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Jon R Lorsch - One of the best experts on this subject based on the ideXlab platform.
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translational initiation factor eif5 replaces eif1 on the 40s ribosomal subunit to promote Start Codon recognition
eLife, 2018Co-Authors: Jose Luis Llacer, Tanweer Hussain, Jagpreet Nanda, Sukhvir Kaur, Adesh K. Saini, Alan G Hinnebusch, Yuliya Gordiyenko, Rakesh Kumar, Jon R LorschAbstract:In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNAi in a ‘PIN’ state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 A, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG Codonsin vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the Codon:antiCodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in Start-Codon selection.
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translational initiation factor eif5 replaces eif1 on the 40s ribosomal subunit to promote Start Codon recognition
bioRxiv, 2018Co-Authors: Jose Luis Llacer, Tanweer Hussain, Jagpreet Nanda, Jon R Lorsch, Sukhvir Kaur, Adesh K. Saini, Alan G Hinnebusch, Yuliya Gordiyenko, Rakesh Kumar, V RamakrishnanAbstract:In eukaryotic translation initiation AUG recognition of the mRNA requires accommodation of Met-tRNAi in a “PIN” state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a 48S pre-initiation complex (PIC), at an overall resolution of 3.0 A, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG Codons in vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the Codon:antiCodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in Start-Codon selection.
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structural changes enable Start Codon recognition by the eukaryotic translation initiation complex
Cell, 2014Co-Authors: Tanweer Hussain, Jose Luis Llacer, Jon R Lorsch, Israel S Fernandez, Antonio M Munoz, Pilar Martinmarcos, Christos G Savva, Alan G HinnebuschAbstract:During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG Start Codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 A resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the Codon-antiCodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2α, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG Codon.
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conserved residues in yeast initiator trna calibrate initiation accuracy by regulating preinitiation complex stability at the Start Codon
Genes & Development, 2014Co-Authors: Jinsheng Dong, Jon R Lorsch, Adesh K. Saini, Antonio M Munoz, Sarah E Kolitz, Wen Ling Chiu, Hafsa Rahman, Alan G HinnebuschAbstract:Eukaryotic initiator tRNA (tRNAi) contains several highly conserved unique sequence features, but their importance in accurate Start Codon selection was unknown. Here we show that conserved bases throughout tRNAi, from the antiCodon stem to acceptor stem, play key roles in ensuring the fidelity of Start Codon recognition in yeast cells. Substituting the conserved G31:C39 base pair in the antiCodon stem with different pairs reduces accuracy (the Sui(-) [suppressor of initiation Codon] phenotype), whereas eliminating base pairing increases accuracy (the Ssu(-) [suppressor of Sui(-)] phenotype). The latter defect is fully suppressed by a Sui(-) substitution of T-loop residue A54. These genetic data are paralleled by opposing effects of Sui(-) and Ssu(-) substitutions on the stability of methionylated tRNAi (Met-tRNA(i)) binding (in the ternary complex [TC] with eIF2-GTP) to reconstituted preinitiation complexes (PICs). Disrupting the C3:G70 base pair in the acceptor stem produces a Sui(-) phenotype and also reduces the rate of TC binding to 40S subunits in vitro and in vivo. Both defects are suppressed by an Ssu(-) substitution in eIF1A that stabilizes the open/P(OUT) conformation of the PIC that exists prior to Start Codon recognition. Our data indicate that these signature sequences of tRNA(i) regulate accuracy by distinct mechanisms, promoting the open/P(OUT) conformation of the PIC (for C3:G70) or destabilizing the closed/P(IN) state (for G31:C39 and A54) that is critical for Start Codon recognition.
