Stem-loop Binding Protein Research Articles

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.

Structure of Histone mRNA Stem-Loop, Human Stem-Loop Binding Protein, and 3′hExo Ternary Complex

Metazoan replication-dependent histone messenger RNAs (mRNAs) have a conserved stem-loop (SL) at their 3'-end. The stem-loop binding protein (SLBP) specifically recognizes the SL to regulate histone mRNA metabolism, and the 3'-5' exonuclease 3'hExo trims its 3'-end after processing. We report the crystal structure of a ternary complex of human SLBP RNA binding domain, human 3'hExo, and a 26-nucleotide SL RNA. Only one base of the SL is recognized specifically by SLBP, and the two proteins primarily recognize the shape of the RNA. SLBP and 3'hExo have no direct contact with each other, and induced structural changes in the loop of the SL mediate their cooperative binding. The 3' flanking sequence is positioned in the 3'hExo active site, but the ternary complex limits the extent of trimming.

Rapid degradation of replication-dependent histone mRNAs largely occurs on mRNAs bound by nuclear cap-binding proteins 80 and 20

The translation of mammalian messenger RNAs (mRNAs) can be driven by either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF)4E. Although CBP80/20-dependent translation (CT) is known to be coupled to an mRNA surveillance mechanism termed nonsense-mediated mRNA decay (NMD), its molecular mechanism and biological role remain obscure. Here, using a yeast two-hybrid screening system, we identify a stem-loop binding protein (SLBP) that binds to a stem-loop structure at the 3′-end of the replication-dependent histone mRNA as a CT initiation factor (CTIF)-interacting protein. SLBP preferentially associates with the CT complex of histone mRNAs, but not with the eIF4E-depedent translation (ET) complex. Several lines of evidence indicate that rapid degradation of histone mRNA on the inhibition of DNA replication largely takes place during CT and not ET, which has been previously unappreciated. Furthermore, the ratio of CBP80/20-bound histone mRNA to eIF4E-bound histone mRNA is larger than the ratio of CBP80/20-bound polyadenylated β-actin or eEF2 mRNA to eIF4E-bound polyadenylated β-actin or eEF2 mRNA, respectively. The collective findings suggest that mRNAs harboring a different 3′-end use a different mechanism of translation initiation, expanding the repertoire of CT as a step for determining the fate of histone mRNAs.

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.

An oocyte‐preferential histone mRNA stem‐loop‐binding protein like is expressed in several mammalian species

During early embryo development, chromatin packaging is sustained by histones of maternal origin. Most histone messenger RNAs are not polyadenylated, but rather end in an evolutionarily conserved stem-loop that controls RNA processing, nucleocytoplasmic transport, stability, and translation via interactions with a specific protein named stem-loop-binding protein (SLBP). In mouse oocytes, mSLBP is synthesized abundantly during maturation and activates histone translation. In Xenopus, xSLBP is present in stage-VI oocytes, but histone mRNA is protected from premature translation by the oocyte-specific Xenopus SLBP2 (xSLBP2) protein; during maturation xSLBP2 replacement by xSLBP results in histone synthesis. Here, we report the first experimental evidence and characterization of a mammalian SLBP2 ortholog. Bovine bSLBP and bSLBP2 display distinct expression patterns throughout oocyte maturation and pre-implantation embryo development. From the immature oocyte to the morula, bSLBP2 is concentrated in the nucleus, while it is homogeneously distributed throughout the cytoplasm in mature oocytes. A putative SLBP2 gene is conserved in the genome of several mammalian species, and the corresponding transcripts were detected in rat, dog, horse, and pig oocytes. By contrast, a pseudogene is found in mouse, human, and rabbit. Altogether, our data suggest that the availability of histones in oocytes is regulated by an alternative mechanism in bovine and other species as compared to mouse and frog.

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.

Interaction of the Histone mRNA Hairpin with Stem–Loop Binding Protein (SLBP) and Regulation of the SLBP–RNA Complex by Phosphorylation and Proline Isomerization

Interaction of the Histone mRNA Hairpin with Stem–Loop Binding Protein (SLBP) and Regulation of the SLBP–RNA Complex by Phosphorylation and Proline Isomerization

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

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

Mutation of the 5′-Untranslated Region Stem-Loop Structure Inhibits α1(I) Collagen Expression in Vivo

