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Stratagene native β globin orf
The sequence of the <t>ORF</t> determines the extension of the AUG-proximity effect. ( A ) Physical maps of the α-globin and the hybrid β/α- and α/β/α-globin mRNAs studied. Dark-gray rectangles represent α-globin exons and light-gray rectangles represent <t>β-globin</t> sequences. Translation initiation codons (AUG) and native termination codons (UAA) or premature translation termination codons (PTCs) are depicted by heavy black vertical lines. The name of each mRNA is indicated to its right. ( B ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments). mRNA levels are normalized to the expression level of the wild-type mRNA (αWT). A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. P -values were estimated using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied β-globin and hybrid α/β- and β/α/β-globin mRNAs, using the same depiction code as for (A) (see above). ( D ) Illustrative RPA of RNA from HeLa cells transiently transfected with the constructs identified beneath each lane. Identification of the β-globin and puro R protected fragments is indicated to the left of the autoradiograph. Average levels (and standard deviations from three independent experiments) of β-globin mRNA (plotted above each respective lane) were measured relatively to the puro R mRNA as in (B) (see above). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control.
Native β Globin Orf, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity"

Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv588

The sequence of the ORF determines the extension of the AUG-proximity effect. ( A ) Physical maps of the α-globin and the hybrid β/α- and α/β/α-globin mRNAs studied. Dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin sequences. Translation initiation codons (AUG) and native termination codons (UAA) or premature translation termination codons (PTCs) are depicted by heavy black vertical lines. The name of each mRNA is indicated to its right. ( B ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments). mRNA levels are normalized to the expression level of the wild-type mRNA (αWT). A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. P -values were estimated using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied β-globin and hybrid α/β- and β/α/β-globin mRNAs, using the same depiction code as for (A) (see above). ( D ) Illustrative RPA of RNA from HeLa cells transiently transfected with the constructs identified beneath each lane. Identification of the β-globin and puro R protected fragments is indicated to the left of the autoradiograph. Average levels (and standard deviations from three independent experiments) of β-globin mRNA (plotted above each respective lane) were measured relatively to the puro R mRNA as in (B) (see above). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control.
Figure Legend Snippet: The sequence of the ORF determines the extension of the AUG-proximity effect. ( A ) Physical maps of the α-globin and the hybrid β/α- and α/β/α-globin mRNAs studied. Dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin sequences. Translation initiation codons (AUG) and native termination codons (UAA) or premature translation termination codons (PTCs) are depicted by heavy black vertical lines. The name of each mRNA is indicated to its right. ( B ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments). mRNA levels are normalized to the expression level of the wild-type mRNA (αWT). A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. P -values were estimated using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied β-globin and hybrid α/β- and β/α/β-globin mRNAs, using the same depiction code as for (A) (see above). ( D ) Illustrative RPA of RNA from HeLa cells transiently transfected with the constructs identified beneath each lane. Identification of the β-globin and puro R protected fragments is indicated to the left of the autoradiograph. Average levels (and standard deviations from three independent experiments) of β-globin mRNA (plotted above each respective lane) were measured relatively to the puro R mRNA as in (B) (see above). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control.

Techniques Used: Sequencing, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing

