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William F. Marzluff - One of the best experts on this subject based on the ideXlab platform.
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interaction of the histone mrna hairpin with stem loop binding protein slbp and regulation of the slbp rna complex by phosphorylation and proline isomerization
Biochemistry, 2012Co-Authors: William F. Marzluff, Minyou Zhang, Marco Tonelli, Roopa ThaparAbstract: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...
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Mutation of the 5′-Untranslated Region Stem-Loop Structure Inhibits α1(I) Collagen Expression in Vivo
The Journal of biological chemistry, 2010Co-Authors: Christopher J. Parsons, William F. Marzluff, Branko Stefanovic, Ekihiro Seki, Tomonori Aoyama, Anne M. Latour, Richard A. Rippe, David A. BrennerAbstract: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.
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The Histone 3′-Terminal Stem-Loop-Binding Protein Enhances Translation through a Functional and Physical Interaction with Eukaryotic Initiation Factor 4G (eIF4G) and eIF3
Molecular and cellular biology, 2002Co-Authors: Jun Ling, Simon J. Morley, Virginia M. Pain, William F. Marzluff, Daniel R. GallieAbstract:Metazoan cell cycle-regulated histone mRNAs are unique cellular mRNAs in that they terminate in a highly conserved Stem-Loop structure instead of a poly(A) tail. Not only is the Stem-Loop structure necessary for 3'-end formation but it regulates the stability and translational efficiency of histone mRNAs. The histone Stem-Loop structure is recognized by the Stem-Loop-binding protein (SLBP), which is required for the regulation of mRNA processing and turnover. In this study, we show that SLBP is required for the translation of mRNAs containing the histone Stem-Loop structure. Moreover, we show that the translation of mRNAs ending in the histone Stem-Loop is stimulated in Saccharomyces cerevisiae cells expressing mammalian SLBP. The translational function of SLBP genetically required eukaryotic initiation factor 4E (eIF4E), eIF4G, and eIF3, and expressed SLBP coisolated with S. cerevisiae initiation factor complexes that bound the 5' cap in a manner dependent on eIF4G and eIF3. Furthermore, eIF4G coimmunoprecipitated with endogenous SLBP in mammalian cell extracts and recombinant SLBP and eIF4G coisolated. These data indicate that SLBP stimulates the translation of histone mRNAs through a functional interaction with both the mRNA Stem-Loop and the 5' cap that is mediated by eIF4G and eIF3.
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the histone 3 terminal stem loop binding protein enhances translation through a functional and physical interaction with eukaryotic initiation factor 4g eif4g and eif3
Molecular and Cellular Biology, 2002Co-Authors: Jun Ling, Simon J. Morley, Virginia M. Pain, William F. Marzluff, Daniel R. GallieAbstract:Metazoan cell cycle-regulated histone mRNAs are unique cellular mRNAs in that they terminate in a highly conserved Stem-Loop structure instead of a poly(A) tail. Not only is the Stem-Loop structure necessary for 3'-end formation but it regulates the stability and translational efficiency of histone mRNAs. The histone Stem-Loop structure is recognized by the Stem-Loop-binding protein (SLBP), which is required for the regulation of mRNA processing and turnover. In this study, we show that SLBP is required for the translation of mRNAs containing the histone Stem-Loop structure. Moreover, we show that the translation of mRNAs ending in the histone Stem-Loop is stimulated in Saccharomyces cerevisiae cells expressing mammalian SLBP. The translational function of SLBP genetically required eukaryotic initiation factor 4E (eIF4E), eIF4G, and eIF3, and expressed SLBP coisolated with S. cerevisiae initiation factor complexes that bound the 5' cap in a manner dependent on eIF4G and eIF3. Furthermore, eIF4G coimmunoprecipitated with endogenous SLBP in mammalian cell extracts and recombinant SLBP and eIF4G coisolated. These data indicate that SLBP stimulates the translation of histone mRNAs through a functional interaction with both the mRNA Stem-Loop and the 5' cap that is mediated by eIF4G and eIF3.
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The Stem-Loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro.
Molecular and cellular biology, 2002Co-Authors: Ricardo Sanchez, William F. MarzluffAbstract: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.
Masaru Tomita - One of the best experts on this subject based on the ideXlab platform.
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Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein.
The Biochemical journal, 2006Co-Authors: Akio Kanai, Asako Sato, Jun Imoto, Masaru TomitaAbstract:Using a Stem-Loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the Stem-Loop structure at a high temperature (75 degrees C) with an apparent dissociation constant of 0.6 microM. A similar Stem-Loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this Stem-Loop structure. In vitro translation analysis using chimaeric constructs containing the Stem-Loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the Stem-Loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.
