RESUMEN
In mammalian cells, splice junctions play a dual role in mRNA quality control: They mediate selective nuclear export of mature mRNA and they serve as a mark for mRNA surveillance, which subjects aberrant mRNAs with premature termination codons to nonsense-mediated decay (NMD). Here, we demonstrate that the protein RNPS1, a component of the postsplicing complex that is deposited 5' to exon-exon junctions, interacts with the evolutionarily conserved human Upf complex, a central component of NMD. Significantly, RNPS1 triggers NMD when tethered to the 3' untranslated region of beta-globin mRNA, demonstrating its role as a subunit of the postsplicing complex directly involved in mRNA surveillance.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Exones/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/metabolismo , Globinas/genética , Células HeLa , Humanos , Sustancias Macromoleculares , Ratones , Modelos Biológicos , Pruebas de Precipitina , Unión Proteica , ARN Helicasas/metabolismo , Empalme del ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores , TransfecciónRESUMEN
U2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5' trimethylated guanosine cap, 13 pseudouridines, and 10 2'-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.
Asunto(s)
Autoantígenos/metabolismo , Núcleo Celular/metabolismo , Oocitos/fisiología , Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas , Transporte Activo de Núcleo Celular , Animales , Autoantígenos/genética , Autorradiografía , Microinyecciones , Conformación de Ácido Nucleico , Oocitos/citología , Unión Proteica , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Xenopus laevis , Proteínas Nucleares snRNPRESUMEN
Nonsense-mediated decay (NMD) rids eukaryotic cells of aberrant mRNAs containing premature termination codons. These are discriminated from true termination codons by downstream cis-elements, such as exon-exon junctions. We describe three novel human proteins involved in NMD, hUpf2, hUpf3a, and hUpf3b. While in HeLa cell extracts these proteins are complexed with hUpf1, in intact cells hUpf3a and hUpf3b are nucleocytoplasmic shuttling proteins, hUpf2 is perinuclear, and hUpf1 cytoplasmic. hUpf3a and hUpf3b associate selectively with spliced beta-globin mRNA in vivo, and tethering of any hUpf protein to the 3'UTR of beta-globin mRNA elicits NMD. These data suggest that assembly of a dynamic hUpf complex initiates in the nucleus at mRNA exon-exon junctions and triggers NMD in the cytoplasm when recognized downstream of a translation termination site.
Asunto(s)
Codón sin Sentido/metabolismo , Proteínas Fúngicas/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Transactivadores/genética , Regiones no Traducidas 3'/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Globinas/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Empalme del ARN/fisiología , Transfección , LevadurasRESUMEN
Among the spliceosomal snRNAs, U2 has the most extensive modifications, including a 5' trimethyl guanosine (TMG) cap, ten 2'-O-methylated residues and 13 pseudouridines. At short times after injection, cellularly derived (modified) U2 but not synthetic (unmodified) U2 rescues splicing in Xenopus oocytes depleted of endogenous U2 by RNase H targeting. After prolonged reconstitution, synthetic U2 regenerates splicing activity; a correlation between the extent of U2 modification and U2 function in splicing is observed. Moreover, 5-fluorouridine-containing U2 RNA, a potent inhibitor of U2 pseudouridylation, specifically abolishes rescue by synthetic U2, while rescue by cellularly derived U2 is not affected. By creating chimeric U2 molecules in which some sequences are from cellularly derived U2 and others are from in vitro transcribed U2, we demonstrate that the functionally important modifications reside within the 27 nucleotides at the 5' end of U2. We further show that 2'-O-methylation and pseudouridylation activities reside in the nucleus and that the 5' TMG cap is not necessary for internal modification but is crucial for splicing activity. Native gel analysis reveals that unmodified U2 is not incorporated into the spliceosome. Examination of the U2 protein profile and glycerol-gradient analysis argue that U2 modifications directly contribute to conversion of the 12S to the 17S U2 snRNP particle, which is essential for spliceosome assembly.
Asunto(s)
Procesamiento Postranscripcional del ARN , Empalme del ARN , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Animales , Secuencia de Bases , Guanosina/análogos & derivados , Datos de Secuencia Molecular , Oocitos , Seudouridina/análogos & derivados , Caperuzas de ARN , Empalmosomas/metabolismo , XenopusRESUMEN
We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.