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coordinated movements of eukaryotic translation initiation factors eif1 eif1a and eif5 trigger phosphate release from eif2 in response to Start Codon recognition by the ribosomal preinitiation complex
Journal of Biological Chemistry, 2013Co-Authors: Jagpreet S Nanda, Alan G Hinnebusch, Adesh K. Saini, Antonio M Munoz, Jon R LorschAbstract:Accurate recognition of the Start Codon in an mRNA by the eukaryotic translation preinitiation complex (PIC) is essential for proper gene expression. The process is mediated by eukaryotic translation initiation factors (eIFs) in conjunction with the 40 S ribosomal subunit and (initiator) tRNAi. Here, we provide evidence that the C-terminal tail (CTT) of eIF1A, which we previously implicated in Start Codon recognition, moves closer to the N-terminal domain of eIF5 when the PIC encounters an AUG Codon. Importantly, this movement is coupled to dissociation of eIF1 from the PIC, a critical event in Start Codon recognition, and is dependent on the scanning enhancer elements in the eIF1A CTT. The data further indicate that eIF1 dissociation must be accompanied by the movement of the eIF1A CTT toward eIF5 in order to trigger release of phosphate from eIF2, which converts the latter to its GDP-bound state. Our results also suggest that release of eIF1 from the PIC and movement of the CTT of eIF1A are triggered by the same event, most likely accommodation of tRNAi in the P site of the 40 S subunit driven by base pairing between the Start Codon in the mRNA and the antiCodon in tRNAi. Finally, we show that the C-terminal domain of eIF5 is responsible for the factor's activity in antagonizing eIF1 binding to the PIC. Together, our data provide a more complete picture of the chain of molecular events that is triggered when the scanning PIC encounters an AUG Start Codon in the mRNA.
Alan G Hinnebusch - One of the best experts on this subject based on the ideXlab platform.
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eif2α interactions with mrna control accurate Start Codon selection by the translation preinitiation complex
Nucleic Acids Research, 2020Co-Authors: Anil Thakur, Swati Gaikwad, Anil K Vijjamarri, Alan G HinnebuschAbstract:In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the -3 nucleotide of the 'Kozak' context. We found that R55/R57 substitutions reduced recognition of a UUG Start Codon at HIS4 in Sui- cells (Ssu- phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG Start Codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG Codon (Sui- phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd- phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG Codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.
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eif3b and eif3i relocate together to the ribosomal subunit interface during translation initiation and modulate Start Codon selection
bioRxiv, 2020Co-Authors: Jose Luis Llacer, Tanweer Hussain, Yuliya Gordiyenko, Jinsheng Dong, Alan G HinnebuschAbstract:During eukaryotic translational initiation, the 48S ribosomal pre-initiation complex (PIC) scans the 59 untranslated region of mRNA until it encounters a Start Codon. We present a single particle electron cryomicroscopy (cryo-EM) reconstruction of a yeast 48S PIC in an open scanning competent state in which eIF3b is observed bound on the 40S subunit interface. eIF3b is relocated with eIF3i from their solvent-interface locations observed in other PIC structures; however, eIF3i is not in contact with the 40S. Re-processing of micrographs of our previous 48S PIC in a closed state using currently available tools reveal a similar re-location of eIF3b and eIF3i from the solvent to subunit interface. Genetic analysis indicates that high fidelity initiation in vivo depends strongly on eIF3b interactions at the subunit interface that either promote the closed conformation of the PIC on Start Codon selection or facilitate subsequent relocation back to the solvent side of the 40S subunit.
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a network of eif2β interactions with eif1 and met trnai promotes accurate Start Codon selection by the translation preinitiation complex
Nucleic Acids Research, 2019Co-Authors: Anil Thakur, Laura Marler, Alan G HinnebuschAbstract:In translation initiation, a 43S preinitiation complex (PIC) containing eIF1 and a ternary complex (TC) of GTP-bound eIF2 and Met-RNAi scans the mRNA for the Start Codon. AUG recognition triggers eIF1 release and rearrangement from an open PIC conformation to a closed state with more tightly-bound Met-tRNAi (PIN state). Cryo-EM models reveal eIF2β contacts with eIF1 and Met-tRNAi exclusive to the open complex that should destabilize the closed state. eIF2β or eIF1 substitutions disrupting these contacts increase initiation at UUG Codons, and compound substitutions also derepress translation of GCN4, indicating slower TC recruitment. The latter substitutions slow TC loading while stabilizing TC binding at UUG Codons in reconstituted PICs, indicating a destabilized open complex and shift to the closed/PIN state. An eIF1 substitution that should strengthen the eIF2β:eIF1 interface has the opposite genetic and biochemical phenotypes. eIF2β is also predicted to restrict Met-tRNAi movement into the closed/PIN state, and substitutions that should diminish this clash increase UUG initiation in vivo and stabilize Met-tRNAi binding at UUG Codons in vitro with little effect on TC loading. Thus, eIF2β anchors eIF1 and TC to the open complex, enhancing PIC assembly and scanning, while impeding rearrangement to the closed conformation at non-AUG Codons.