Type I collagen is a heterotrimeric extracellular matrix protein consisting of two α1(I) chains and one α2(I) chain. During liver fibrosis, activated hepatic stellate cells (HSCs) are the major source of the type I collagen that accumulates in the damaged tissue. Expression of α1(I) and α2(I) collagen mRNA is increased 60-fold compared with quiescent stellate cells and is due predominantly to post-transcriptional message regulation. Specifically, a stem-loop structure in the 5'-untranslated region of α1(I) collagen mRNA may regulate mRNA expression in activated HSCs through its interaction with stem-loop binding proteins. The stem-loop may also be necessary for efficient production and folding of the type I collagen heterotrimer. To assess the role of the stem-loop in type I collagen expression in vivo, we generated a knock-in mouse harboring a mutation that abolished the stem-loop structure. Heterozygous and homozygous knock-in mice exhibited a normal phenotype. However, steady-state levels of α1(I) collagen mRNA decreased significantly in homozygous mutant MEFs as well as HSCs; intracellular and secreted type I collagen protein levels also decreased. Homozygous mutant mice developed less liver fibrosis. These results confirm an important role of the 5' stem-loop in regulating type I collagen mRNA and protein expression and provide a mouse model for further study of collagen-associated diseases.

Muscle wasted: a novel component of theDrosophilahistone locus body required for muscle integrity

Skeletal muscles arise by cellular differentiation and regulated gene expression. Terminal differentiation programmes such as muscle growth, extension and attachment to the epidermis, lead to maturation of the muscles. These events require changes in chromatin organization as genes are differentially regulated. Here, we identify and characterise muscle wasted (mute), a novel component of the Drosophila histone locus body (HLB). We demonstrate that a mutation in mute leads to severe loss of muscle mass and an increase in levels of normal histone transcripts. Importantly, Drosophila Myocyte enhancer factor 2 (Mef2), a central myogenic differentiation factor, and how, an RNA binding protein required for muscle and tendon cell differentiation, are downregulated. Mef2 targets are, in turn, misregulated. Notably, the degenerating muscles in mute mutants show aberrant localisation of heterochromatin protein 1 (HP1). We further show a genetic interaction between mute and the Stem-loop binding protein (Slbp) and a loss of muscle striations in Lsm11 mutants. These data demonstrate a novel role of HLB components and histone processing factors in the maintenance of muscle integrity. We speculate that mute regulates terminal muscle differentiation possibly through heterochromatic reorganisation.

Crystal structure of an eIF4G-like protein from Danio rerio

The gene LOC791917 Danio rerio (zebrafish) encodes a protein annotated in the UniProt knowledgebase1 as the “middle domain of eukaryotic initiation factor 4G domain containing protein b” (MIF4Gdb). Its molecular weight is 25.8 kDa, and it comprises 222 amino acid residues. BLAST searches revealed homologues of D. rerio MIF4Gdb in many eukaryotes including humans.2 The homologues and MIF4Gdb were identified as members of the Pfam family, MIF4G (PF02854), which is named after the middle domain of eukaryotic initiation factor 4G (eIF4G).3-5 eIF4G is a component of eukaryotic translational initiation complex, and contains binding sites for other initiation factors, suggesting its critical role in translational initiation.6 The MIF4G domain also occurs in several other proteins involved in RNA metabolism, including the Nonsense-mediated mRNA decay 2 protein (NMD2/UPF2), and the nuclear cap-binding protein 80-kD subunit (CBP80).5 Sequence and structure analysis of the MIF4G domains in many proteins indicates that the domain assumes all helical fold and has tandem repeated motifs.5,7 The zebrafish protein described here has homology to domains of other proteins variously referred to as NIC-containing proteins (NMD2, eIF4G, CBP80). The biological function of D. rerio MIF4Gdb has not yet been experimentally characterized, and the annotation is based on amino acid sequence comparison. D. rerio MIF4Gdb did not share more than 25% sequence identity with any protein for which the three-dimensional structure is known and was selected as a target for structure determination by the Center for Eukaryotic Structural Genomics (CESG). Here, we report the crystal structure of D. rerio MIF4Gdb (UniGene code Dr.79360, UniProt code {"type":"entrez-protein","attrs":{"text":"Q5EAQ1","term_id":"82178873"}}Q5EAQ1, CESG target number GO.79294).

Loss of the Histone Pre-mRNA Processing Factor Stem-Loop Binding Protein in Drosophila Causes Genomic Instability and Impaired Cellular Proliferation

BackgroundMetazoan replication-dependent histone mRNAs terminate in a conserved stem-loop structure rather than a polyA tail. Formation of this unique mRNA 3′ end requires Stem-loop Binding Protein (SLBP), which directly binds histone pre-mRNA and stimulates 3′ end processing. The 3′ end stem-loop is necessary for all aspects of histone mRNA metabolism, including replication coupling, but its importance to organism fitness and genome maintenance in vivo have not been characterized.Methodology/Principal FindingsIn Drosophila, disruption of the Slbp gene prevents normal histone pre-mRNA processing and causes histone pre-mRNAs to utilize the canonical 3′ end processing pathway, resulting in polyadenylated histone mRNAs that are no longer properly regulated. Here we show that Slbp mutants display genomic instability, including loss of heterozygosity (LOH), increased presence of chromosome breaks, tetraploidy, and changes in position effect variegation (PEV). During imaginal disc growth, Slbp mutant cells show defects in S phase and proliferate more slowly than control cells.Conclusions/SignificanceThese data are consistent with a model in which changing the 3′ end of histone mRNA disrupts normal replication-coupled histone mRNA biosynthesis and alters chromatin assembly, resulting in genomic instability, inhibition of cell proliferation, and impaired development.

