RESUMO
Small nucleolar RNAs (snoRNAs) use base pairing to guide modification of conserved nucleotides in functionally important regions of ribosomal RNA. The box C/D snoRNAs direct 2'-O-methylation and the box H/ACA snoRNAs direct pseudouridylation. Each snoRNA interacts with proteins, many of them newly identified. Progress in understanding how snoRNA sequences are stored within genomes, liberated from precursor molecules and targeted to the nucleolus has begun to elucidate each step in the biogenesis of these critical contributors to ribosome formation.
Assuntos
Nucléolo Celular/metabolismo , Precursores de RNA/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Humanos , Precursores de RNA/genética , Transcrição GênicaRESUMO
AU-rich elements (AREs) present in the 3' untranslated regions of many protooncogene, cytokine, and lymphokine messages target them for rapid degradation. HuR, a ubiquitously expressed member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins, selectively binds AREs and stabilizes ARE-containing mRNAs in transiently transfected cells. Here, we identify four mammalian proteins that bind regions of HuR known to be essential for its ability to shuttle between the nucleus and the cytoplasm and to stabilize mRNA: SETalpha, SETbeta, pp32, and acidic protein rich in leucine (APRIL). Three have been reported to be protein phosphatase 2A inhibitors. All four ligands contain long, acidic COOH-terminal tails, while pp32 and APRIL share a second motif: rev-like leucine-rich repeats in their NH(2)-terminal regions. We show that pp32 and APRIL are nucleocytoplasmic shuttling proteins that interact with the nuclear export factor CRM1 (chromosomal region maintenance protein 1). The inhibition of CRM1 by leptomycin B leads to the nuclear retention of pp32 and APRIL, their increased association with HuR, and an increase in HuR's association with nuclear poly(A)+ RNA. Furthermore, transcripts from the ARE-containing c-fos gene are selectively retained in the nucleus, while the cytoplasmic distribution of total poly(A)+ RNA is not altered. These data provide evidence that interaction of its ligands with HuR modulate HuR's ability to bind its target mRNAs in vivo and suggest that CRM1 is instrumental in the export of at least some cellular mRNAs under certain conditions. We discuss the possible role of these ligands upstream of HuR in pathways that govern the stability of ARE-containing mRNAs.
Assuntos
Antígenos de Superfície , Carioferinas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade , Citoplasma/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ácidos Graxos Insaturados/farmacologia , Células HeLa , Humanos , Ligantes , Dados de Sequência Molecular , Neuropeptídeos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas/metabolismo , Ligação Proteica , Proteína Fosfatase 2 , Transporte Proteico , Análise de Sequência de Proteína , Proteína Exportina 1RESUMO
A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.
Assuntos
Nucléolo Celular/análise , Precursores de RNA/fisiologia , RNA Ribossômico 5S/fisiologia , RNA Ribossômico/fisiologia , Ribossomos/metabolismo , Animais , Autoanticorpos/imunologia , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Vírus da Leucemia Murina de Friend , Células HeLa , Humanos , Imunoensaio , Leucemia Eritroblástica Aguda , Precursores de RNA/análise , RNA Ribossômico 5S/análise , Ribonucleoproteínas/análise , Ribonucleoproteínas/imunologia , Proteínas Ribossômicas/análise , Células Tumorais CultivadasRESUMO
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.
Assuntos
Autoantígenos/metabolismo , Núcleo Celular/metabolismo , Oócitos/fisiologia , Processamento Pós-Transcricional do RNA , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas , Transporte Ativo do Núcleo Celular , Animais , Autoantígenos/genética , Autorradiografia , Microinjeções , Conformação de Ácido Nucleico , Oócitos/citologia , Ligação Proteica , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Xenopus laevis , Proteínas Centrais de snRNPRESUMO
In eukaryotic cells, the conversion of gene transcripts into messenger RNA's involves multiple factors, including the highly abundant small nuclear ribonucleoprotein (snRNP) complexes that mediate the splicing reaction. Separable factors are also required for the 3' end processing of histone pre-mRNA's. The two conserved signals flanking the 3' cleavage site are recognized by discrete components present in active HeLa cell extracts: the upstream stem loop associates with a nuclease-insensitive factor, while binding to the downstream element is mediated by a component having the properties of a snRNP. The sequence of the RNA moiety of the low abundance human U7 snRNP suggests how the relatively degenerate downstream element of mammalian pre-mRNA's could be recognized by RNA base-pairing.
