RESUMO
Replication-associated histone genes encode the only metazoan mRNAs that lack polyA tails, ending instead in a conserved 26-nt sequence that forms a stem-loop. Most of the regulation of mammalian histone mRNA is posttranscriptional and mediated by this unique 3' end. Stem-loop-binding protein (SLBP) binds to the histone mRNA 3' end and is thought to participate in all aspects of histone mRNA metabolism, including cell cycle regulation. To examine SLBP function genetically, we have cloned the gene encoding Drosophila SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations in dSLBP. dSLBP function is required both zygotically and maternally. Strong dSLBP alleles cause zygotic lethality late in development and result in production of stable histone mRNA that accumulates in nonreplicating cells. These histone mRNAs are cytoplasmic and have polyadenylated 3' ends like other polymerase II transcripts. Hypomorphic dSLBP alleles support zygotic development but cause female sterility. Eggs from these females contain dramatically reduced levels of histone mRNA, and mutant embryos are not able to complete the syncytial embryonic cycles. This is in part because of a failure of chromosome condensation at mitosis that blocks normal anaphase. These data demonstrate that dSLBP is required in vivo for 3' end processing of histone pre-mRNA, and that this is an essential function for development. Moreover, dSLBP-dependent processing plays an important role in coupling histone mRNA production with the cell cycle.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Drosophila/metabolismo , Histonas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas Nucleares , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus , Fatores de Poliadenilação e Clivagem de mRNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Ciclo Celular , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Drosophila/citologia , Drosophila/embriologia , Feminino , Genes de Insetos , Dados de Sequência Molecular , Mutação , Oócitos/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , XenopusRESUMO
The replication-dependent histone mRNAs end in a conserved 26-nt sequence that forms a stem-loop structure. This sequence is required for histone pre-mRNA processing and plays a role in multiple aspects of histone mRNA metabolism. Two proteins that bind the 3' end of histone mRNA are found in Xenopus oocytes. xSLBP1 is found in the nucleus, where it functions in histone pre-mRNA processing, and in the cytoplasm, where it may control histone mRNA translation and stability. xSLBP2 is a cytoplasmic protein, inactive in histone pre-mRNA processing, whose expression is restricted to oogenesis and early development. These proteins are similar only in their RNA-binding domains (RBD). A chimeric protein (1-2-1) in which the RBD of xSLBP1 has been replaced with the RBD of xSLBP2 binds the stem-loop with an affinity similar to the original protein. The 1-2-1 protein efficiently localizes to the nucleus of the frog oocyte, but is not active in processing of histone pre-mRNA in vivo. This protein does not support processing in a nuclear extract, but inhibits processing by competing with the active SLBP by binding to the substrate. The 1-2-1 protein also inhibits processing of synthetic histone pre-mRNA injected into frog oocytes, but has no effect on processing of histone pre-mRNA transcribed from an injected histone gene. This result suggests that sequences in the RBD of xSLBP1 give it preferential access to histone pre-mRNA transcribed in vivo.
Assuntos
Histonas/genética , Proteínas Nucleares , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus , Fatores de Poliadenilação e Clivagem de mRNA , Sequência de Aminoácidos , Animais , Sítios de Ligação , Feminino , Dados de Sequência Molecular , Oócitos/fisiologia , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle-regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for chi2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.
Assuntos
Cromossomos Bacterianos , Organelas/metabolismo , RNA Nuclear Pequeno/genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Cromossomos Humanos , Colágeno/genética , Dosagem de Genes , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Interfase/genética , Modelos Biológicos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3' end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3' end of histone mRNA.
Assuntos
Histonas/genética , Proteínas Nucleares , Oogênese/fisiologia , Biossíntese de Proteínas , RNA Mensageiro , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus , Fatores de Poliadenilação e Clivagem de mRNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Regulação da Expressão Gênica no Desenvolvimento , Histonas/biossíntese , Dados de Sequência Molecular , Oócitos , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , XenopusRESUMO
Replication-dependent histone mRNAs are not polyadenylated but end in a conserved 26-nucleotide structure that contains a stem-loop. Much of the cell cycle regulation of histone mRNA is post-transcriptional and is mediated by the 3' end of histone mRNA. The stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA is a candidate for the factor that participates in most, if not all, of the post-transcriptional regulatory events. We have cloned the cDNA for the SLBP from humans, mice, and frogs, using the recently developed yeast three-hybrid system. The human SLBP is a 31-kD protein and contains a novel RNA-binding domain, which has been mapped to a 73-amino-acid region of the protein. The cloned SLBP is the protein bound to the 3' end of histone mRNA as antibodies specific for the SLBP remove all specific binding activity from nuclear and polyribosomal extracts. These depleted extracts do not cleave histone pre-mRNA efficiently, demonstrating that the SLBP is required for efficient histone pre-mRNA processing.
Assuntos
DNA Complementar/genética , Histonas/metabolismo , Proteínas Nucleares , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transcrição Gênica/genética , Fatores de Poliadenilação e Clivagem de mRNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Precursores de RNA/biossíntese , Proteínas de Ligação a RNA/metabolismo , Ranidae , Análise de Sequência de DNA , Transfecção , Leveduras/genéticaRESUMO
Chimeric genes which contained the mouse U1b snRNA promoter, portions of the histone H2a or globin coding regions and the U1b 3'-end followed by a histone 3'-end were constructed. The distance between the U1 promoter and the U1 3' box was varied between 146 and 670 nt. The chimeric genes were introduced into CHO cells by stable transfection or into Xenopus oocytes by microinjection. The efficiency of utilization of the U1 3' box, as measured by the relative amounts of transcripts that ended at the U1 3' box and the histone 3'-end, was dependent on the distance between the promoter and 3'-end box. U1 3'-ends were formed with >90% efficiency on transcripts shorter than 200 nt, with 50-70% efficiency on transcripts of 280-400 nt and with only 10-20% efficiency on transcripts >500 nt. Essentially identical results were obtained after stable transfection of CHO cells or after injecting the genes into Xenopus oocytes. The distance between the U1 promoter and the U1 3' box must be <280 nt for efficient transcription termination at the U1 3' box, regardless of the sequence transcribed.
Assuntos
Regiões Promotoras Genéticas , RNA Nuclear Pequeno/genética , Animais , Sequência de Bases , Células CHO , Cricetinae , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Transcrição Gênica , TransfecçãoRESUMO
The stem-loop structure at the 3' end of replication-dependent histone mRNA is required for efficient pre-mRNA processing, localization of histone mRNA to the polyribosomes, and regulation of histone mRNA degradation. A protein, the stem-loop binding protein (SLBP), binds the 3' end of histone mRNA and is thought to mediate some or all of these processes. A mutant histone mRNA with two nucleotide changes in the loop was constructed and found to be transported inefficiently to the cytoplasm. The mutant histone mRNA, unlike the wild-type histone mRNA, was not rapidly degraded when DNA synthesis is inhibited, and was not stabilized upon inhibition of protein synthesis. The stem-loop binding protein (SLBP) has between a 20-50 fold greater affinity for the wild type histone stem-loop structure than for the mutant stem-loop structure, suggesting that the alteration in the efficiency of transport and the normal degradation pathway in histone mRNA may be due to the reduced affinity of the mutant stem-loop for the SLBP.