Proteasomal Activity Is Required to Initiate and to Sustain Translational Activation of Messenger RNA Encoding the Stem-Loop-Binding Protein During Meiotic Maturation in Mice1

Developmentally regulated translation plays a key role in controlling gene expression during oogenesis. In particular, numerous mRNA species are translationally repressed in growing oocytes and become translationally activated during meiotic maturation. While many studies have focused on a U-rich sequence, termed the cytoplasmic polyadenylation element (CPE), located in the 3'-untranslated region (UTR) and the CPE-binding protein (CPEB) 1, multiple mechanisms likely contribute to translational control in oocytes. The stem-loop-binding protein (SLBP) is expressed in growing oocytes, where it is required for the accumulation of nonpolyadenylated histone mRNAs, and then accumulates substantially during meiotic maturation. We report that, in immature oocytes, Slbp mRNA carries a short poly(A) tail, and is weakly translated, and that a CPE-like sequence in the 3'-UTR is required to maintain this low activity. During maturation, Slbp mRNA becomes polyadenylated and translationally activated. Unexpectedly, proteasomal activity is required both to initiate and to sustain translational activation. This proteasomal activity is not required for the polyadenylation of Slbp mRNA during early maturation; however, it is required for a subsequent deadenylation of the mRNA that occurs during late maturation. Moreover, although CPEB1 is degraded during maturation, inhibiting its degradation by blocking mitogen-activated protein kinase 1/3 activity does not prevent the accumulation of SLBP, indicating that CPEB1 is not the protein whose degradation is required for translational activation of Slbp mRNA. These results identify a new role for proteasomal activity in initiating and sustaining translational activation during meiotic maturation.

Three Proteins of the U7-Specific Sm Ring Function as the Molecular Ruler To Determine the Site of 3′-End Processing in Mammalian Histone Pre-mRNA

Cleavage of histone pre-mRNAs at the 3' end is guided by the U7 snRNP, which is a component of a larger 3'-end processing complex. To identify other components of this complex, we isolated proteins that stably associate with a fragment of histone pre-mRNA containing all necessary processing elements and a biotin affinity tag at the 5' end. Among the isolated proteins, we identified three well-characterized processing factors: the stem-loop binding protein (SLBP), which interacts with the stem-loop structure upstream of the cleavage site, and both Lsm11 and SmB, which are components of the U7-specific Sm ring. We also identified 3'hExo/Eri-1, a multifunctional 3' exonuclease that is known to trim the 3' end of 5.8S rRNA. 3'hExo primarily binds to the downstream portion of the stem-loop structure in mature histone mRNA, with the upstream portion being occupied by SLBP. The two proteins bind their respective RNA sites in a cooperative manner, and 3'hExo can recruit SLBP to a mutant stem-loop that itself does not interact with SLBP. UV-cross-linking studies used to characterize interactions within the processing complex demonstrated that 3'hExo also interacts in a U7-dependent manner with unprocessed histone pre-mRNA. However, this interaction is not required for the cleavage reaction. The region between the cleavage site and the U7-binding site interacts with three low-molecular-weight proteins, which were identified as components of the U7-specific Sm core: SmB, SmD3, and Lsm10. These proteins likely rigidify the substrate and function as the molecular ruler in determining the site of cleavage.

Decapping of mRNA containing the histone 3′‐stem loop requires recruitment of stem loop binding protein (SLBP)

Modifications at the 5' and 3' ends of eukaryotic mRNA determine both mRNA stability and translational efficiency. One pathway for mRNA degradation involves decapping by Dcp2. We previously developed a phosphorothioate cap analog, m27.2'−OGppspG (S‐ARCA), that is resistant to Dcp2 and confers a longer in vivo half‐life when incorporated into mRNA, indicating that decapping contributes to degradation of polyadenylated mRNAs (Grudzien et al., RNA 13, 1745, 2007). We have now extended this to the 3′‐terminal stem loop (SL) of histone mRNA. The SL is recognized by the stem loop binding protein (SLBP), which is required for the regulation of mRNA processing, turnover, and translation. Luciferase mRNA containing the 5′‐S‐ARCA and 3′‐SL (S‐ARCA‐Luc‐SL) had a 1.8‐fold longer half‐life when introduced into HeLa cells than ARCA‐Luc‐SL, indicating that degradation of SL‐containing mRNAs also involves decapping. mRNA containing a mutated SL that cannot bind SLBP could not be translated yet had a half‐life that was ~3 fold longer than that of S‐ARCA‐Luc‐SL. Interestingly, the increase in stability was independent of the cap, since both mRNAs had similar half‐lives. This result is consistent with translation being required for rapid degradation of histone mRNA, and with SLBP playing a role in the recruitment of the decapping machinery. (Supported by grants No. GM20818 and GM29832 from the NIGMS and No. 55005604 from the HHMI)

