The sequence of the stem and flanking sequences at the 3' end of histone mRNA are critical determinants for the binding of the stem-loop binding protein.

Complexes of different electrophoretic mobility containing the stem-loop binding protein, a 45 kDa protein, bound to the stem-loop at the 3' end of histone mRNA, are present in both nuclear and cytoplasmic extracts from mammalian cells. We have determined the effect of changes in the loop, in the stem and in the flanking sequences on the affinity of the SLBP for the 3' end of histone mRNA. The sequence of the stem is particularly critical for SLBP binding. Specific sequences both 5' and 3' of the stem-loop are also required for high-affinity binding. Expanding the four base loop by one or two uridines reduced but did not abolish SLBP binding. RNA footprinting experiments show that the flanking sequences on both sides of the stem-loop are critical for efficient binding, but that cleavages in the loop do not abolish binding. Thus all three regions of the RNA sequence contribute to SLBP binding, suggesting that the 26 nt at the 3' end of histone mRNA forms a defined tertiary structure recognized by the SLBP.     

NMR structure and dynamics of the RNA-binding site for the histone mRNA stem-loop binding protein

The 3' end of replication-dependent histone mRNAs terminate in a conserved sequence containing a stem-loop. This 26-nt sequence is the binding site for a protein, stem-loop binding protein (SLBP), that is involved in multiple aspects of histone mRNA metabolism and regulation. We have determined the structure of the 26-nt sequence by multidimensional NMR spectroscopy. There is a 16-nt stem-loop motif, with a conserved 6-bp stem and a 4-nt loop. The loop is closed by a conserved U.A base pair that terminates the canonical A-form stem. The pyrimidine-rich 4-nt loop, UUUC, is well organized with the three uridines stacking on the helix, and the fourth base extending across the major groove into the solvent. The flanking nucleotides at the base of the hairpin stem do not assume a unique conformation, despite the fact that the 5' flanking nucleotides are a critical component of the SLBP binding site.     

Interaction of the histone mRNA hairpin with stem-loop binding protein (SLBP) and regulation of the SLBP-RNA complex by phosphorylation and proline isomerization

In metazoans, the majority of histone proteins are generated from replication-dependent histone mRNAs. These mRNAs are unique in that they are not polyadenylated but have a stem-loop structure in their 3' untranslated region. An early event in 3' end formation of histone mRNAs is the binding of stem-loop binding protein (SLBP) to the stem-loop structure. Here we provide insight into the mechanism by which SLBP contacts the histone mRNA. There are two binding sites in the SLBP RNA binding domain for the histone mRNA hairpin. The first binding site (Glu129-Val158) consists of a helix-turn-helix motif that likely recognizes the unpaired uridines in the loop of the histone hairpin and, upon binding, destabilizes the first G-C base pair at the base of the stem. The second binding site lies between residues Arg180 and Pro200, which appears to recognize the second G-C base pair from the base of the stem and possibly regions flanking the stem-loop structure. We show that the SLBP-histone mRNA complex is regulated by threonine phosphorylation and proline isomerization in a conserved TPNK sequence that lies between the two binding sites. Threonine phosphorylation increases the affinity of SLBP for histone mRNA by slowing the off rate for complex dissociation, whereas the adjacent proline acts as a critical hinge that may orient the second binding site for formation of a stable SLBP-histone mRNA complex. The nuclear magnetic resonance and kinetic studies presented here provide a framework for understanding how SLBP recognizes histone mRNA and highlight possible structural roles of phosphorylation and proline isomerization in RNA binding proteins in remodeling ribonucleoprotein complexes.     

Changes in the stem-loop at the 3' terminus of histone mRNA affects its nucleocytoplasmic transport and cytoplasmic regulation.

