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.
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.
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.
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. Replicationdependent 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. Replicationdependent 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.Eukaryotic chromosomes are composed of equal amounts of DNA and histone proteins. Each time a eukaryotic cell divides, it must not only properly replicate its DNA but also synthesize large amounts of histones to package the DNA into chromatin properly. In order to coordinate the synthesis of histones and DNA, mammalian cells tightly regulate the concentrations of the replication-dependent histone mRNAs with DNA synthesis. Most of this regulation occurs at posttranscriptional levels, and in mammalian cells a major regulatory step is the regulation of the half-life of histone mRNA. The levels of replication-dependent histone mRNAs are cell cycle regulated. Histone mRNA levels increase 35-fold as cells enter S phase, and they are rapidly degraded at the end of S phase (12). In addition, replication-dependent histone mRNAs are rapidly degraded when cells are treated with inhibitors of DNA replication (29).Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end in a conserved stem-loop structure (5), which is necessary and sufficient for the rapid degradation of histone mRNAs after the inhibition of DNA replication (24). The stem-loop structure is recognized by a 31-kDa protein called the stem-loop binding protein (SLBP). SLBP is necessary for histone premRNA processing (7) as well as translation of histone mRNAs, and a 15-amino-acid region of SLBP required for translational activation of histone mRNAs has been identified (27).Histone mRNAs are rapidly degraded following treatment of cells with inhibitors of DNA synthesis. In contrast, treatment of cells with inhibitors of protein synthesis prevents rapid histone mRNA degradation after the inhibition of DNA synthesis (2) and increases histone mRNA level...
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.
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