Stem-loop Binding Protein Research Articles

The stem–loop binding protein stimulates histone translation at an early step in the initiation pathway

Metazoan replication-dependent histone mRNAs do not have a poly(A) tail but end instead in a conserved stem-loop structure. Efficient translation of these mRNAs is dependent on the stem-loop binding protein (SLBP). Here we explore the mechanism by which SLBP stimulates translation in vertebrate cells, using the tethered function assay and analyzing protein-protein interactions. We show for the first time that translational stimulation by SLBP increases during oocyte maturation and that SLBP stimulates translation at the level of initiation. We demonstrate that SLBP can interact directly with subunit h of eIF3 and with Paip1; however, neither of these interactions is sufficient to mediate its effects on translation. We find that Xenopus SLBP1 functions primarily at an early stage in the cap-dependent initiation pathway, targeting small ribosomal subunit recruitment. Analysis of IRES-driven translation in Xenopus oocytes suggests that SLBP activity requires eIF4E. We propose a model in which a novel factor contacts eIF4E bound to the 5' cap and SLBP bound to the 3' end simultaneously, mediating formation of an alternative end-to-end complex.

Expression of an RNAi-resistant SLBP restores proper S-phase progression

Metazoan histone mRNAs are unique in that they lack a 3'-polyadenylated tail, but instead end in a conserved stem loop that is bound by SLBP (stem-loop binding protein). SLBP is required for efficient histone mRNA synthesis and translation. Removal of SLBP by RNA interference causes an increase in the number of cells in S-phase and this effect can be reversed by expressing an exogenous SLBP resistant to the small interfering RNA. Cells with decreased SLBP levels progress slowly through S-phase when released from a double-thymidine block. Thus SLBP is required for efficient DNA replication probably because a decreased ability to assemble chromatin results in a decrease in the rate of DNA replication.

Expression of metazoan replication-dependent histone genes

Histone proteins are essential components of eukaryotic chromosomes. In metazoans, they are produced from the so-called replication-dependent histone genes. The biogenesis of histones is tightly coupled to DNA replication in a stoichiometric manner because an excess of histones is highly toxic for the cell. Therefore, a strict cell cycle-regulation of critical factors required for histone expression ensures exclusive S-phase expression. This review focuses on the molecular mechanisms responsible for such a fine expression regulation. Among these, a large part will be dedicated to post-transcriptional events occurring on histone mRNA, like histone mRNA 3′ end processing, nucleo-cytoplasmic mRNA export, translation and mRNA degradation. Many factors are involved, including an RNA-binding protein called HBP, also called SLBP (for hairpin- or stem-loop-binding protein) that binds to a conserved hairpin located in the 3′ UTR part of histone mRNA. HBP plays a pivotal role in the expression of histone genes since it is necessary for most of the steps of histone mRNA metabolism in the cell. Moreover, the strict S-phase expression pattern of histones is achieved through a fine cell cycle-regulation of HBP. A large part of the discussion will be centered on the critical role of HBP in histone biogenesis.

Nuclear Import of the Stem–Loop Binding Protein and Localization during the Cell Cycle

A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem-loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Impalpha/Impbeta and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Impalpha/Impbeta binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Impalpha/Impbeta pathway contributes to SLBP nuclear import in HeLa cells.

The stem-loop binding protein regulates translation of histone mRNA during mammalian oogenesis

The stem-loop binding protein regulates translation of histone mRNA during mammalian oogenesis

Nuclear Import of the Stem-Loop Binding Protein and Localization during the Cell Cycle

Nuclear Import of the Stem-Loop Binding Protein and Localization during the Cell Cycle

Nuclear export of metazoan replication-dependent histone mRNAs is dependent on RNA length and is mediated by TAP

Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated, ending instead in a conserved stem-loop sequence. Histone pre-mRNAs lack introns and are processed in the nucleus by a single cleavage step, which produces the mature 3' end of the mRNA. We have systematically examined the requirements for the nuclear export of a mouse histone mRNA using the Xenopus oocyte system. Histone mRNAs were efficiently exported when injected as mature mRNAs, demonstrating that the process of 3' end cleavage is not required for export factor binding. Export also does not depend on the stem-loop binding protein (SLBP) since mutations of the stem-loop that prevent SLBP binding and competition with a stem-loop RNA did not affect export. Only the length of the region upstream of the stem-loop, but not its sequence, was important for efficient export. Histone mRNA export was blocked by competition with constitutive transport element (CTE) RNA, indicating that the mRNA export receptor TAP is involved in histone mRNA export. Consistent with this observation, depletion of TAP from Drosophila cells by RNAi resulted in the restriction of mature histone mRNAs to the nucleus.