Maternally encoded stem-loop-binding protein is degraded in 2-cell mouse embryos by the co-ordinated activity of two separately regulated pathways

Oocytes accumulate mRNAs and proteins that direct early embryonic development. Although subsequent development requires the timely degradation of these maternal products, little is known of the underlying mechanisms. The stem-loop-binding protein (SLBP), which regulates the stability and translation of mRNAs encoding histones and is synthesized during S-phase and degraded during G2 in somatic cells, accumulates during oogenesis. Maternal SLBP is required for mouse embryos to develop beyond the 2-cell stage, but must be degraded to allow the cell-cycle-regulated expression of somatic cells to be established. We report that the quantity of maternal SLBP changes little following fertilization until 44–52 h post-hCG, corresponding to mid-/late G2 of the 2-cell stage, when it decreases by 75%. Efficient degradation requires two pathways. The first requires activity of cyclin-dependent kinases (cdk) and embryonic transcription, preferentially targets nuclear SLBP, and likely corresponds to the pathway that degrades SLBP at G2 in somatic cells. The second does not require cdk activity or transcription and becomes active at 44–52 h post-hCG independently of cell-cycle progression to mid-/late G2, but is not solely regulated by the time elapsed since hCG injection. Thus, the co-ordinated activity of two separately regulated pathways eliminates maternally encoded SLBP from early mouse embryos.

Knockdown of SLBP results in nuclear retention of histone mRNA

Histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated. Synthesis of mature histone mRNA requires only a single processing reaction: an endonucleolytic cleavage between a conserved stem-loop and a purine-rich downstream element to form the 3' end. The stem-loop binding protein (SLBP) is required for processing, and following processing, histone mRNA is transported to the cytoplasm, where SLBP participates in translation of the histone mRNA and is also involved in regulation of histone mRNA degradation. Here we present an analysis of histone mRNA metabolism in cells with highly reduced levels of SLBP using RNA interference. Knocking down SLBP in U2OS cells results in a reduction in the rate of cell growth and an accumulation of cells in S-phase. Surprisingly, there is only a modest (twofold) decrease in histone mRNA levels. Much of histone mRNA in the SLBP knockdown cells is properly processed but is retained in the nucleus. The processed histone mRNA in SLBP knockdown cells is not rapidly degraded when DNA replication is inhibited. These results suggest a previously undescribed role for SLBP in histone mRNA export.

Loss of the Histone Pre-mRNA Processing Factor Stem-Loop Binding Protein in Drosophila Causes Genomic Instability and Impaired Cellular Proliferation

Loss of the Histone Pre-mRNA Processing Factor Stem-Loop Binding Protein in Drosophila Causes Genomic Instability and Impaired Cellular Proliferation

Phosphorylation of Threonine 61 by Cyclin A/Cdk1 Triggers Degradation of Stem-Loop Binding Protein at the End of S Phase

Histone mRNA levels are cell cycle regulated, and a major regulatory mechanism is restriction of stem-loop binding protein (SLBP) to S phase. Degradation of SLBP at the end of S phase results in cessation of histone mRNA biosynthesis, preventing accumulation of histone mRNA until SLBP is synthesized just before entry into the next S phase. Degradation of SLBP requires an SFTTP (58 to 62) and KRKL (95 to 98) sequence, which is a putative cyclin binding site. A fusion protein with the 58-amino-acid sequence of SLBP (amino acids 51 to 108) fused to glutathione S-transferase (GST) is sufficient to mimic SLBP degradation at late S phase. Using GST-SLBP fusion proteins as a substrate, we show that cyclin A/Cdk1 phosphorylates Thr61. Furthermore, knockdown of Cdk1 by RNA interference stabilizes SLBP at the end of S phase. Phosphorylation of Thr61 is necessary for subsequent phosphorylation of Thr60 by CK2 in vitro. Inhibitors of CK2 also prevent degradation of SLBP at the end of S phase. Thus, phosphorylation of Thr61 by cyclin A/Cdk1 primes phosphorylation of Thr60 by CK2 and is responsible for initiating SLBP degradation. We conclude that the increase in cyclin A/Cdk1 activity at the end of S phase triggers degradation of SLBP at S/G(2).