Asunto(s)
Nucléolo Celular/química , Mitosis/fisiología , Fosfoproteínas/análisis , Ribonucleoproteínas Nucleares Pequeñas/análisis , Ribonucleoproteínas/análisis , Secuencia de Aminoácidos , Secuencia de Bases , Fraccionamiento Celular , Proteínas Cromosómicas no Histona/análisis , Clonación Molecular , ADN Complementario/genética , Dactinomicina/farmacología , Células HeLa , Humanos , Punto Isoeléctrico , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , ARN Nuclear Pequeño/análisis , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
2'-O-methylation of eukaryotic ribosomal RNAs occurs in the cell nucleoli. At least 100 modification sites that are highly conserved among vertebrate rRNAs have been mapped. However, in part because of the insensitivity of current approaches, there are 2'-O-methylated sites that remain unidentified. We have developed an extremely sensitive method for detecting 2'-O-methylated residues that are predicted within a long RNA molecule. Utilizing RNase H cleavage directed by a 2'-O-methyl RNA-DNA chimeric oligonucleotide, this method has allowed identification of two methylated nucleotides, G1448 in Xenopus 18S rRNA and A394 in Xenopus 28S rRNA. The latter (A394 in 28S) had not been detected before. We have confirmed that the methylation at G1448 in 18S is dependent upon Xenopus U25 snoRNA and have demonstrated that the methylation at A394 in 28S requires U26 snoRNA. One advantage of this technique is that it can examine specific rRNA and precursor molecules. We show that about 30% of the 40S pre-rRNA has been methylated at these two sites and their methylation is complete at the stage of 20S (immediate precursor to 18S) and 32S (immediate precursor to 28S). We also show that methylation at these two sites is not essential for rRNA transport from the nucleus to the cytoplasm.
Asunto(s)
ARN Ribosómico 18S/química , ARN Ribosómico 28S/química , Animales , Secuencia de Bases , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Metilación , Datos de Secuencia Molecular , Oocitos/citología , ARN Ribosómico 18S/metabolismo , ARN Ribosómico 28S/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa H/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , XenopusRESUMEN
Vertebrate cells contain a large number of small nucleolar RNA (snoRNA) species, the vast majority of which bind fibrillarin. Most of the fibrillarin-associated snoRNAs can form 10- to 21-nt duplexes with rRNA and are thought to guide 2'-O-methylation of selected nucleotides in rRNA. These include mammalian UHG (U22 host gene)-encoded U25-U31 snoRNAs. We have characterized two novel human snoRNA species, U62 and U63, which similarly exhibit 15- (with one interruption) and 12-nt complementarities and are therefore predicted to direct 2'-O-methylation of A590 in 18S and A4531 in 28S rRNA, respectively. To establish the function of antisense snoRNAs in vertebrates, we exploited the Xenopus oocyte system. Cloning of the Xenopus U25-U31 snoRNA genes indicated that they are encoded within multiple homologs of mammalian UHG. Depletion of U25 from the Xenopus oocyte abolished 2'-O-methylation of G1448 in 18S rRNA; methylation could be restored by injecting either the Xenopus or human U25 transcript into U25-depleted oocytes. Comparison of Xenopus and human U25 sequences revealed that only boxes C, D, and D', as well as the 18S rRNA complement, were invariant, suggesting that they may be the only elements required for U25 snoRNA stability and function.
Asunto(s)
ARN Ribosómico 28S/genética , ARN Nuclear Pequeño/genética , Ribosa/genética , Xenopus/genética , Animales , Secuencia de Bases , Humanos , Metilación , Datos de Secuencia Molecular , ARN Ribosómico 28S/metabolismo , ARN Nuclear Pequeño/metabolismo , Ribosa/metabolismo , Análisis de Secuencia de ADNRESUMEN
The nucleoli of eukaryotic cells are the sites of ribosomal RNA transcription and processing and of ribosomal subunit assembly. They contain multiple small nucleolar RNAs (snoRNAs), several of which are essential for rRNA maturation. The U3, U8 and U13 snoRNA genes are transcribed independently, whereas U14-U24, as well as E3, are located within introns of protein-coding genes, most of whose functions are linked to translation. These snoRNAs are co-transcribed with their host pre-mRNAs and released by processing from excised introns. Here we show that, in addition to U22, seven novel fibrillarin-associated snoRNAs, named U25-U31, are encoded within different introns of the unusually compact mammalian U22 host gene (UHG). All seven RNAs exhibit extensive (12-15 nucleotides) complementarity to different segments of the mature rRNAs, followed by a C/AUGA ('U-turn') sequence. The spliced UHG RNA, although it is associated with polysomes, has little potential for protein coding, is short-lived, and is poorly conserved between human and mouse. Thus, the introns rather than the exons specify the functional products of UHG.
