The Experts below are selected from a list of 2382 Experts worldwide ranked by ideXlab platform
Stephen A Liebhaber - One of the best experts on this subject based on the ideXlab platform.
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the KH Domain protein αcp has a direct role in mrna stabilization independent of its cognate binding site
Molecular and Cellular Biology, 2003Co-Authors: Jian Kong, Xinjun Ji, Stephen A LiebhaberAbstract:Previous studies suggest that high-level stability of a subset of mammalian mRNAs is linked to a C-rich motif in the 3′ untranslated region (3′UTR). High-level expression of human α-globin mRNA (hα-globin mRNA) in erythroid cells has been specifically attributed to formation of an RNA-protein complex comprised of a 3′UTR C-rich motif and an associated 39-kDa poly(C) binding protein, αCP. Documentation of this RNA-protein α-complex has been limited to in vitro binding studies, and its impact has been monitored by alterations in steady-state mRNA. Here we demonstrate that αCP is stably bound to hα-globin mRNA in vivo, that α-complex assembly on the hα-globin mRNA is restricted to the 3′UTR C-rich motif, and that α-complex assembly extends the physical half-life of hα-globin mRNA selectively in erythroid cells. Significantly, these studies also reveal that an artificially tethered αCP has the same mRNA-stabilizing activity as the native α-complex. These data demonstrate a unique contribution of the α-complex to hα-globin mRNA stability and support a model in which the sole function of the C-rich motif is to selectively tether αCP to a subset of mRNAs. Once bound, αCP appears to be fully sufficient to trigger downstream events in the stabilization pathway.
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targeting a KH Domain protein with rna decoys
RNA, 2002Co-Authors: Aleksandr V Makeyev, Dawn L Eastmond, Stephen A LiebhaberAbstract:RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA–protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the αCP subgroup of KH-Domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to αCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for αCP decoy action. Because αCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of αCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.
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optimized rna targets of two closely related triple KH Domain proteins heterogeneous nuclear ribonucleoprotein k and αcp 2kl suggest distinct modes of rna recognition
Journal of Biological Chemistry, 2001Co-Authors: Thomas Thisted, Dmitry L Lyakhov, Stephen A LiebhaberAbstract:Abstract The KH Domain mediates RNA binding in a wide range of proteins. Here we investigate the RNA-binding properties of two abundant RNA-binding proteins, αCP-2KL and heterogeneous nuclear ribonucleoprotein (hnRNP) K. These proteins constitute the major poly(C) binding activity in mammalian cells, are closely related on the basis of the structures and positioning of their respective triplicated KH Domains, and have been implicated in a variety of post-transcriptional controls. By using SELEX, we have obtained sets of high affinity RNA targets for both proteins. The primary and secondary structures necessary for optimal protein binding were inferred in each case from SELEX RNA sequence comparisons and confirmed by mutagenesis and structural mapping. The target sites for αCP-2KL and hnRNP K were both enriched for cytosine bases and were presented in a single-stranded conformation. In contrast to these shared characteristics, the optimal target sequence for hnRNP K is composed of a single short “C-patch” compatible with recognition by a single KH Domain whereas that for αCP-2KL encompassed three such C-patches suggesting more extensive interactions. The binding specificities of the respective SELEX RNAs were confirmed by testing their interactions with native proteins in cell extracts, and the importance of the secondary structure in establishing an optimized αCP-2KL-binding site was supported by comparison of SELEX target structure with that of the native human α-globin 3′-untranslated region. These data indicate that modes of macromolecular interactions of arrayed KH Domains can differ even among closely related KH proteins and that binding affinities are substantially dependent on the presentation of the target site within the RNA secondary structure.
