RESUMO
Replication-dependent histone mRNAs are coordinately regulated in parallel with DNA replication. Histone mRNAs accumulate to high levels only in S-phase cells and are degraded rapidly at the end of S phase or when DNA replication is inhibited in S-phase cells. The unique 3' end on histone mRNAs is the cis element responsible for the regulation of histone mRNA degradation. This chapter describes the approaches used to demonstrate the connection between translation of histone mRNA and its degradation as well as the pathway of histone mRNA degradation in mammalian cells. In particular, the initial step in histone mRNA degradation is attachment of an oligo(U) tail to the 3' end of histone mRNA, providing a platform for binding factors that trigger mRNA degradation.
Assuntos
Ciclo Celular/genética , Ciclo Celular/fisiologia , Histonas/genética , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Hibridização de Ácido Nucleico , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Eukaryotic cells coordinately regulate histone and DNA synthesis. In mammalian cells, most of the regulation of histone synthesis occurs post-transcriptionally by regulating the concentrations of histone mRNA. As cells enter S phase, histone mRNA levels increase, and at the end of S phase they are rapidly degraded. Moreover, inhibition of DNA synthesis causes rapid degradation of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is the only cis-acting element required for coupling regulation of histone mRNA half-life with DNA synthesis. Here we show that regulated degradation of histone mRNAs requires Upf1, a key regulator of the nonsense-mediated decay pathway, and ATR, a key regulator of the DNA damage checkpoint pathway activated during replication stress.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , Histonas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/farmacologia , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Hidroxiureia/farmacologia , Lisina/genética , Lisina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , RNA Helicases , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Transativadores/genéticaRESUMO
The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3' untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.
Assuntos
Histonas/química , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Códon , Códon de Terminação , Regulação da Expressão Gênica , Células HeLa , Histonas/metabolismo , Humanos , Camundongos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Poliadenilação , RNA/química , Interferência de RNA , Ribossomos/química , Fatores de Tempo , TransfecçãoRESUMO
The stem-loop binding protein (SLBP) binds the 3' end of histone mRNA and is present both in nucleus, and in the cytoplasm on the polyribosomes. SLBP participates in the processing of the histone pre-mRNA and in translation of the mature message. Histone mRNAs are rapidly degraded when cells are treated with inhibitors of DNA replication and are stabilized by inhibitors of translation, resulting in an increase in histone mRNA levels. Here, we show that SLBP is a component of the histone messenger ribonucleoprotein particle (mRNP). Histone mRNA from polyribosomes is immunoprecipitated with anti-SLBP. Most of the SLBP in cycloheximide-treated cells is present on polyribosomes as a result of continued synthesis and transport of the histone mRNP to the cytoplasm. When cells are treated with inhibitors of DNA replication, histone mRNAs are rapidly degraded but SLBP levels remain constant and SLBP is relocalized to the nucleus. SLBP remains active both in RNA binding and histone pre-mRNA processing when DNA replication is inhibited.
Assuntos
Histonas/genética , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Polirribossomos/metabolismo , Ribonucleoproteínas/química , Fatores de Poliadenilação e Clivagem de mRNA/análise , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Células CHO , Núcleo Celular/química , Cricetinae , Cricetulus , Cicloeximida/farmacologia , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Histonas/metabolismo , Humanos , Camundongos , Proteínas Nucleares/imunologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Testes de Precipitina , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Células Tumorais Cultivadas , Fatores de Poliadenilação e Clivagem de mRNA/imunologiaRESUMO
Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.
Assuntos
Exonucleases/química , Histonas/metabolismo , Proteínas Nucleares , RNA Mensageiro/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA , Sequência de Aminoácidos , Animais , Baculoviridae/metabolismo , Clonagem Molecular , DNA/metabolismo , Exonucleases/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Modelos Genéticos , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/química , Coelhos , Fatores de Tempo , Técnicas do Sistema de Duplo-HíbridoRESUMO
In metazoans, the 3' end of histone mRNA is not polyadenylated but instead ends with a stem-loop structure that is required for cell cycle-regulated expression. The sequence of the stem-loop in the Drosophila melanogaster histone H2b, H3, and H4 genes is identical to the consensus sequence of other metazoan histone mRNAs, but the sequence of the stem-loop in the D. melanogaster histone H2a and H1 genes is novel. dSLBP binds to these novel stem-loop sequences as well as the canonical stem-loop with similar affinity. Eggs derived from females containing a viable, hypomorphic mutation in dSLBP store greatly reduced amounts of all five histone mRNAs in the egg, indicating that dSLBP is required in the maternal germ line for production of each histone mRNA. Embryos deficient in zygotic dSLBP function accumulate poly(A)(+) versions of all five histone mRNAs as a result of usage of polyadenylation signals located 3' of the stem-loop in each histone gene. Since the 3' ends of adjacent histone genes are close together, these polyadenylation signals may ensure the termination of transcription in order to prevent read-through into the next gene, which could possibly disrupt transcription or produce antisense histone mRNA that might trigger RNA interference. During early wild-type embryogenesis, ubiquitous zygotic histone gene transcription is activated at the end of the syncytial nuclear cycles during S phase of cycle 14, silenced during the subsequent G(2) phase, and then reactivated near the end of that G(2) phase in the well-described mitotic domain pattern. There is little or no dSLBP protein provided maternally in wild-type embryos, and zygotic expression of dSLBP is immediately required to process newly made histone pre-mRNA.