The boundary of the AUG-proximity effect for NMD resistance is determined by the mRNA secondary structure. ( A ) Schematic representation of the studied human β-globin mRNAs. Heavy black vertical lines indicate the positions of translation initiation codons (AUG) and natural (UAA) or premature termination codons (PTCs). Diagonally-striped rectangles represent 25 consecutive ‘CAA’ triplets [(CAA) 25 ]. The identification of each transcript is indicated to the right. ( B ) Representative RPA of RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of β-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of β-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments) normalized to the expression level of βWT mRNA. A 2-fold RNA sample (2x βWT) from HeLa cells transfected with the βWT gene was also analyzed to ascertain that the RPA was carried out in probe excess. P -values were determined using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied human α-globin mRNAs, using the same depiction code as for (A) (see above). Loop shapes in dark rectangles represent pseudoknot (pk) structures. The alteration of the two potential re-initiating AUGs, at codons 32 and 76, to ACG triplets is indicated by short and thick vertical lines (32–76Met→Thr). The identification of each mRNA is indicated to the right of the diagram. ( D ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The positions of α-globin and the puromycin-resistance (puro R ) protected fragments are indicated to the right of the autoradiograph. Resulting levels of α-globin mRNA were quantified as in (B). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( E ) Luminometry assay to test translation re-initiation after translation of a highly structured (pseudoknot; pk) or unstructured [(CAA) 26 ] ORF. The 5′-part of α-globin gene upstream of codon 32 (AUG), was fused with the firefly luciferase ORF at its translation initiation codon. Volume-shaped rectangles represent luciferase exons, dark-gray rectangles represent α-globin sequences. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (UAG)] codons, are represented as black vertical lines. A control construct was created, in which the α-globin translation initiation codon was mutated to AUA allowing to control for the maximum of luciferase activity. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids and protein levels were analyzed as above for Figure 2C .
Figure Legend Snippet: The boundary of the AUG-proximity effect for NMD resistance is determined by the mRNA secondary structure. ( A ) Schematic representation of the studied human β-globin mRNAs. Heavy black vertical lines indicate the positions of translation initiation codons (AUG) and natural (UAA) or premature termination codons (PTCs). Diagonally-striped rectangles represent 25 consecutive ‘CAA’ triplets [(CAA) 25 ]. The identification of each transcript is indicated to the right. ( B ) Representative RPA of RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of β-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of β-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments) normalized to the expression level of βWT mRNA. A 2-fold RNA sample (2x βWT) from HeLa cells transfected with the βWT gene was also analyzed to ascertain that the RPA was carried out in probe excess. P -values were determined using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied human α-globin mRNAs, using the same depiction code as for (A) (see above). Loop shapes in dark rectangles represent pseudoknot (pk) structures. The alteration of the two potential re-initiating AUGs, at codons 32 and 76, to ACG triplets is indicated by short and thick vertical lines (32–76Met→Thr). The identification of each mRNA is indicated to the right of the diagram. ( D ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The positions of α-globin and the puromycin-resistance (puro R ) protected fragments are indicated to the right of the autoradiograph. Resulting levels of α-globin mRNA were quantified as in (B). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( E ) Luminometry assay to test translation re-initiation after translation of a highly structured (pseudoknot; pk) or unstructured [(CAA) 26 ] ORF. The 5′-part of α-globin gene upstream of codon 32 (AUG), was fused with the firefly luciferase ORF at its translation initiation codon. Volume-shaped rectangles represent luciferase exons, dark-gray rectangles represent α-globin sequences. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (UAG)] codons, are represented as black vertical lines. A control construct was created, in which the α-globin translation initiation codon was mutated to AUA allowing to control for the maximum of luciferase activity. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids and protein levels were analyzed as above for Figure 2C .

Techniques Used: Cellular Antioxidant Activity Assay, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing, Luciferase, Activity Assay