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identification of two novel mammalian genes establishes a subfamily of KH Domain rna binding proteins
Genomics, 2000Co-Authors: Aleksandr V Makeyev, Stephen A LiebhaberAbstract:Abstract We have identified two novel human genes encoding proteins with a high level of sequence identity to two previously characterized RNA-binding proteins, αCP-1 and αCP-2. Both of these novel genes, αCP-3 and αCP-4, are predicted to encode proteins with triplicated KH Domains. The number and organization of the KH Domains, their sequences, and the sequences of the contiguous regions are conserved among all four αCP proteins. The common evolutionary origin of these proteins is substantiated by conservation of exon–intron organization in the corresponding genes. The map positions of αCP-1 and αCP-2 (previously reported) and those of αCP-3 and αCP-4 (present report) reveal that the four αCP loci are dispersed in the human genome; αCP-3 and αCP–4 mapped to 21q22.3 and 3p21, and the respective mouse orthologues mapped to syntenic regions of the mouse genome, 10B5 and 9F1-F2, respectively. Two additional loci in the human genome were identified as αCP-2 processed pseudogenes (PCBP2P1, 21q22.3, and PCBP2P2, 8q21–q22). Although the overall levels of αCP-3 and αCP-4 mRNAs are substantially lower than those of αCP-1 and αCP-2, transcripts of αCP-3 and αCP-4 were found in all mouse tissues tested. These data establish a new subfamily of genes predicted to encode closely related KH-containing RNA-binding proteins with potential functions in posttranscriptional controls.
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identification of two KH Domain proteins in the alpha globin mrnp stability complex
The EMBO Journal, 1995Co-Authors: Megerditch Kiledjian, Xiaoming Wang, Stephen A LiebhaberAbstract:Abstract Accumulation of globin mRNAs during erythroid differentiation is dependent on their extraordinary stability. The longevity of human alpha-globin mRNA is associated with a ribonucleoprotein complex (alpha-complex) formed on the 3' untranslated region (3'UTR). One or more of the proteins within this alpha-complex contain strong polycytosine [poly(C)] binding (alpha PCB) activity. In the present report we purify alpha PCB activity from human erythroid K562 cells. Although not able to bind the alpha-globin 3'UTR directly, alpha PCB activity is sufficient to complement alpha-complex formation in a cytosolic extract depleted of poly(C) binding activity. Peptide microsequencing demonstrates that alpha PCB activity contains two structurally related poly(C) binding proteins. These two proteins, alpha-complex protein (alpha CP)-1 and -2, have an overall structural identity of 80% and contain three repeats of the K homology (KH) Domain which is found in a subset of RNA binding proteins. Epitope-tagged recombinant alpha CP-1 and alpha CP-2 expressed in cells are each incorporated into the alpha-complex. We conclude that alpha CP-1 and alpha CP-2, members of the KH Domain RNA binding protein family, are involved in formation of a sequence-specific alpha-globin mRNP complex associated with alpha-globin mRNA stability. As such this represents the first example of a specific function for this class of proteins and suggests potential roles for other members of this protein family.
Thomas L James - One of the best experts on this subject based on the ideXlab platform.
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X-ray crystallographic and NMR studies of protein-protein and protein-nucleic acid interactions involving the KH Domains from human poly(C)-binding protein-2.
RNA, 2007Co-Authors: Zhihua Du, Sebastian Fenn, Richard Tjhen, Robert M Stroud, Thomas L JamesAbstract:Poly(C)-binding proteins (PCBPs) are KH (hnRNP K homology) Domain-containing proteins that recognize poly(C) DNA and RNA sequences in mammalian cells. Binding poly(C) sequences via the KH Domains is critical for PCBP functions. To reveal the mechanisms of KH Domain-D/RNA recognition and its functional importance, we have determined the crystal structures of PCBP2 KH1 Domain in complex with a 12-nucleotide DNA corresponding to two repeats of the human C-rich strand telomeric DNA and its RNA equivalent. The crystal structures reveal molecular details for not only KH1-DNA/RNA interaction but also protein–protein interaction between two KH1 Domains. NMR studies on a protein construct containing two KH Domains (KH1 + KH2) of PCBP2 indicate that KH1 interacts with KH2 in a way similar to the KH1–KH1 interaction. The crystal structures and NMR data suggest possible ways by which binding certain nucleic acid targets containing tandem poly(C) motifs may induce structural rearrangement of the KH Domains in PCBPs; such structural rearrangement may be crucial for some PCBP functions.