Assuntos
Histonas/genética , Precursores de RNA/genética , Ribonucleoproteínas/genética , Sequência de Bases , Endorribonucleases , Células HeLa/metabolismo , Humanos , Dados de Sequência Molecular , Processamento Pós-Transcricional do RNA , Ribonuclease H , Ribonucleoproteínas Nucleares PequenasRESUMO
Five small nuclear RNAs (U1, U2, U4, U5, and U6) participate in precursor messenger RNA (pre-mRNA) splicing. To probe their interactions within the active center of the mammalian spliceosome, substrates containing a single photoactivatable 4-thiouridine residue adjacent to either splice site were synthesized, and crosslinks were induced during the course of in vitro splicing. An invariant loop sequence in U5 small nuclear RNA contacts exon 1 before and after the first step of splicing because a crosslink between U5 and the last residue of exon 1 appeared in the pre-mRNA and then in the cutoff exon 1 intermediate. Both of these crosslinked species could undergo subsequent splicing, indicating that the crosslinks reflect a functional interaction that is maintained through both reaction steps. The same U5 loop aligns the two exons for ligation since the first residue of exon 2 also became crosslinked to U5 in the lariat intermediate. An invariant sequence in U6 RNA became crosslinked to the conserved second position of the intron within both the lariat intermediate and the lariat intron product. On the basis of these results, several conformational arrangements of small nuclear RNAs within the spliceosomal active center can be distinguished, and additional mechanistic parallels between the spliceosome and self-splicing introns can be drawn.
Assuntos
Splicing de RNA/fisiologia , RNA Nuclear Pequeno/fisiologia , Spliceossomos/fisiologia , Adenoviridae/genética , Sequência de Bases , Sítios de Ligação , Catálise , Éxons/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Precursores de RNA/metabolismo , RNA Viral/fisiologia , TiouridinaRESUMO
The transport of messenger RNAs (mRNAs) from the nucleus to the cytoplasm involves adapter proteins that bind the mRNA as well as receptor proteins that interact with the nuclear pore complex. We demonstrate the utility of cell-permeable peptides designed to interfere with interactions between potential adapter and receptor proteins to define the pathways accessed by particular mRNAs. We show that HuR, a protein implicated in the stabilization of short-lived mRNAs containing AU-rich elements (AREs), serves as an adapter for c-fos mRNA export through two pathways. One involves the HuR shuttling domain, HNS, which exhibits a heat shock-sensitive interaction with transportin 2 (Trn2); the other involves two protein ligands of HuR-pp32 and APRIL-which contain leucine-rich nuclear export signals (NES) recognized by the export receptor CRM1. Heterokaryon and in situ hybridization experiments reveal that the peptides selectively block the nucleocytoplasmic shuttling of their respective adapter proteins without perturbing the overall cellular distribution of polyadenylated mRNAs.
Assuntos
Antígenos de Superfície , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Genes fos/genética , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Proteína do Homeodomínio de Antennapedia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Resposta ao Choque Térmico , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Carioferinas/metabolismo , Dados de Sequência Molecular , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Estabilidade de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Sequências Reguladoras de Ácido Nucleico/genética , Reprodutibilidade dos Testes , Tetra-Hidrofolato Desidrogenase/genética , Proteína Exportina 1RESUMO
Precursor messenger RNA splicing requires multiple factors including U1, U2, U4, U5, and U6 small nuclear RNA's. The crosslinking reagent psoralen was used to analyze the interactions of these RNA's with an adenovirus precursor messenger RNA in HeLa nuclear extract. An endogenous U2-U4-U6 crosslinkable complex dissociated upon incubation with precursor messenger RNA. During splicing, U1, U2, U5, and U6 became crosslinked to precursor messenger RNA and U2, U5, and U6 became crosslinked to excised lariat intron. U2 also formed a doubly crosslinked complex with U6 and precursor messenger RNA. The U1, U5, and U6 crosslinks to the precursor messenger RNA mapped to intron sequences near the 5' splice site, whereas the U2 crosslink mapped to the branch site. The kinetics of crosslink formation and disappearance delineates a temporal pathway for the action of small RNA's in the spliceosome. Potential base pairing interactions between conserved sequences in the small nuclear RNA's and precursor messenger RNA at the sites of crosslinking suggest that the 5' splice site is defined in several steps prior to the first cleavage event.