The stem-loop structure at the 3' end of replication-dependent histone mRNA is required for efficient pre-mRNA processing, localization of histone mRNA to the polyribosomes, and regulation of histone mRNA degradation. A protein, the stem-loop binding protein (SLBP), binds the 3' end of histone mRNA and is thought to mediate some or all of these processes. A mutant histone mRNA with two nucleotide changes in the loop was constructed and found to be transported inefficiently to the cytoplasm. The mutant histone mRNA, unlike the wild-type histone mRNA, was not rapidly degraded when DNA synthesis is inhibited, and was not stabilized upon inhibition of protein synthesis. The stem-loop binding protein (SLBP) has between a 20-50 fold greater affinity for the wild type histone stem-loop structure than for the mutant stem-loop structure, suggesting that the alteration in the efficiency of transport and the normal degradation pathway in histone mRNA may be due to the reduced affinity of the mutant stem-loop for the SLBP.     

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 polyribosomal protein bound to the 3' end of histone mRNA can function in histone pre-mRNA processing.

Cell cycle-regulated histone mRNAs end in a conserved 26-nt sequence that can form a stem-loop with a six-base stem and a four-base loop. The 3' end of histone mRNA has distinct functions in the nucleus and in the cytoplasm. In the nucleus it functions in pre-mRNA processing and transport, whereas in the cytoplasm it functions in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), present in both nuclei and polyribosomes, is likely the trans-acting factor that binds to the 3' end of mature histone mRNA and mediates its function. A nuclear extract that efficiently processes histone pre-mRNA was prepared from mouse myeloma cells. The factor(s) that bind to the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide containing the conserved stem-loop sequence. Using this depleted extract which is deficient in histone pre-mRNA processing, we show that SLBP found in polyribosomes can restore processing, suggesting that SLBP associates with histone pre-mRNA in the nucleus, participates in processing, and then accompanies the mature mRNA to the cytoplasm.     

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.     

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.     

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.     

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.     

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.     

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.     

Early evolution of histone mRNA 3' end processing

The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.     

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.     

Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

Previous studies indicate that the 3' terminal 46 nt of the RNA genome of hepatitis C virus (HCV) are highly conserved among different viral strains and essential for RNA replication. Here, we describe a mutational analysis of the 3' terminal hairpin (stem-loop I) that is putatively formed by this sequence and demonstrate its role in replication of the viral RNA. We show that single base substitutions within the 6-nt loop at positions adjacent to the stem abrogate replication of a subgenomic RNA, whereas substitutions in the three apical nucleotides were well tolerated without loss of replication competence. Single point mutations were also well tolerated within the middle section of the duplex, but not at the penultimate nucleotide positions near either end of the stem. However, complementary substitutions at the -19 and -28 positions (from the 3' end) restored replication competence, providing strong evidence for the existence of the structure and its involvement in RNA replication. This was confirmed by rescue of replicating RNAs from mutants containing complementary 10-nt block substitutions at the base of the stem. Each of these RNAs contained an additional U at the 3' terminus. Further experiments indicated a strong preference for U at the 3' terminal position (followed in order by C, A, and G), and a G at the -2 position. These features of stem-loop I are likely to facilitate recognition of the 3' end of the viral RNA by the viral RNA replicase.     

Formation of the 3' end of histone mRNA

All metazoan messenger RNAs, with the exception of the replication-dependent histone mRNAs, terminate at the 3' end with a poly(A) tail. Replication-dependent histone mRNAs end instead in a conserved 26-nucleotide sequence that contains a 16-nucleotide stem-loop. Formation of the 3' end of histone mRNA occurs by endonucleolytic cleavage of pre-mRNA releasing the mature mRNA from the chromatin template. Cleavage requires several trans-acting factors, including a protein, the stem-loop binding protein (SLBP), which binds the 26-nucleotide sequence; and a small nuclear RNP, U7 snRNP. There are probably additional factors also required for cleavage. One of the functions of the SLBP is to stabilize binding of the U7 snRNP to the histone pre-mRNA. In the nucleus, both U7 snRNP and SLBP are present in coiled bodies, structures that are associated with histone genes and may play a direct role in histone pre-mRNA processing in vivo. One of the major regulatory events in the cell cycle is regulation of histone pre-mRNA processing, which is at least partially mediated by cell-cycle regulation of the levels of the SLBP protein.