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translational initiation factor eif5 replaces eif1 on the 40s ribosomal subunit to promote Start Codon recognition
eLife, 2018Co-Authors: Jose Luis Llacer, Tanweer Hussain, Jagpreet Nanda, Sukhvir Kaur, Adesh K. Saini, Alan G Hinnebusch, Yuliya Gordiyenko, Rakesh Kumar, Jon R LorschAbstract:In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNAi in a ‘PIN’ state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 A, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG Codonsin vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the Codon:antiCodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in Start-Codon selection.
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translational initiation factor eif5 replaces eif1 on the 40s ribosomal subunit to promote Start Codon recognition
bioRxiv, 2018Co-Authors: Jose Luis Llacer, Tanweer Hussain, Jagpreet Nanda, Jon R Lorsch, Sukhvir Kaur, Adesh K. Saini, Alan G Hinnebusch, Yuliya Gordiyenko, Rakesh Kumar, V RamakrishnanAbstract:In eukaryotic translation initiation AUG recognition of the mRNA requires accommodation of Met-tRNAi in a “PIN” state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a 48S pre-initiation complex (PIC), at an overall resolution of 3.0 A, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG Codons in vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the Codon:antiCodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in Start-Codon selection.
Lara S. Carroll - One of the best experts on this subject based on the ideXlab platform.
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Start Codon disruption with crispr cas9 prevents murine fuchs endothelial corneal dystrophy
eLife, 2021Co-Authors: Hironori Uehara, Felipe Zibetti Pereira, Siddharth Narendran, Susie Choi, Sai Bhuvanagiri, Sangeetha Ravi Kumar, Austin Bohner, Xiaohui Zhang, Jinlu Liu, Lara S. CarrollAbstract:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 Start Codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.
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Start Codon disruption with crispr cas9 prevents murine fuchs endothelial corneal dystrophy
bioRxiv, 2020Co-Authors: Hironori Uehara, Felipe Zibetti Pereira, Siddharth Narendran, Susie Choi, Sai Bhuvanagiri, Sangeetha Ravi Kumar, Austin Bohner, Lara S. Carroll, Xiaohui Zhang, Bonnie ArcherAbstract:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs9 endothelial corneal dystrophy (FECD), which progressively impairs vision through loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA), efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 Start Codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.
Xiaohui Zhang - One of the best experts on this subject based on the ideXlab platform.
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Start Codon disruption with crispr cas9 prevents murine fuchs endothelial corneal dystrophy
eLife, 2021Co-Authors: Hironori Uehara, Felipe Zibetti Pereira, Siddharth Narendran, Susie Choi, Sai Bhuvanagiri, Sangeetha Ravi Kumar, Austin Bohner, Xiaohui Zhang, Jinlu Liu, Lara S. CarrollAbstract:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 Start Codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.
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Start Codon disruption with CRISPR/Cas9 prevents murine Fuchs’ endothelial corneal dystrophy
'eLife Sciences Publications Ltd', 2021Co-Authors: Hironori Uehara, Siddharth Narendran, Susie Choi, Sai Bhuvanagiri, Sangeetha Ravi Kumar, Austin Bohner, Xiaohui Zhang, Felipe Pereira, Jinlu Liu, Lara CarrollAbstract:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs’ endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 Start Codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells
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amelioration of an inherited metabolic liver disease through creation of a de novo Start Codon by cytidine base editing
Molecular Therapy, 2020Co-Authors: Lei Yang, Xiaohui Zhang, Liren Wang, Yanan Huo, Xi Chen, Shuming Yin, Rui Zheng, Hongquan Geng, Honghui Han, Meizhen LiuAbstract:Base editing technology efficiently generates nucleotide conversions without inducing excessive double-strand breaks (DSBs), which makes it a promising approach for genetic disease therapy. In this study, we generated a novel hereditary tyrosinemia type 1 (HT1) mouse model, which contains a Start Codon mutation in the fumarylacetoacetate hydrolase (Fah) gene by using an adenine base editor (ABE7.10). To investigate the feasibility of base editing for recombinant adeno-associated virus (rAAV)-mediated gene therapy, an intein-split cytosine base editor (BE4max) was developed. BE4max efficiently induced C-to-T conversion and restored the Start Codon to ameliorate HT1 in mice, but an undesired bystander mutation abolished the effect of on-target editing. To solve this problem, an upstream sequence was targeted to generate a de novo in-frame Start Codon to initiate the translation of FAH. After treatment, almost all C-to-T conversions created a Start Codon and restored Fah expression, which efficiently ameliorated the disease without inducing off-target mutations. Our study demonstrated that base editing-mediated creation of de novo functional elements would be an applicable new strategy for genetic disease therapy.