Assuntos
Splicing de RNA , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Núcleo Celular/metabolismo , Reagentes de Ligações Cruzadas , Células HeLa , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Dados de Sequência Molecular , Precursores de RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/ultraestruturaRESUMO
Removal of a rare class of metazoan precursor messenger RNA introns with AU-AC at their termini is catalyzed by a spliceosome that contains U11, U12, and U5 small nuclear ribonucleoproteins. Two previously unidentified, low-abundance human small nuclear RNAs (snRNAs), U4atac and U6atac, were characterized as associated with the AT-AC spliceosome and necessary for AT-AC intron splicing. The excision of AT-AC introns therefore requires four snRNAs not found in the major spliceosome. With the use of psoralen crosslinking, a U6atac interaction with U12 was identified that is similar to a U6-U2 helix believed to contribute to the spliceosomal active center. The conservation of only limited U6atac sequences in the neighborhood of this interaction and the potential of U6atac to base pair with the 5' splice site consensus for AT-AC introns provide support for current models of the core of the spliceosome.
Assuntos
Íntrons , Precursores de RNA/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Composição de Bases , Sequência de Bases , Sequência Conservada , Reagentes de Ligações Cruzadas , Éxons , Furocumarinas , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Nuclear Pequeno/química , Spliceossomos/químicaRESUMO
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.
Assuntos
Nucléolo Celular/química , Íntrons , Precursores de RNA/metabolismo , RNA Nuclear/genética , RNA Nuclear/fisiologia , RNA Ribossômico 18S/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/fisiologia , Animais , Sequência de Bases , Northern Blotting , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Oócitos/metabolismo , Processamento Pós-Transcricional do RNA , RNA Nuclear/química , RNA Nuclear Pequeno/química , XenopusRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Éxons/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/metabolismo , Globinas/genética , Células HeLa , Humanos , Substâncias Macromoleculares , Camundongos , Modelos Biológicos , Testes de Precipitina , Ligação Proteica , RNA Helicases/metabolismo , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores , TransfecçãoRESUMO
A component present in splicing extracts selectively binds the 3' splice site of a precursor messenger RNA (pre-mRNA) transcript of a human beta-globin gene. Since this component can be immunoprecipitated by either autoantibodies of the Sm class or antibodies specifically directed against trimethylguanosine, it is a small nuclear ribonucleoprotein (snRNP). Its interaction with the 3' splice site occurs rapidly even at 0 degrees C, does not require adenosine triphosphate, and is altered by certain mutations in the 3' splice site region. Binding is surprisingly insensitive to treatment of the extract with micrococcal nuclease. The U5 particle is the only abundant Sm snRNP with a capped 5' end that is equally resistant to micrococcal nuclease. This suggests that, in addition to the U1 and U2 snRNP's, U5 snRNP's participate in pre-mRNA splicing.
Assuntos
Precursores de Ácido Nucleico/genética , Splicing de RNA , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Sequência de Bases , Sítios de Ligação , Globinas/genética , Humanos , Conformação de Ácido Nucleico , Precursores de Ácido Nucleico/metabolismo , Ligação Proteica , Precursores de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas Nucleares Pequenas , Transcrição GênicaRESUMO
The RNP and Sm antigens recognized by lupus erythematosus antibodies are located on discrete particles containing single small nuclear RNA's complexed with proteins. The antigens Ro and La are also on ribonucleoproteins. The small RNA's in ribonucleoproteins with Ro are discrete, like those associated with RNP and Sm; in contrast, ribonucleoproteins with La contain a striking highly banded spectrum of small RNA's from uninfected cells as well as virus-associated RNA from adenovirus-infected cells.
Assuntos
Antígenos/análise , Autoanticorpos , Autoantígenos/análise , Lúpus Eritematoso Sistêmico/imunologia , Nucleoproteínas/imunologia , Ribonucleoproteínas/imunologia , Animais , Carcinoma de Ehrlich , Epitopos , Células HeLa , Humanos , RNA/imunologia , RNA Neoplásico/imunologia , RNA Nuclear PequenoRESUMO
A rare class of pre-mRNA introns with non-canonical consensus sequences has been identified in metazoan genes. The novel, low-abundance spliceosome that excises these introns contains one small nuclear ribonucleoprotein (snRNP) in common with the major spliceosome (U5) and four snRNPs that are distinct from, but structurally and functionally analogous to U1, U2 and U4-U6. The architecture of RNA components at the presumptive core of the AT-AC splicesome supports current models of the spliceosomal active center and raises tantalizing questions about spliceosome evolution.