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Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein
Biochemical Journal, 2005Co-Authors: Akio Kanai, Asako Sato, Jun Imoto, Masaru TomitaAbstract:Using a stem–loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem–loop structure at a high temperature (75 °C) with an apparent dissociation constant of 0.6 μM. A similar stem–loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem–loop structure. In vitro translation analysis using chimaeric constructs containing the stem–loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem–loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine–Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.
Akio Kanai - One of the best experts on this subject based on the ideXlab platform.
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Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein.
The Biochemical journal, 2006Co-Authors: Akio Kanai, Asako Sato, Jun Imoto, Masaru TomitaAbstract:Using a Stem-Loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the Stem-Loop structure at a high temperature (75 degrees C) with an apparent dissociation constant of 0.6 microM. A similar Stem-Loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this Stem-Loop structure. In vitro translation analysis using chimaeric constructs containing the Stem-Loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the Stem-Loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.
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Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein
Biochemical Journal, 2005Co-Authors: Akio Kanai, Asako Sato, Jun Imoto, Masaru TomitaAbstract:Using a stem–loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem–loop structure at a high temperature (75 °C) with an apparent dissociation constant of 0.6 μM. A similar stem–loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem–loop structure. In vitro translation analysis using chimaeric constructs containing the stem–loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem–loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine–Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.
Daniel R. Gallie - One of the best experts on this subject based on the ideXlab platform.
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The Histone 3′-Terminal Stem-Loop-Binding Protein Enhances Translation through a Functional and Physical Interaction with Eukaryotic Initiation Factor 4G (eIF4G) and eIF3
Molecular and cellular biology, 2002Co-Authors: Jun Ling, Simon J. Morley, Virginia M. Pain, William F. Marzluff, Daniel R. GallieAbstract:Metazoan cell cycle-regulated histone mRNAs are unique cellular mRNAs in that they terminate in a highly conserved Stem-Loop structure instead of a poly(A) tail. Not only is the Stem-Loop structure necessary for 3'-end formation but it regulates the stability and translational efficiency of histone mRNAs. The histone Stem-Loop structure is recognized by the Stem-Loop-binding protein (SLBP), which is required for the regulation of mRNA processing and turnover. In this study, we show that SLBP is required for the translation of mRNAs containing the histone Stem-Loop structure. Moreover, we show that the translation of mRNAs ending in the histone Stem-Loop is stimulated in Saccharomyces cerevisiae cells expressing mammalian SLBP. The translational function of SLBP genetically required eukaryotic initiation factor 4E (eIF4E), eIF4G, and eIF3, and expressed SLBP coisolated with S. cerevisiae initiation factor complexes that bound the 5' cap in a manner dependent on eIF4G and eIF3. Furthermore, eIF4G coimmunoprecipitated with endogenous SLBP in mammalian cell extracts and recombinant SLBP and eIF4G coisolated. These data indicate that SLBP stimulates the translation of histone mRNAs through a functional interaction with both the mRNA Stem-Loop and the 5' cap that is mediated by eIF4G and eIF3.
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the histone 3 terminal stem loop binding protein enhances translation through a functional and physical interaction with eukaryotic initiation factor 4g eif4g and eif3
Molecular and Cellular Biology, 2002Co-Authors: Jun Ling, Simon J. Morley, Virginia M. Pain, William F. Marzluff, Daniel R. GallieAbstract:Metazoan cell cycle-regulated histone mRNAs are unique cellular mRNAs in that they terminate in a highly conserved Stem-Loop structure instead of a poly(A) tail. Not only is the Stem-Loop structure necessary for 3'-end formation but it regulates the stability and translational efficiency of histone mRNAs. The histone Stem-Loop structure is recognized by the Stem-Loop-binding protein (SLBP), which is required for the regulation of mRNA processing and turnover. In this study, we show that SLBP is required for the translation of mRNAs containing the histone Stem-Loop structure. Moreover, we show that the translation of mRNAs ending in the histone Stem-Loop is stimulated in Saccharomyces cerevisiae cells expressing mammalian SLBP. The translational function of SLBP genetically required eukaryotic initiation factor 4E (eIF4E), eIF4G, and eIF3, and expressed SLBP coisolated with S. cerevisiae initiation factor complexes that bound the 5' cap in a manner dependent on eIF4G and eIF3. Furthermore, eIF4G coimmunoprecipitated with endogenous SLBP in mammalian cell extracts and recombinant SLBP and eIF4G coisolated. These data indicate that SLBP stimulates the translation of histone mRNAs through a functional interaction with both the mRNA Stem-Loop and the 5' cap that is mediated by eIF4G and eIF3.