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crystal structure of the third KH Domain of human poly c binding protein 2 in complex with a c rich strand of human telomeric dna at 1 6 a resolution
Nucleic Acids Research, 2007Co-Authors: Sebastian Fenn, Zhihua Du, Richard Tjhen, Robert M Stroud, Thomas L JamesAbstract:KH (hnRNP K homology) Domains, consisting of ~70 amino acid residues, are present in a variety of nucleic-acid-binding proteins. Among these are poly(C)-binding proteins (PCBPs), which are important regulators of mRNA stability and posttranscriptional regulation in general. All PCBPs contain three different KH Domains and recognize poly(C)-sequences with high affinity and specificity. To reveal the molecular basis of poly(C)-sequence recognition, we have determined the crystal structure, at 1.6 A resolution, of PCBP2 KH3 Domain in complex with a 7-nt DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the C-rich strand of human telomeric DNA. The Domain assumes a type-I KH fold in a βααββα configuration. The protein-DNA interface could be studied in unprecedented detail and is made up of a series of direct and water-mediated hydrogen bonds between the protein and the DNA, revealing an especially dense network involving several structural water molecules for the last 2 nt in the core recognition sequence. Unlike published KH Domain structures, the protein crystallizes without protein-protein contacts, yielding new insights into the dimerization properties of different KH Domains. A nucleotide platform, an interesting feature found in some RNA molecules, was identified, evidently for the first time in DNA.
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specific recognition of the c rich strand of human telomeric dna and the rna template of human telomerase by the first KH Domain of human poly c binding protein 2
Journal of Biological Chemistry, 2004Co-Authors: Zhihua Du, Jinghua Yu, Yihong Chen, Raul Andino, Thomas L JamesAbstract:Abstract Poly(C)-binding proteins (PCBPs) constitute a family of nucleic acid-binding proteins that play important roles in a wide spectrum of regulatory mechanisms. The diverse functions of PCBPs are dependent on the ability of the PCBPs to recognize poly(C) sequences with high affinity and specificity. PCBPs contain three copies of KH (hnRNP K homology) Domains, which are responsible for binding nucleic acids. We have determined the NMR structure of the first KH Domain (KH1) from PCBP2. The PCBP2 KH1 Domain adopts a structure with three α-helices packed against one side of a three-stranded antiparallel β-sheet. Specific binding of PCBP2 KH1 to a number of poly(C) RNA and DNA sequences, including the C-rich strand of the human telomeric DNA repeat, the RNA template region of human telomerase, and regulatory recognition motifs in the poliovirus-1 5′-untranslated region, was established by monitoring chemical shift changes in protein 15N-HSQC spectra. The nucleic acid binding groove was further mapped by chemical shift perturbation upon binding to a six-nucleotide human telomeric DNA. The binding groove is an α/β platform formed by the juxtaposition of two α-helices, one β-strand, and two flanking loops. Whereas there is a groove in common with all of the DNA and RNA binders with a hydrophobic floor accommodating a three-residue stretch of C residues, nuances in recognizing flanking residues are provided by hydrogen bonding partners in the KH Domain. Specific interactions of PCBP2 KH1 with telomeric DNA and telomerase RNA suggest that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.
Stephane Richard - One of the best experts on this subject based on the ideXlab platform.
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Reaching for the stars: Linking RNA binding proteins to diseases.