Assuntos
Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética , Spliceossomos/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Humanos , Dados de Sequência Molecular , Filogenia , Precursores de RNA/química , RNA Mensageiro/química , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
We characterized the RNA-containing antigens precipitated by sera from 260 patients with positive antinuclear antibodies. 49 individuals, most of whom had systemic lupus erythematosus or Sjögren's syndrome, possessed antibodies that precipitated the previously identified RNP, Sm, Ro, and La antigens either singly or in combinations. These antigens, which are located on discrete sets of small nuclear or cytoplasmic RNA-protein particles, exhibited a number of antigenic interrelationships. One patient's serum recognized a new particle containing a small RNA which we have called Th; it also precipitated the Ro complexes. Other patients with systemic lupus erythematosus, hepatitis B virus infection, juvenile rheumatoid arthritis, myositis, and rheumatoid arthritis had antibodies that precipitated specific subsets of ribosomal RNA and transfer RNA. One patient's serum contained a monoclonal immunoglobulin G that precipitated ribosomes. Most of these antibodies identified antigenic determinants constituted at least in part of protein. The specificity of the proteins bound to particular cellular RNA, probably explains the exquisite precision with which antibodies from rheumatic disease patients discriminate among RNA subsets. Such sera should be useful probes for investigating specific roles that different RNA and RNA-protein complexes play in cellular metabolism.
Assuntos
Anticorpos Antinucleares , Sítios de Ligação de Anticorpos , Proteínas de Transporte/metabolismo , Doenças do Tecido Conjuntivo/imunologia , Especificidade de Anticorpos , Antígenos de Grupos Sanguíneos , Proteínas de Transporte/análise , Proteínas de Transporte/classificação , Células HeLa/análise , Células HeLa/imunologia , Hepatite B/imunologia , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Proteínas de Ligação a RNARESUMO
We have identified gas5 (growth arrest-specific transcript 5) as a non-protein-coding multiple small nucleolar RNA (snoRNA) host gene similar to UHG (U22 host gene). Encoded within the 11 introns of the mouse gas5 gene are nine (10 in human) box C/D snoRNAs predicted to function in the 2'-O-methylation of rRNA. The only regions of conservation between mouse and human gas5 genes are their snoRNAs and 5'-end sequences. Mapping the 5' end of the mouse gas5 transcript demonstrates that it possesses an oligopyrimidine tract characteristic of the 5'-terminal oligopyrimidine (5'TOP) class of genes. Arrest of cell growth or inhibition of translation by cycloheximide, pactamycin, or rapamycin-which specifically inhibits the translation of 5'TOP mRNAs-results in accumulation of the gas5 spliced RNA. Classification of gas5 as a 5'TOP gene provides an explanation for why it is a growth arrest specific transcript: while the spliced gas5 RNA is normally associated with ribosomes and rapidly degraded, during arrested cell growth it accumulates in mRNP particles, as has been reported for other 5'TOP messages. Strikingly, inspection of the 5'-end sequences of currently known snoRNA host gene transcripts reveals that they all exhibit features of the 5'TOP gene family.
Assuntos
Nucléolo Celular/genética , Proteínas de Membrana/genética , Família Multigênica/genética , RNA Nuclear Pequeno/genética , RNA Nucleolar Pequeno , Células 3T3 , Animais , Sequência de Bases , Divisão Celular/genética , Clonagem Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas/genética , Splicing de RNA/genética , RNA Antissenso/genética , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Ribossomos/genética , Transcrição Gênica/genéticaRESUMO
To study the regulation of AUUUA-mediated RNA deadenylation and destabilization during Xenopus early development, we microinjected chimeric mRNAs containing Xenopus or mammalian 3' untranslated region (3'-UTR) sequences into Xenopus oocytes, mature eggs, or fertilized embryos. We found that the AU-rich elements (ARE) of Xenopus c-myc II and the human granulocyte-macrophage colony-stimulating factor gene (GMCSF) both direct deadenylation of chimeric mRNAs in an AUUUA-dependent manner. In the case of the Xenopus c-myc II ARE, mutation of a single AUUUA within an absolutely conserved 11-nucleotide region in c-myc 3'-UTRs prevents ARE-mediated deadenylation. AUUUA-specific deadenylation appears to be developmentally regulated: low deadenylation activity is observed in the oocyte, whereas rapid deadenylation occurs following egg activation or fertilization. Deadenylation results in the accumulation of stable deadenylated RNAs that become degraded only following mid-blastula transition. We conclude that ARE-mediated mRNA deadenylation can be uncoupled from ARE-mediated mRNA decay and that AUUUAs directly signal deadenylation during Xenopus early development.