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The Histone 3′-Terminal Stem-Loop is Necessary for Translation in Chinese Hamster Ovary Cells
Nucleic acids research, 1996Co-Authors: Daniel R. Gallie, Nancy J. Lewis, William F. MarzluffAbstract:The metazoan cell cycle-regulated histone mRNAs are the only known cellular mRNAs that do not terminate in a poly(A) tall. Instead, mammalian histone mRNAs terminate in a highly conserved Stem-Loop structure which is required for 3'-end processing and regulates mRNA stability. The poly(A) tail not only regulates translational efficiency and mRNA stability but is required for the function of the cap in translation (m(7)GpppN). We show that the histone terminal Stem-Loop is functionally similar to a poly(A) tail in that it enhances translational efficiency and is co-dependent on a cap in order to establish an efficient level of translation. The histone Stem-Loop is sufficient and necessary to increase the translation of reporter mRNA in transfected Chinese hamster ovary cells but must be positioned at the 3'-terminus in order to function optimally. Mutations within the conserved stem or loop regions reduced its ability to facilitate translation. All histone mRNAs in higher plants are polyadenylated. The histone Stem-Loop did not function to influence translational efficiency or mRNA stability in plant protoplasts. These data demonstrate that the histone stem/loop directs efficient translation and that it is functionally analogous to a poly(A) tail.
Pascale Legault - One of the best experts on this subject based on the ideXlab platform.
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NMR structure of varkud satellite ribozyme Stem-Loop V in the presence of magnesium ions and localization of metal-binding sites.
Biochemistry, 2006Co-Authors: Dean O Campbell, Geneviève Desjardins, Patricia Bouchard, Pascale LegaultAbstract:In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop-loop interaction between Stem-Loops I and V, which is important for recognition and activation of the Stem-Loop I substrate. Here, we present the high-resolution NMR structure of Stem-Loop V (SL5) in the presence of Mg 2+ (SL5 Mg ) and demonstrate that Mg 2+ induces a conformational change in which the SL5 loop adopts a compact structure with most characteristics of canonical U-turn structures. Divalent cation-binding sites were probed with Mn 2+ -induced paramagnetic line broadening and intermolecular NOEs to Co(NH 3 ) 6 3+ . Structural modeling of Mn(H 2 O) 6 2+ in SL5 Mg revealed four divalent cation-binding sites in the loop. Sites 1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent to N7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1-3 in SL5 are present in other U-turn motifs, and these metal-binding sites may represent a common feature of the U-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change the conformation of residues 697-699 involved in the proposed Watson-Crick base pairs with Stem-Loop I. In both the presence and the absence of Mg 2+ , G697, A698, and C699 adopt an A-form structure that exposes their Watson-Crick faces, and this is compatible with their proposed interaction with Stem-Loop I. In SL5 Mg , however, U700 becomes exposed on the minor groove face of the loop in the proximity of the bases of G697, A698, and C699, suggesting that the Mg 2+ -bound conformation of Stem-Loop V allows additional contacts with Stem-Loop I. These studies improve our understanding of the role of Mg 2+ in U-turn structures and in substrate recognition by the VS ribozyme.
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Nuclear magnetic resonance structure of the Varkud satellite ribozyme Stem-Loop V RNA and magnesium-ion binding from chemical-shift mapping.
Biochemistry, 2005Co-Authors: Dean O Campbell, Pascale LegaultAbstract:An important step in the substrate recognition of the Neurospora Varkud Satellite (VS) ribozyme is the formation of a magnesium-dependent loop/loop interaction between the terminal loops of Stem-Loops I and V. We have studied the structure of Stem-Loop V by nuclear magnetic resonance spectroscopy and shown that it adopts a U-turn conformation, a common motif found in RNA. Structural comparisons indicate that the U-turn of Stem-Loop V fulfills some but not all of the structural characteristics found in canonical U-turn structures. This U-turn conformation exposes the Watson-Crick faces of the bases within Stem-Loop V (G697, A698, and C699) and makes them accessible for interaction with Stem-Loop I. Using chemical-shift mapping, we show that magnesium ions interact with the loop of the isolated Stem-Loop V and induce a conformational change that may be important for interaction with Stem-Loop I. This study expands our understanding of the role of U-turn motifs in RNA structure and function and provides insights into the mechanism of substrate recognition in the VS ribozyme.