Asunto(s)
Intrones , ARN Nuclear Pequeño/genética , Animales , Secuencia de Bases , Northern Blotting , Sondas de ADN , Dactinomicina/farmacología , Exones , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Nuclear Pequeño/efectos de los fármacos , ARN Nuclear Pequeño/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The nucleoli of vertebrate cells contain a number of small RNAs that are generated by the processing of intron fragments of protein-coding gene transcripts. The host gene (UHG) for intro-encoded human U22 is unusual in that it specifies a polyadenylated but apparently noncoding RNA. Depletion of U22 from Xenopus oocytes by oligonucleotide-directed ribonuclease H targeting prevented the processing of 18S ribosomal RNA (rRNA) at both ends. The appearance of 18S rRNA was restored by injection of in vitro-synthesized U22 RNA. These results identify a cellular function for an intron-encoded small RNA.
Asunto(s)
Nucléolo Celular/química , Intrones , Precursores del ARN/metabolismo , ARN Nuclear/genética , ARN Nuclear/fisiología , ARN Ribosómico 18S/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/fisiología , Animales , Secuencia de Bases , Northern Blotting , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Oocitos/metabolismo , Procesamiento Postranscripcional del ARN , ARN Nuclear/química , ARN Nuclear Pequeño/química , XenopusRESUMEN
A human small nucleolar RNA, identified previously in HeLa cells by anti-fibrillarin autoantibody precipitation and termed RNA X, has been characterized. It comprises two uridine-rich variants (148 and 146 nucleotides), which we refer to as snRNA U15A and U15B. Secondary structure models predict for both variants a U15-specific stem-loop structure, as well as a new structural motif that contains conserved sequences and can also be recognized in the other fibrillarin-associated nucleolar snRNAs, U3, U14, and RNA Y. The single-copy gene for human U15A has been found unexpectedly to reside in intron 1 of the ribosomal protein S3 gene; the U15A sequence appears on the same strand as the S3 mRNA and does not exhibit canonical transcription signals for nuclear RNA polymerases. U15A RNA is processed in vitro from S3 intron 1 transcripts to yield the correct 5' end with a 5'-monophosphate; the in vitro system requires ATP for 3' cleavage, which occurs a few nucleotides downstream of the mature end. The production of a single primary transcript specifying the mRNA for a ribosomal or nucleolar protein and a nucleolar snRNA may constitute a general mechanism for balancing the levels of nucleolar components in vertebrate cells.
Asunto(s)
Nucléolo Celular/química , Intrones , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Proteínas Ribosómicas/genética , Secuencia de Bases , Sistema Libre de Células , Secuencia Conservada , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Precursores del ARN , Procesamiento Postranscripcional del ARN , Mapeo Restrictivo , Proteínas Ribosómicas/química , Análisis de Secuencia de ARN , Nucleótidos de Uracilo/análisisRESUMEN
The Epstein-Barr virus-encoded small RNA (EBER) genes are transcribed by RNA polymerase III, but their transcription unit appears to contain both class II and class III promoter elements. One of these promoter element, a TATA-like box which we call the EBER TATA box, or ETAB, is located in a position typical for a class II TATA box but contains G/C residues in the normal T/A motif and a conserved thymidine doublet. Experiments using chloramphenicol acetyltransferase constructs and mutations in the TATA box of the adenovirus major late promoter showed that the ETAB promoter element does not substitute for a class II TATA box. However, when the ETAB promoter element sequence was changed to a class II TATA box consensus sequence, the EBER 2 gene was transcribed in vitro by both RNA polymerases II and III. From these results, we conclude that the ETAB promoter element is important for the exclusive transcription of the EBER 2 gene by RNA polymerase III.
