Five RFLPS of X-linked coagulation factor IX were evaluated in more than 500 normal persons (723-804 X chromosomes) of both sexes who belonged to eight ethnic groups: Anglo-Americans, Basques, Swedes, African-Americans, East Africans, East Indians, Chinese, and Malays. The polymorphisms, 5' to 3', were BamHI, XmnI, TaqI, MnlI, and HhaI. A PCR procedure was developed for three previously described RFLPs-XmnI, TaqI, and MnlI; a PCRP procedure was developed for BamHI, and a PCRP which had been described by others was used for HhaI. Europeans were the most polymorphic, African-Americans and East Africans were intermediate, and Orientals were the least polymorphic. Extragenic 3' HhaI was highly polymorphic in most groups, and extragenic 5' BamHI was polymorphic only in persons with African ancestry. Two major haplotypes predominated among 247 men, and the expected and observed heterozygosities were concordant among women. Allelic association was very strong between the three intragenic PCRPs; it was present but weak between 5' extragenic BamHI and XmnI. No association was found between 3' extragenic HhaI and MnlI.

Asunto(s)

Etnicidad , Factor IX/genética , Polimorfismo de Longitud del Fragmento de Restricción , Cromosoma X , Pueblo Asiatico/genética , Secuencia de Bases , Población Negra/genética , Southern Blotting , Endodesoxirribonucleasas/metabolismo , Femenino , Ligamiento Genético/genética , Genética de Población , Humanos , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Población Blanca/genética

Asunto(s)

Desoxirribonucleasas de Localización Especificada Tipo II , Polimorfismo Genético , Factor de von Willebrand/genética , Alelos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 12 , Codón , Heterocigoto , Humanos , Datos de Secuencia Molecular

Asunto(s)

ARN Polimerasa III/genética , Transcripción Genética , Animales , Secuencia de Bases , Humanos , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Especificidad por Sustrato , Xenopus

RESUMEN

A previously described HindIII restriction fragment length polymorphism (RFLP) of factor VIII (FVIII) has its polymorphic site in the unsequenced nineteenth intron. We have located the polymorphic site, as well as an invariant site, by amplifying and sequencing IVS 19 using the polymerase chain reaction (PCR). The oligonucleotide primers were synthesized from known FVIII sequence on either side of the 19-20 splice junction. The amplified product was cloned into a plasmid and sequenced by the dideoxy chain termination method. The polymorphic HindIII site was 103 bp and the invariant site 184 bp from the 3' end of the nineteenth exon. The frequency of the polymorphism was determined in 457 subjects (643 chromosomes) of seven ethnic groups on whom frequency of the BclI RFLP of IVS 18 was also assessed. The HindIII site is highly polymorphic in all groups, approximately 0.25:0.75, the expected heterozygosity averaging 37.6%, and the observed number of heterozygotes did not differ significantly from expectation. The (+):(-) allelic ratio is similar in all groups, except African-Americans in whom it is reversed. Strong allelic association (linkage disequilibrium) is present between the HindIII polymorphism of IVS 19 and the BclI polymorphism of IVS 18.

Asunto(s)

Factor VIII/genética , Polimorfismo de Longitud del Fragmento de Restricción , Grupos Raciales/genética , Alelos , Secuencia de Bases , Southern Blotting , Femenino , Humanos , Desequilibrio de Ligamiento , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa

Asunto(s)

Cromosomas Humanos Par 12 , Codón , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero , Factor de von Willebrand/genética , Secuencia de Bases , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos

