Translation termination is involved in histone mRNA degradation when DNA replication is inhibited

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.     

The Prolyl Isomerase Pin1 Targets Stem-Loop Binding Protein (SLBP) To Dissociate the SLBP-Histone mRNA Complex Linking Histone mRNA Decay with SLBP Ubiquitination

Histone mRNAs are rapidly degraded at the end of S phase, and a 26-nucleotide stem-loop in the 3′ untranslated region is a key determinant of histone mRNA stability. This sequence is the binding site for stem-loop binding protein (SLBP), which helps to recruit components of the RNA degradation machinery to the histone mRNA 3′ end. SLBP is the only protein whose expression is cell cycle regulated during S phase and whose degradation is temporally correlated with histone mRNA degradation. Here we report that chemical inhibition of the prolyl isomerase Pin1 or downregulation of Pin1 by small interfering RNA (siRNA) increases the mRNA stability of all five core histone mRNAs and the stability of SLBP. Pin1 regulates SLBP polyubiquitination via the Ser20/Ser23 phosphodegron in the N terminus. siRNA knockdown of Pin1 results in accumulation of SLBP in the nucleus. We show that Pin1 can act along with protein phosphatase 2A (PP2A) in vitro to dephosphorylate a phosphothreonine in a conserved TPNK sequence in the SLBP RNA binding domain, thereby dissociating SLBP from the histone mRNA hairpin. Our data suggest that Pin1 and PP2A act to coordinate the degradation of SLBP by the ubiquitin proteasome system and the exosome-mediated degradation of the histone mRNA by regulating complex dissociation.     

SLBP is associated with histone mRNA on polyribosomes as a component of the histone mRNP

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.     

The C-terminal extension of Lsm4 interacts directly with the 3′ end of the histone mRNP and is required for efficient histone mRNA degradation

Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem–loop sequence, which is bound to the stem–loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3′ end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3′hExo, forms a ternary complex with SLBP and the stem–loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3′hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3′hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.     

Structural and biochemical studies of SLIP1–SLBP identify DBP5 and eIF3g as SLIP1-binding proteins

In metazoans, replication-dependent histone mRNAs end in a stem-loop structure instead of the poly(A) tail characteristic of all other mature mRNAs. This specialized 3′ end is bound by stem-loop binding protein (SLBP), a protein that participates in the nuclear export and translation of histone mRNAs. The translational activity of SLBP is mediated by interaction with SLIP1, a middle domain of initiation factor 4G (MIF4G)-like protein that connects to translation initiation. We determined the 2.5 Å resolution crystal structure of zebrafish SLIP1 bound to the translation–activation domain of SLBP and identified the determinants of the recognition. We discovered a SLIP1-binding motif (SBM) in two additional proteins: the translation initiation factor eIF3g and the mRNA-export factor DBP5. We confirmed the binding of SLIP1 to DBP5 and eIF3g by pull-down assays and determined the 3.25 Å resolution structure of SLIP1 bound to the DBP5 SBM. The SBM-binding and homodimerization residues of SLIP1 are conserved in the MIF4G domain of CBP80/20-dependent translation initiation factor (CTIF). The results suggest how the SLIP1 homodimer or a SLIP1–CTIF heterodimer can function as platforms to bridge SLBP with SBM-containing proteins involved in different steps of mRNA metabolism.     

Coordinate regulation of histone mRNA metabolism and DNA replication

S phase is characterized by the replication of DNA and assembly of chromatin. This requires the synthesis of large amounts of histone proteins to package the newly replicated DNA. Histone mRNAs are the only mRNAs that do not have polyA tails, ending instead in a conserved stemloop sequence. The stemloop binding protein (SLBP) that binds the 3' end of histone mRNA is cell cycle regulated and SLBP is required in all steps of histone mRNA metabolism. Activation of cyclin E/cdk2 prior to entry into S phase is critical for initiation of DNA replication and histone mRNA accumulation. At the end of S phase SLBP is rapidly degraded as a result of phosphorylation of SLBP by cyclin A/cdk1 and CK2 effectively shutting off histone mRNA biosynthesis. E2F1, which is required for expression of many S-phase genes, is regulated in parallel with SLBP and its degradation also requires a cyclin binding site, suggesting that it may also be regulated by the same pathway. It is likely that activation of cyclin A/cdk1 so helps inhibit both DNA replication and histone mRNA accumulation, marking the end of S phase and entry into G2 phase.     