Asunto(s)
Genes Virales , Herpesvirus Humano 4/genética , Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , ARN Viral/genética , TATA Box , Transcripción Genética , Secuencia de Bases , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
The Epstein-Barr virus-encoded small RNA (EBER) genes appear to comprise an interesting subset of class III genes different from any previously identified, including U6 and 7SK. EBER genes have functional A and B box intragenic control regions. In addition, they contain three upstream elements that together stimulate in vivo expression 50-fold and resemble sites associated with typical class II promoters. DNAase I footprinting analyses using purified proteins or oligonucleotide competition demonstrate that nucleotides -40 to -55 bind activating transcription factor (ATF) or a related protein, while nucleotides -56 to -77 bind Sp1 protein or a related protein. The element between positions -23 and -28 resembles a TATA box. EBERs are unusual RNA polymerase III transcripts shown to be controlled by ATF- and Sp1-like promoter elements, suggesting mechanisms for their high level expression in EBV-transformed lymphocytes.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Genes Virales , Genes , Herpesvirus Humano 4/genética , Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , ARN Viral/genética , Transcripción Genética , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , TransfecciónRESUMEN
Genes for the Epstein-Barr virus-encoded RNAs (EBERs), two low-molecular-weight RNAs encoded by the human gammaherpesvirus Epstein-Barr virus (EBV), hybridize to two small RNAs in a baboon cell line that contains a similar virus, herpesvirus papio (HVP). The genes for the HVP RNAs (HVP-1 and HVP-2) are located together in the small unique region at the left end of the viral genome and are transcribed by RNA polymerase III in a rightward direction, similar to the EBERs. There is significant similarity between EBER1 and HVP-1 RNA, except for an insert of 22 nucleotides which increases the length of HVP-1 RNA to 190 nucleotides. There is less similarity between the sequences of EBER2 and HVP-2 RNA, but both have a length of about 170 nucleotides. The predicted secondary structure of each HVP RNA is remarkably similar to that of the respective EBER, implying that the secondary structures are important for function. Upstream from the initiation sites of all four RNA genes are several highly conserved sequences which may function in the regulation of transcription. The HVP RNAs, together with the EBERs, are highly abundant in transformed cells and are efficiently bound by the cellular La protein.
Asunto(s)
Genes Virales , Herpesviridae/genética , ARN Viral/genética , Autoantígenos/metabolismo , Secuencia de Bases , Clonación Molecular , Herpesvirus Humano 4/genética , Enlace de Hidrógeno , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Polimerasa III/metabolismo , Ribonucleoproteínas/genética , Relación Estructura-Actividad , Transcripción Genética , Antígeno SS-BRESUMEN
We have studied the ability of poly-2-vinylpyridine-N-oxide (PVNO), a lysosomal stabilizing agent, to abrogate the cytotoxic effects of silica on macrophages. Male C3H/HeN mice were pretreated with PVNO and inoculated intravenously with silica particles. At 24 h after silica injection, silica-treated and -untreated mice were challenged intravenously with varying doses of live yeast cells of Histoplasma capsulatum. All mice receiving silica died when challenged with 5 X 10(5) yeast cells of Histoplasma sp. compared with no deaths in PVNO-pretreated animals and 10% mortality in controls not receiving PVNO or silica. When animals were given 2.5 X 10(5) yeast cells (a sublethal dose), the protective effect of PVNO was seen by a reduction in splenomegaly and viable Histoplasma sp. present in the spleen. Furthermore, PVNO alone showed a significant protective effect (P less than 0.05) against a lethal challenge with Histoplasma sp. Prior treatment with PVNO also protected mouse peritoneal macrophages from the cytotoxic effects of silica particles in vitro. These results indicate that PVNO abrogates the cytotoxicity of silica particles on macrophages and also increases the resistance of mice to histoplasmosis.
Asunto(s)
Histoplasmosis/prevención & control , Inmunidad Innata/efectos de los fármacos , N-Óxido de Polivinilpiridina/uso terapéutico , Polivinilos/uso terapéutico , Animales , Histoplasma/patogenicidad , Histoplasmosis/etiología , Histoplasmosis/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos , Dióxido de SilicioRESUMEN
The role of macrophages in the innate immunity of mice to histoplasmosis was investigated using silica, which selectively inactivates macrophages. Mice given silica IV 1 day prior to challenge with live yeast cells of Histoplasma capsulatum were more susceptible to infection than were untreated controls. This increased susceptibility to Histoplasma was observed when mice were given silica at 1, 14, and 21 days prior to infection but not at 3 and 7 days. Silica treated mice that survived 30 days after challenge with a sublethal dose of Histoplasma had 23 times more viable organisms in their spleens than in those of untreated controls. The blastogenic response of spleen cells to concanavalin A and phytohemagglutinin was unaffected at 12 hr after silica injection but was significantly depressed between 1 and 21 days. In contrast, silica treatment did not affect the blastogenic response of spleen cells to lipopolysaccharide. Silica particles were cytotoxic for mouse peritoneal macrophages but not to lymphocytes in vitro. These results indicate that macrophages play an essential role in natural immunity to histoplasmosis.