Assuntos
Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Xenopus/crescimento & desenvolvimento , Regiões 3' não Traduzidas/genética , Animais , Sequência Conservada/genética , Fertilização/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Microinjeções , Oligodesoxirribonucleotídeos/genética , Oócitos/metabolismo , Poli A/genética , Xenopus/embriologiaRESUMO
The human U3 ribonucleoprotein (RNP) has been analyzed to determine its protein constituents, sites of protein-RNA interaction, and RNA secondary structure. By using anti-U3 RNP antibodies and extracts prepared from HeLa cells labeled in vivo, the RNP was found to contain four nonphosphorylated proteins of 36, 30, 13, and 12.5 kilodaltons and two phosphorylated proteins of 74 and 59 kilodaltons. U3 nucleotides 72-90, 106-121, 154-166, and 190-217 must contain sites that interact with proteins since these regions are immunoprecipitated after treatment of the RNP with RNase A or T1. The secondary structure was probed with specific nucleases and by chemical modification with single-strand-specific reagents that block subsequent reverse transcription. Regions that are single stranded (and therefore potentially able to interact with a substrate RNA) include an evolutionarily conserved sequence at nucleotides 104-112 and nonconserved sequences at nucleotides 65-74, 80-84, and 88-93. Nucleotides 159-168 do not appear to be highly accessible, thus making it unlikely that this U3 sequence base pairs with sequences near the 5.8S rRNA-internal transcribed spacer II junction, as previously proposed. Alternative functions of the U3 RNP are discussed, including the possibility that U3 may participate in a processing event near the 3' end of 28S rRNA.
Assuntos
Precursores de RNA/genética , Ribonucleoproteínas/genética , Animais , Composição de Bases , Sequência de Bases , Bombyx/genética , Dictyostelium/genética , Drosophila/genética , Células HeLa , Humanos , Conformação de Ácido Nucleico , Filogenia , Ribonucleoproteínas Nucleares Pequenas , Termodinâmica , Transcrição GênicaRESUMO
Pre-mRNA substrates containing sequences from human and mouse histone genes are accurately processed in a HeLa cell nuclear extract to generate mature 3' termini. When in vitro processing reactions containing either human histone H3 or mouse histone H3 transcripts are treated with RNase T1 and probed with antibodies specific for the Sm protein determinants or for the trimethylguanosine cap structure unique to the U RNAs present in small nuclear ribonucleoproteins, RNA fragments that encompass the site of 3' end formation on the pre-mRNA transcript are selectively recovered. Several different interactions are detected: at time zero, the protected region contains the upstream conserved hairpin loop structure; at later times during the reaction, protection extends beyond the site of 3' end formation to include the downstream conserved sequence element and the 5' cap of the transcript is bound as well. Possible interactions between Sm small nuclear ribonucleoproteins and these conserved sequence elements in histone pre-mRNAs are discussed.
Assuntos
Histonas/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Ribonucleoproteínas/fisiologia , Autoantígenos/imunologia , Sítios de Ligação , Técnicas Imunológicas , Capuzes de RNA/imunologia , Ribonucleoproteínas Nucleares Pequenas , Fatores de Tempo , Proteínas Centrais de snRNPRESUMO
We examined the ability of U1 small nuclear ribonucleoproteins (U1 snRNPs) to recognize mutant and cryptic 5' splice sites on beta-globin pre-mRNA substrates using an RNase T1 protection assay. When U1 snRNPs were prebound to anti-(U1)RNP antibodies, we detected binding to mutant but not to cryptic 5' splice sites on several substrates. By contrast, in a splicing extract at 0 degree C, neither the mutated nor cryptic 5' splice sites of a human beta-globin transcript were selected as protected fragments with the same antibodies. However, after incubation of the transcript in the extract to yield splicing intermediates, fragments that included a cryptic 5' splice site were detected. The results of our analyses suggest that U1 snRNPs are involved in recognizing cryptic 5' splice sites but that interactions with other splicing components are required to stabilize the association.