SLIP1, a factor required for activation of histone mRNA translation by the stem-loop binding protein

Replication-dependent histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated, ending instead in a conserved stem-loop. The 3′ end of histone mRNA is required for histone mRNA translation, as is the stem-loop binding protein (SLBP), which binds the 3′ end of histone mRNA. We have identified five conserved residues in a 15-amino-acid region in the amino-terminal portion of SLBP, each of which is required for translation. Using a yeast two-hybrid screen, we identified a novel protein, SLBP-interacting protein 1 (SLIP1), that specifically interacts with this region. Mutations in any of the residues required for translation reduces SLIP1 binding to SLBP. The expression of SLIP1 in Xenopus oocytes together with human SLBP stimulates translation of a reporter mRNA ending in the stem-loop but not a reporter with a poly(A) tail. The expression of SLIP1 in HeLa cells also stimulates the expression of a green fluorescent protein reporter mRNA ending in a stem-loop. RNA interference-mediated downregulation of endogenous SLIP1 reduces the rate of translation of endogenous histone mRNA and also reduces cell viability. SLIP1 may function by bridging the 3′ end of the histone mRNA with the 5′ end of the mRNA, similar to the mechanism of translation of polyadenylated mRNAs.     

Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

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.     

A 3' exonuclease that specifically interacts with the 3' end of histone 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 stem-loop binding protein regulates translation of histone mRNA during mammalian oogenesis

Although messenger RNAs encoding the histone proteins are among the most abundant in mammalian oocytes, the mechanism regulating their translation has not been identified. The stem-loop binding protein (SLBP) binds to a highly conserved sequence in the 3'-untranslated region (utr) of the non-polyadenylated histone mRNAs in somatic cells and mediates their stabilization and translation. We previously showed that SLBP, which is expressed only during S-phase of proliferating cells, is expressed in growing oocytes at G2 of the cell cycle and accumulates substantially during meiotic maturation. We report here that elevating the amount of SLBP in immature (G2) oocytes is sufficient to increase translation of a reporter mRNA bearing the histone 3'-utr and endogenous histone synthesis and that this effect is not mediated through increased stability of the encoding mRNAs. We further report that translation of the reporter mRNA increases dramatically during meiotic maturation coincident with the accumulation of SLBP. Conversely, when SLBP accumulation during maturation is prevented using RNA interference, both translation of the reporter mRNA and synthesis of endogenous histones are significantly reduced. This effect is not mediated by a loss of the encoding mRNAs. Moreover, following fertilization, SLBP-depleted oocytes also show a significant decrease in pronuclear size and in the amount of acetylated histone detectable on the chromatin. These results demonstrate that histone synthesis in immature and maturing oocytes is governed by a translational control mechanism that is directly regulated by changes in the amount of SLBP.     

The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro

Metazoan replication-dependent histone mRNAs end in a conserved stem-loop rather than in the poly(A) tail found on all other mRNAs. The 3' end of histone mRNA binds a single class of proteins, the stem-loop binding proteins (SLBP). In Xenopus, there are two SLBPs: xSLBP1, the homologue of the mammalian SLBP, which is required for processing of histone pre-mRNA, and xSLBP2, which is expressed only during oogenesis and is bound to the stored histone mRNA in Xenopus oocytes. The stem-loop is required for efficient translation of histone mRNAs and substitutes for the poly(A) tail, which is required for efficient translation of other eucaryotic mRNAs. When a rabbit reticulocyte lysate is programmed with uncapped luciferase mRNA ending in the histone stem-loop, there is a three- to sixfold increase in translation in the presence of xSLBP1 while xSLBP2 has no effect on translation. Neither SLBP affected the translation of a luciferase mRNA ending in a mutant stem-loop that does not bind SLBP. Capped luciferase mRNAs ending in the stem-loop were injected into Xenopus oocytes after overexpression of either xSLBP1 or xSLBP2. Overexpression of xSLBP1 in the oocytes stimulated translation, while overexpression of xSLBP2 reduced translation of the luciferase mRNA ending in the histone stem-loop. A small region in the N-terminal portion of xSLBP1 is required to stimulate translation both in vivo and in vitro. An MS2-human SLBP1 fusion protein can activate translation of a reporter mRNA ending in an MS2 binding site, indicating that xSLBP1 only needs to be recruited to the 3' end of the mRNA but does not need to be directly bound to the histone stem-loop to activate translation.