Crystallographic Structure of the Nuclease Domain of 3′hExo, a DEDDh Family Member, Bound to rAMP

A human 3'-5'-exoribonuclease (3'hExo) has recently been identified and shown to be responsible for histone mRNA degradation. Functionally, 3'hExo and a stem-loop binding protein (SLBP) target opposite faces of a unique highly conserved stem-loop RNA scaffold towards the 3' end of histone mRNA, which is composed of a 6 bp stem and a 4 nt loop, followed by an ACCCA sequence. Its Caenorhabditis elegans homologue, ERI-1, has been shown to degrade small interfering RNA in vitro and to function as a negative regulator of RNA interference in neuronal cells. We have determined the structure of the nuclease domain (Nuc) of 3'hExo complexed with rAMP in the presence of Mg2+ at 1.6 A resolution. The Nuc domain adopts an alpha/beta globular fold, with four acidic residues coordinating a binuclear metal cluster within the active site, whose topology is related to DEDDh exonuclease family members, despite a very low level of primary sequence identity. The two magnesium cations in the Nuc active site are coordinated to D134, E136, D234 and D298, and together with H293, which can potentially act as a general base, provide a platform for hydrolytic cleavage of bound RNA in the 3' --> 5' direction. The bound rAMP is positioned within a deep active-site pocket, with its purine ring close-packed with the hydrophobic F185 and L189 side-chains and its sugar 2'-OH and 3'-OH groups hydrogen bonded to backbone atoms of Nuc. There are striking similarities between the active sites of Nuc and epsilon186, an Escherichia coli DNA polymerase III proofreading domain, providing a common hydrolytic cleavage mechanism for RNA degradation and DNA editing, respectively.

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 N-terminal domain of the Drosophila histone mRNA binding protein, SLBP, is intrinsically disordered with nascent helical structure.

Stem-loop binding protein (SLBP) is a 31 kDa protein that is central to the regulation of histone mRNAs and is highly conserved in metazoans. In vertebrates, the N-terminal domain of SLBP has sequence determinants necessary for histone mRNA translation, SLBP degradation, cyclin binding, and histone mRNA import. We have used high-resolution NMR spectroscopy and circular dichroism to characterize the structural and dynamic features of this domain of SLBP from Drosophila (dSLBP). We report that the N-terminal domain of dSLBP is stably unfolded but has nascent helical structure at physiological pH and native-like solution conditions. The conformational and dynamic properties of the isolated domain are mimicked in a longer 175-residue region of the N-terminus, as well as in the full-length protein. Complete resonance assignments, secondary structure propensity, and motional properties of a 91-residue N-terminal domain (G17-K108) of dSLBP are reported here. The deviation of (1)H(alpha), (13)C(alpha), and (13)C(beta) chemical shifts from random coil reveals that there are four regions between residues I28-A45, S50-L57, S66-G75, and F91-N96 that have helical propensity. These regions also have small but positive heteronuclear NOEs, interresidue d(NN) NOEs, and small but significant protection from solvent exchange. However the lack of medium- and long-range NOEs in 3D (15)N- and (13)C-edited spectra, fast amide proton exchange rates (all greater than 1 s(-1)), and long (15)N relaxation (T(1), T(2)) times suggest that the domain from dSLBP does not adopt a well-defined tertiary fold. The backbone residual dipolar couplings (RDCs) for this domain are small and lie close to 0 Hz (+/-2 Hz) for most residues with no well-defined periodicity. The implications of this unfolded state for the function of dSLBP in regulating histone metabolism are discussed.

Electrostatic Contribution of Serine Phosphorylation to the Drosophila SLBP−Histone mRNA Complex

Unlike all other metazoan mRNAs, mRNAs encoding the replication-dependent histones are not polyadenylated but end in a unique 26 nucleotide stem-loop structure. The protein that binds the 3' end of histone mRNA, the stem-loop binding protein (SLBP), is essential for histone pre-mRNA processing, mRNA translation, and mRNA degradation. Using biochemical, biophysical, and nuclear magnetic resonance (NMR) experiments, we report the first structural insight into the mechanism of SLBP-RNA recognition. In the absence of RNA, phosphorylated and unphosphorylated forms of the RNA binding and processing domain (RPD) of Drosophila SLBP (dSLBP) possess helical secondary structure but no well-defined tertiary fold. Drosophila SLBP is phosphorylated at four out of five potential serine or threonine sites in the sequence DTAKDSNSDSDSD at the extreme C-terminus, and phosphorylation at these sites is necessary for histone pre-mRNA processing. Here, we provide NMR evidence for serine phosphorylation of the C-terminus using (31)P direct-detect experiments and show that both serine phosphorylation and RNA binding are necessary for proper folding of the RPD. The electrostatic effect of protein phosphorylation can be partially mimicked by a mutant form of SLBP wherein four C-terminal serines are replaced with glutamic acids. Hence, both RNA binding and protein phosphorylation are necessary for stabilization of the SLBP RPD.

