RESUMO
The expression of the replication-dependent histone mRNAs is tightly regulated during the cell cycle. As cells progress from G(1) to S phase, histone mRNA levels increase 35-fold, and they decrease again during G(2) phase. Replication-dependent histone mRNAs are the only metazoan mRNAs that lack polyadenylated tails, ending instead in a conserved stem-loop. Much of the cell cycle regulation is posttranscriptional and is mediated by the 3' stem-loop. A 31-kDa stem-loop binding protein (SLBP) binds the 3' end of histone mRNA. The SLBP is necessary for pre-mRNA processing and accompanies the histone mRNA to the cytoplasm, where it is a component of the histone messenger RNP. We used synchronous CHO cells selected by mitotic shakeoff and HeLa cells synchronized at the G(1)/S or the M/G(1) boundary to study the regulation of SLBP during the cell cycle. In each system the amount of SLBP is regulated during the cell cycle, increasing 10- to 20-fold in the late G(1) and then decreasing in the S/G(2) border. SLBP mRNA levels are constant during the cell cycle. SLBP is regulated at the level of translation as cells progress from G(1) to S phase, and the protein is rapidly degraded as they progress into G(2). Regulation of SLBP may account for the posttranscriptional component of the cell cycle regulation of histone mRNA.
Assuntos
Ciclo Celular , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Células CHO , Cricetinae , Replicação do DNA , Histonas/genética , Histonas/metabolismo , Proteínas Nucleares/genética , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genéticaRESUMO
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
Histone mRNAs end in a conserved 26 nt sequence which can form a stem-loop with a six-base stem and a four base loop. The 3' end of histone mRNA functions in the nucleus in pre-mRNA processing and mRNA transport and in the cytoplasm in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), found in both the polyribosomes and the nucleus, binds to the 3' end of histone mRNA. A nuclear extract which efficiently processes histone pre-mRNA has been prepared from mouse myeloma cells. The factors which bind the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide. Using the depleted extract, we show that the SLBP found in the polyribosomes can function in histone pre-mRNA processing, suggesting that the SLBP associates with histone pre-mRNA in the nucleus and accompanies the mature mRNA to the cytoplasm.