Advances in Experimental Medicine and Biology, 2020Co-Authors: Stephane RichardAbstract:The prototype STAR (Signal Transduction and Activation of RNA) protein is Sam68, the Src-associated substrate during mitosis of 68 kDa. Sam68, like all other STAR proteins, belongs to the large class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) Domain family of RNA-binding proteins. The KH Domain is an evolutionarily conserved RNA binding Domain that consists of 70–100 amino acids. The KH Domain is one of the most prevalent RNA binding Domains that directly contacts single-stranded RNA with a signature topology. Sam68 contains a single KH Domain that harbors additional conserved N- and C-terminal sequences also required for RNA binding specificity and dimerization. Sam68 frequently contains post-translational modifications including serine/threonine, tyrosine phosphorylation, lysine acetylation, arginine methylation and sumoylation. The phosphorylation of Sam68 or its association with SH3 Domain containing proteins has been shown to influence its RNA binding activity. Hence Sam68 behaves as a STAR protein, whereby extracellular signals influence its ability to regulate RNA metabolism. Studies in mice have revealed physiological roles linking Sam68 to osteoporosis, cancer, infertility and ataxia. The role of Sam68, a closely related family member quaking (QKI), the KH Domain and their links with human disease will be discussed in the present chapter.
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quaking KH Domain proteins as regulators of glial cell fate and myelination
RNA Biology, 2005Co-Authors: Daniel Larocque, Stephane RichardAbstract:The quaking viable (qkv) mice have always attracted attention because of their characteristic tremor caused by their dysmyelination. In the central nervous system, qkv mice fail to develop mature myelinating oligodendrocytes and display uncompacted myelin. The genetic defect in the qkv mice prevents the proper expression of alternatively spliced KH-type QKI RNA binding proteins. Thus qkv mice provide a unique animal model linking RNA binding proteins to defects in oligodendrocyte cell fate and myelination. The fact that QKI proteins are modified post-translationally makes them Signal Transduction Activiators of RNA (STAR) proteins. We have used a gain-of-function approach with the ectopic expression of the separate QKI isoforms using adenoviruses and retroviruses to determine their separate roles in cell fate and myelination. Herein, we discuss the recent advances in characterizing the QKI KH-type proteins as glial cell fate and myelin regulators.
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sam68 the KH Domain containing superstar
Biochimica et Biophysica Acta, 2003Co-Authors: Kiven E Lukong, Stephane RichardAbstract:Sam68 is one of the most studied members of the STAR family of RNA-binding proteins since its identification over a decade ago. Since its ascension into prominence, enormous progress has been made to unmask the link between the RNA-binding properties of Sam68 and the regulation of cellular processes including signal transduction, cell cycle regulation and tumorigenesis and RNA biogenesis in general. In this review we provide a detailed description of the functional Domains of Sam68 and the possible biological roles that justify its superSTAR status.
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self association of the single KH Domain family members sam68 grp33 gld 1 and qk1 role of the KH Domain
Molecular and Cellular Biology, 1997Co-Authors: Taiping Chen, Bassam B Damaj, Constance Herrera, Paul Lasko, Stephane RichardAbstract:Sam68 is a member of a growing family of proteins that contain a single KH Domain embedded in a larger conserved Domain of approximately 170 amino acids. Loops 1 and 4 of this KH Domain family are longer than the corresponding loops in other KH Domains and contain conserved residues. KH Domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH Domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 Domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH Domain proteins such as Bicaudal C. These observations indicate that the single KH Domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.
Nico Tjandra - One of the best experts on this subject based on the ideXlab platform.
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chemical shift mapped dna binding sites and 15n relaxation analysis of the c terminal KH Domain of heterogeneous nuclear ribonucleoprotein k
Biochemistry, 2000Co-Authors: James L Baber, David Levens, Daniel Libutti, Nico TjandraAbstract:The K homology (KH) motif is one of the major classes of nucleic acid binding proteins. Some members of this family have been shown to interact with DNA while others have RNA targets. There have been no reports containing direct experimental evidence regarding the nature of KH module−DNA interaction. In this study, the interaction of the C-terminal KH Domain of heterogeneous nuclear ribonucleoprotein K (KH3) with it's cognate single-stranded DNA (ssDNA) are investigated. Chemical shift perturbation mapping indicates that the first two helices, the conserved GxxG loop, β1, and β2, are the primary regions involved in DNA binding for KH3. The nature of the KH3−ssDNA interaction is further illuminated by a comparison of backbone 15N relaxation data for the bound and unbound KH3. Relaxation data are also used to confirm that the backbone of wild-type KH3 is structurally identical to that of the G26R mutant KH3, which was previously published. Amide proton exchange experiments indicate that the two helices invo...