TNFα-induced hepatocyte apoptosis is associated with alterations of the cell cycle and decreased stem loop binding protein

Background Inhibition of nuclear factor kappa B (NFκB) during liver regeneration induces hepatocyte apoptosis associated with normal DNA synthesis but decreased mitosis, suggesting that inhibition of NFκB impairs progression from S-phase through the G 2/M phase of the cell cycle. Our aim was to determine if inhibition of NFκB alters cell cycle characteristics in hepatocytes treated with tumor necrosis factor alpha (TNFα). Methods Primary hepatocytes from BALB/c mice were infected with adenoviruses expressing luciferase (control; AdLuc) or the IκB super-repressor (AdIκB) and treated with or without TNFα (30 ng/ml). Flow cytometry was performed (0 to 40 hours) to determine apoptosis and cell cycle progression. Reverse transcriptase-polymerase chain reaction and immunoblots assessed changes in cell cycle mediators and antiapoptotic factors. Results Primary hepatocytes treated with AdIκB and TNFα demonstrated significantly more S-phase cells (14% ± 3% vs 6% ± 2%, P<.05) at 14 hours compared with controls. Inhibition of NFκB with or without TNFα was associated with decreased expression of stem loop bind protein, a marker of cell cycle progression through S-phase. The NFκB-induced antiapoptotic proteins, iNOS and TRAF2, had decreased message at 9 and 12 hours, respectively, in TNFα- and AdIκB-treated cells. Conclusion Inhibition of NFκB in TNFα-treated primary mouse hepatocytes is associated with increased S-phase cell cycle retention and decreased stem loop bind protein.

The sea urchin stem-loop-binding protein: a maternally expressed protein that probably functions in expression of multiple classes of histone mRNA.

Following the completion of oogenesis and oocyte maturation, histone mRNAs are synthesized and stored in the sea urchin egg pronucleus. Histone mRNAs are the only mRNAs that are not polyadenylated but instead end in a stem-loop which has been conserved in evolution. The 3' end binds the stem-loop-binding protein (SLBP), and SLBP is required for histone pre-mRNA processing as well as translation of the histone mRNAs. A cDNA encoding a 59 kDa sea urchin SLBP (suSLBP) has been cloned from an oocyte cDNA library. The suSLBP contains an RNA-binding domain that is similar to the RNA-binding domain found in SLBPs from other species, although there is no similarity between the rest of the suSLBP and other SLBPs. The suSLBP is present at constant levels in eggs and for the first 12 h of development. The levels of suSLBP then decline and remain at a low level for the rest of embryogenesis. The suSLBP is concentrated in the egg pronucleus and is released from the nucleus only when cells enter the first mitosis. SuSLBP expressed by in vitro translation does not bind the stem-loop RNA, suggesting that suSLBP is modified to activate RNA binding in sea urchin embryos.

Drosophila stem-loop binding protein intracellular localization is mediated by phosphorylation and is required for cell cycle-regulated histone mRNA expression.

Stem-loop binding protein (SLBP) is an essential component of the histone pre-mRNA processing machinery. SLBP protein expression was examined during Drosophila development by using transgenes expressing hemagglutinin (HA) epitope-tagged proteins expressed from the endogenous Slbp promoter. Full-length HA-dSLBP complemented a Slbp null mutation, demonstrating that it was fully functional. dSLBP protein accumulates throughout the cell cycle, in contrast to the observed restriction of mammalian SLBP to S phase. dSLBP is located in both nucleus and cytoplasm in replicating cells, but it becomes predominantly nuclear during G2. dSLBP is present in mitotic cells and is down-regulated in G1 when cells exit the cell cycle. We determined whether mutation at previously identified phosphorylation sites, T120 and T230, affected the ability of the protein to restore viability and histone mRNA processing to dSLBP null mutants. The T120A SLBP restored viability and histone pre-mRNA processing. However, the T230A mutant, located in a conserved TPNK sequence in the RNA binding domain, did not restore viability and histone mRNA processing in vivo, although it had full activity in histone mRNA processing in vitro. The T230A protein is concentrated in the cytoplasm, suggesting that it is defective in nuclear targeting, and accounting for its failure to function in histone pre-mRNA processing in vivo.