RESUMEN

Most eukaryotic genes transcribed by RNA polymerase III contain internal control regions. U6 small nuclear RNA genes are transcribed by RNA polymerase III but are unusual in that, at least in vitro, their expression does not require intragenic sequences. Here we show that this is true as well in vivo. A human U6 gene devoid of all but the first 6 and last 10 base-pairs was expressed efficiently after transfection into human 293 cells. We also report data extending the previous identification of 5' flanking sequences important for human U6 gene transcription. Deletion-substitution of a 10 base-pair upstream sequence encompassing the TATATA element (-29 to -24) abolished U6 transcription. A double point mutation in the middle of this element (TATATA-TAGCTA) reduced U6 transcription but not to the extent brought about by TATATA deletion-substitution. These results establish that, in vivo, transcription of human U6 small nuclear RNA is independent of intragenic sequences between nucleotides 6 and 98, and requires the upstream TATATA box.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/fisiología , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , ARN Polimerasa III/fisiología , ARN Nuclear Pequeño/genética , Transcripción Genética , Análisis Mutacional de ADN , Humanos , Técnicas In Vitro , Secuencias Reguladoras de Ácidos Nucleicos

RESUMEN

A DNA polymorphism in the coding region of coagulation factor IX--potentially valuable for carrier detection, prenatal diagnosis, and population studies--was described in 1985. It had been discovered with monoclonal antibodies that distinguish between threonine and alanine as the 148th residue of the peptide. Its use as a diagnostic tool has been limited because threonine-containing factor IX (Malmö A) is dominant to alanine-containing factor IX (Malmö B) in immunoassays of plasma; therefore, detection of Malmö heterozygotes is not possible in all instances. A DNA method for recognizing all heterozygotes has been developed, but it also has limitations. We report the development of another DNA procedure based on amplification of the relevant DNA with the polymerase chain reaction (PCR). This method is quick, avoids the use of isotopes and x-ray film, and specifically identifies all the Malmö genotypes: hemizygotes, homozygotes, and heterozygotes. The procedure can be performed satisfactorily on small samples of blood (less than 1 mL) as suggested by Kogan et al (N Engl J Med 317:985, 1987). The method described is applicable to any genetic polymorphism that overlaps a restriction enzyme recognition site.

Asunto(s)

Factor IX/genética , Genotipo , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Secuencia de Bases , Exones , Femenino , Amplificación de Genes , Frecuencia de los Genes , Hemofilia B/genética , Humanos , Masculino , Datos de Secuencia Molecular , Linaje

RESUMEN

U6 small nuclear RNA is transcribed by a different polymerase than U1-U5 RNAs, likely to be RNA polymerase III. Transcription from human U6 gene deletion-substitution templates in a HeLa S100 extract delineated the 5' border of a control element lying between 67 and 43 bp upstream from the initiation site. This region matches the location of, and shows considerable sequence similarity with, the proximal control element of U1 and U2 RNA genes, which are transcribed by RNA polymerase II. Transfection of human 293 cells with 5'-flanking deletion-substitution mutants of a U6 maxigene revealed a dominant control element between 245 and 149 bp upstream of the transcription start site. An octamer motif was found in this region in an inverted orientation relative to that of the human U1 and U2 RNA gene enhancers but in the same orientation as a human U4 RNA gene, the transcript of which functions together with U6 RNA in a single small nuclear ribonucleoprotein (snRNP) particle. The human U2 gene enhancer joined to the U6 maxigene was able to functionally replace the U6 distal control element(s).

Asunto(s)

Genes Reguladores , ARN Nuclear Pequeño/genética , Transcripción Genética , Secuencia de Bases , Elementos de Facilitación Genéticos , Células HeLa/metabolismo , Humanos , Datos de Secuencia Molecular , ARN Polimerasa III/metabolismo , ARN Nuclear Pequeño/metabolismo

RESUMEN

A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to alpha-amanitin at 1 microgram/ml but was completely inhibited at 200 micrograms/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to alpha-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited greater than 90% at 1 microgram/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/fisiología , ARN Polimerasa III/fisiología , ARN Nuclear Pequeño/metabolismo , Transcripción Genética , Amanitinas/farmacología , Secuencia de Bases , Clonación Molecular , Células HeLa , Humanos , ARN Nuclear Pequeño/genética , Homología de Secuencia de Ácido Nucleico

RESUMEN

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

Asunto(s)

ARN Nuclear Pequeño/genética , Secuencia de Bases , Núcleo Celular , Células HeLa , Humanos , Hibridación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/biosíntesis , Ribonucleoproteínas/análisis , Ribonucleoproteínas Nucleares Pequeñas , Transcripción Genética