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high precision solution structure of the c terminal KH Domain of heterogeneous nuclear ribonucleoprotein k a c myc transcription factor
Journal of Molecular Biology, 1999Co-Authors: James L Baber, David Levens, Daniel Libutti, Nico TjandraAbstract:Abstract Among it's many reported functions, heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transcription factor for the c- myc gene, a proto-oncogene critical for the regulation of cell growth and differentiation. We have determined the solution structure of the Gly26→Arg mutant of the C-terminal K-homology (KH) Domain of hnRNP K by NMR spectroscopy. This is the first structure investigation of hnRNP K. Backbone residual dipolar couplings, which provide information that is fundamentally different from the standard NOE-derived distance restraints, were employed to improve structure quality. An independent assessment of structure quality was achieved by comparing the backbone15N T1/T2ratios to the calculated structures. The C-terminal KH module of hnRNP K (KH3) is revealed to be a three-stranded β-sheet stacked against three α-helices, two of which are nearly parallel to the strands of the β-sheet. The Gly26→Arg mutation abolishes single-stranded DNA binding without altering the overall fold of the protein. This provides a clue to possible nucleotide binding sites of KH3. It appears unlikely that the solvent-exposed side of the β-sheet will be the site of protein-nucleic acid complex formation. This is in contrast to the earlier theme for protein-RNA complexes incorporating proteins structurally similar to KH3. We propose that the surface of KH3 that interacts with nucleic acid is comparable to the region of DNA interaction for the double-stranded DNA-binding Domain of bovine papillomavirus-1 E2 that has a three-dimensional fold similar to that of KH3.
Craig P Hunter - One of the best experts on this subject based on the ideXlab platform.
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the star maxi KH Domain protein gld 1 mediates a developmental switch in the translational control of c elegans pal 1
Development, 2004Co-Authors: Darcy Mootz, Diana M Ho, Craig P HunterAbstract:Translational control is an essential mechanism of gene control utilized throughout development, yet the molecular mechanisms underlying translational activation and repression are poorly understood. We have investigated the translational control of the C. elegans caudal homolog, pal-1, and found that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH Domain family, acts through a minimal pal-1 3′ UTR element to repress pal-1 translation in the distal germline. We also provide data suggesting that GLD-1 may repress pal-1 translation after initiation. Finally, we show that GLD-1 represses the distal germline expression of the KH Domain protein MEX-3, which was previously shown to repress PAL-1 expression in the proximal germline and which appears specialized to control PAL-1 expression patterns in the embryo. Hence, GLD-1 mediates a developmental switch in the control of PAL-1 repression, allowing MEX-3 to accumulate and take over the task of PAL-1 repression in the proximal germline, where GLD-1 protein levels decline.
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The STAR/Maxi-KH Domain protein GLD-1 mediates a developmental switch in the translational control of C. elegans PAL-1
Development, 2004Co-Authors: Darcy Mootz, Diana M Ho, Craig P HunterAbstract:Translational control is an essential mechanism of gene control utilized throughout development, yet the molecular mechanisms underlying translational activation and repression are poorly understood. We have investigated the translational control of the C. elegans caudal homolog, pal-1, and found that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH Domain family, acts through a minimal pal-1 3′ UTR element to repress pal-1 translation in the distal germline. We also provide data suggesting that GLD-1 may repress pal-1 translation after initiation. Finally, we show that GLD-1 represses the distal germline expression of the KH Domain protein MEX-3, which was previously shown to repress PAL-1 expression in the proximal germline and which appears specialized to control PAL-1 expression patterns in the embryo. Hence, GLD-1 mediates a developmental switch in the control of PAL-1 repression, allowing MEX-3 to accumulate and take over the task of PAL-1 repression in the proximal germline, where GLD-1 protein levels decline.