Critical residues for RNA discrimination of the histone hairpin binding protein (HBP) investigated by the yeast three-hybrid system

The histone hairpin binding protein (HBP, also called SLBP, which stands for stem-loop binding protein) binds specifically to a highly conserved hairpin structure located in the 3′ UTR of the cell-cycle-dependent histone mRNAs. HBP consists of a minimal central RNA binding domain (RBD) flanked by an N- and C-terminal domain. The yeast three-hybrid system has been used to investigate the critical residues of the human HBP involved in the binding of its target hairpin structure. By means of negative selections followed by positive selections, we isolated mutant HBP species. Our results indicate tight relationships between the RBD and the N- and C-terminal domains.

CSP41a, a multifunctional RNA-binding protein, initiates mRNA turnover in tobacco chloroplasts.

Expression of chloroplast stem-loop binding protein (CSP)41a, a highly conserved chloroplast endoribonuclease, was reduced >90% by the expression of antisense RNA in Nicotiana tabacum. The most striking effects of this silencing were two- to sevenfold decreases in the degradation rates of rbcL, psbA, and petD transcripts in lysed chloroplast extracts. These results are consistent with the hypothesis that CSP41a participates in initiating mRNA turnover through endonucleolytic cleavages. Surprisingly, rbcL and psbA mRNAs accumulated to similar levels in wild-type and antisense lines. This suggested that decreased degradation was compensated by reduced transcription, which was confirmed using run-on transcription assays. The collective accumulation of petD-containing mRNAs in antisense plants decreased by 25% compared to wild-type controls. However, the relative levels of petD processing intermediates in wild-type and antisense plants did not differ, and there were no changes in petD 3'-end maturation, suggesting that CSP41a is not required for petD RNA processing. CSP41a is a Mg2+-dependent enzyme; therefore, extracts from antisense plants were tested at different Mg2+ concentrations. These experiments showed that the half-life of rbcL decreased as the Mg2+ concentration was reduced, and at <1 mm free Mg2+, conditions where CSP41a is nearly inactive in vitro, the rbcL degradation rate was similar in wild-type and antisense extracts, suggesting that CSP41a is normally bypassed under these conditions. Mg2+ has been shown to mediate RNA stability during chloroplast biogenesis, and our data suggest that regulation of CSP41a activity by Mg2+ is a component of this process.

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.

Read-through histone transcripts containing 3 ′ adenylate tails are zygotically expressed in Xenopus embryos and undergo processing to mature transcripts when introduced into oocyte nuclei

Messages encoding replication-dependent histone genes generally terminate with a stem-loop structure and lack polyadenylate tails. Adenylated histone transcripts were identified in Xenopus oocytes, though the role of the adenylate tracts is unknown. We report isolation of cDNAs from Xenopus embryos encoding histone mRNAs with 3 ′ adenylate tracts. They also contain targets for stem-loop binding protein and U7 snRNA, which are required for histone RNA processing. One sequence is a read-through transcript containing a complete version of the downstream gene from the anti-parallel strand, similar to the RNAs from lampbrush loops of Notophthalmus oocytes. We injected read-through transcripts into Xenopus oocyte nuclei and they were processed to mature histone RNAs. Our results suggest that addition of 3 ′ adenylate sequences might be a normal part of histone RNA synthesis. Also, these results shed light on the enigma of the developmental regulation of adenylated histone transcripts in Xenopus oocytes.

TNFα-induced hepatocyte apoptosis is associated with S-phase block and decreased stem-loop binding protein

TNFα-induced hepatocyte apoptosis is associated with S-phase block and decreased stem-loop binding protein

Phosphorylation of stem-loop binding protein (SLBP) on two threonines triggers degradation of SLBP, the sole cell cycle-regulated factor required for regulation of histone mRNA processing, at the end of S phase.

The replication-dependent histone mRNAs, the only eukaryotic mRNAs that do not have poly(A) tails, are present only in S-phase cells. Coordinate posttranscriptional regulation of histone mRNAs is mediated by the stem-loop at the 3' end of histone mRNAs. The protein that binds the 3' end of histone mRNA, stem-loop binding protein (SLBP), is required for histone pre-mRNA processing and is involved in multiple aspects of histone mRNA metabolism. SLBP is also regulated during the cell cycle, accumulating as cells enter S phase and being rapidly degraded as cells exit S phase. Mutation of any residues in a TTP sequence (amino acids 60 to 62) or mutation of a consensus cyclin binding site (amino acids 99 to 104) stabilizes SLBP in G2 and mitosis. These two threonines are phosphorylated in late S phase, as determined by mass spectrometry (MS) of purified SLBP from late S-phase cells, triggering SLBP degradation. Cells that express a stable SLBP still degrade histone mRNA at the end of S phase, demonstrating that degradation of SLBP is not required for histone mRNA degradation. Nuclear extracts from G1 and G2 cells are deficient in histone pre-mRNA processing, which is restored by addition of recombinant SLBP, indicating that SLBP is the only cell cycle-regulated factor required for histone pre-mRNA processing.