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Start Codon disruption with crispr cas9 prevents murine fuchs endothelial corneal dystrophy
bioRxiv, 2020Co-Authors: Hironori Uehara, Felipe Zibetti Pereira, Siddharth Narendran, Susie Choi, Sai Bhuvanagiri, Sangeetha Ravi Kumar, Austin Bohner, Lara S. Carroll, Xiaohui Zhang, Bonnie ArcherAbstract:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs9 endothelial corneal dystrophy (FECD), which progressively impairs vision through loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA), efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 Start Codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.
David Feldman - One of the best experts on this subject based on the ideXlab platform.
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the vitamin d receptor gene Start Codon polymorphism a functional analysis of foki variants
Journal of Bone and Mineral Research, 1998Co-Authors: Coleman Gross, Aruna V Krishnan, Peter J Malloy, Ross T Eccleshall, Xiaoyan Zhao, David FeldmanAbstract:The vitamin D receptor (VDR) gene contains a Start Codon polymorphism (SCP) which is three Codons upstream of a second Start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where “f” indicates the presence of the restriction site and the first ATG, while “F” indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F-VDR) being three amino acids shorter than the f variant (f-VDR). To determine whether there are functional differences between the f-VDR and the F-VDR, we studied the two VDR forms expressed in COS-7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f-VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]-1,25-dihydroxyvitamin D3 ([3H]-1,25(OH)2D3) (Kd = 131 ± 78 pM, f-VDR; Kd = 237 ± 190 pM, F-VDR; p = 0.24); however, a 2-fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24-hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose-response (0.1–10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF,Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24-hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f-VDR and F-VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.
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the vitamin d receptor Start Codon polymorphism foki and bone mineral density in premenopausal american black and white women
Journal of Bone and Mineral Research, 1997Co-Authors: Susan S Harris, Coleman Gross, Ross T Eccleshall, Bess Dawsonhughes, David FeldmanAbstract:This study examines the association between bone mineral density (BMD) and a Start Codon polymorphism (SCP) at the translation initiation site of the vitamin D receptor (VDR) gene. The thymine/cytosine (T/C) polymorphism in the first of two Start (ATG) Codons can be detected by a restriction fragment length polymorphism (RFLP) using the endonuclease FokI, which recognizes ATG as part of its restriction site. F indicates absence of the first ATG and a VDR that is shorter by three amino acids. The FokI genotype was determined in 154 premenopausal American women (72 black and 82 white) who were 20–40 years old. BMD of the total body, femoral neck, and lumbar spine were measured by dual-energy X-ray absorptiometry. The distribution of the SCP genotypes differed significantly by race (p < 0.001): 4% of blacks versus 18% of whites were ff homozygous and 65% of blacks versus 37% of whites were FF homozygous. There was no statistically significant interaction between race and SCP genotype in analyses of BMD at any skeletal site. In the group as a whole, the ff women had femoral neck BMD that was 7.4% lower than that of the FF women. The ff white women had total body BMD values that were 4.3% lower and femoral neck values that were 12.1% lower than FF white women. Total body and femoral neck BMD did not differ significantly by genotype in black women, and spine BMD did not differ by genotype in either race. Addition of the SCP genotype to analysis of covariance models comparing BMD of the black and white women reduced estimated differences in femoral neck BMD between the two groups by about 35%. In conclusion, the SCP polymorphism, detected with the endonuclease FokI, appears to influence peak bone density, particularly at the femoral neck. Racial differences in its distribution may explain some of the racial difference in femoral neck BMD.