Contrary to what occurs in β-globin mRNA, α-globin mRNAs allow for efficient translation re-initiation, but only partially contributes to NMD inhibition. ( A ) Schematic representation of the studied human α-globin mRNAs. Vertical lines represent translation initiation (AUG) or termination [native (UAA) or premature (PTC)] codons. The conversion of the two potential re-initiation AUGs, at codons 32 and 76, to ACG codons is indicated by short and thick vertical lines. The name of each transcript is specified on the right. ( B ) A representative RPA of RNA isolated from HeLa cells transiently transfected with each gene construct is shown. Identification of the α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the right of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations) normalized to the expression level of the αWT gene. A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. For each case, three independent experiments were performed. P -values were determined using a student's t -test and refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) The 5′-part of α-globin gene upstream of codon 32 (AUG), or upstream of codon 55 (AUG) of β-globin gene, was fused with the firefly luciferase open reading frame (ORF). Volume-shaped rectangles represent luciferase ORFs, dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin exons. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (PTC)] codons, are represented as black vertical lines. A positive control construct was created for each hybrid gene, in which the translation initiation codon for α- or β-globin gene was mutated to AUA. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids. Luciferase activity was analyzed 16 h after transfection. In parallel, both firefly and Renilla luciferase mRNA levels were determined by semi-quantitative RT-PCR. The activities of firefly and Renilla luciferase were calculated relative to their respective mRNA levels (Relative mRNA Levels, left bars), and then the resulting values for firefly luciferase activity/mRNA were quantified relative to those of Renilla luciferase activity/mRNA. The obtained luciferase relative activities from hybrid mRNAs were normalized to the relative activity of the uncoupled firefly luciferase (Luc). The average values and standard deviations from three independent experiments corresponding to three independent sets of transfections are shown.
Figure Legend Snippet: Contrary to what occurs in β-globin mRNA, α-globin mRNAs allow for efficient translation re-initiation, but only partially contributes to NMD inhibition. ( A ) Schematic representation of the studied human α-globin mRNAs. Vertical lines represent translation initiation (AUG) or termination [native (UAA) or premature (PTC)] codons. The conversion of the two potential re-initiation AUGs, at codons 32 and 76, to ACG codons is indicated by short and thick vertical lines. The name of each transcript is specified on the right. ( B ) A representative RPA of RNA isolated from HeLa cells transiently transfected with each gene construct is shown. Identification of the α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the right of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations) normalized to the expression level of the αWT gene. A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. For each case, three independent experiments were performed. P -values were determined using a student's t -test and refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) The 5′-part of α-globin gene upstream of codon 32 (AUG), or upstream of codon 55 (AUG) of β-globin gene, was fused with the firefly luciferase open reading frame (ORF). Volume-shaped rectangles represent luciferase ORFs, dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin exons. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (PTC)] codons, are represented as black vertical lines. A positive control construct was created for each hybrid gene, in which the translation initiation codon for α- or β-globin gene was mutated to AUA. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids. Luciferase activity was analyzed 16 h after transfection. In parallel, both firefly and Renilla luciferase mRNA levels were determined by semi-quantitative RT-PCR. The activities of firefly and Renilla luciferase were calculated relative to their respective mRNA levels (Relative mRNA Levels, left bars), and then the resulting values for firefly luciferase activity/mRNA were quantified relative to those of Renilla luciferase activity/mRNA. The obtained luciferase relative activities from hybrid mRNAs were normalized to the relative activity of the uncoupled firefly luciferase (Luc). The average values and standard deviations from three independent experiments corresponding to three independent sets of transfections are shown.

Techniques Used: Inhibition, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing, Luciferase, Positive Control, Activity Assay, Quantitative RT-PCR

A model for the effect of an AUG-proximal premature termination codon (PTC or stop codon). ( A ) The effect in the human β-globin transcript. During cap-mediated translation initiation, cytoplasmic poly(A) binding protein 1 (PABPC1) interacts with the eukaryotic initiation factor 4G (eIF4G). This interaction indirectly tethers PABPC1 to the 40S ribosomal subunit via the interaction of eIF4G with eIF3, which interacts with the 40S. The resulting configuration brings PABPC1 into the vicinity of the AUG initiation codon as a consequence of 43S scanning. The maintenance of the PABPC1-eIF4G-eIF3 interactions with the 40S during the first steps of translation elongation brings PABPC1 into close proximity with the termination complex at an AUG-proximal PTC. This proximity allows PABPC1 to interact with the release factor eRF3, thus impairing the UPF1-eRF3 interaction, resulting in efficient translation termination and inhibition of NMD—this was called the ‘AUG-proximity effect’. ( B ) In the human α-globin transcript, the AUG-proximity effect is observed for PTCs located further downstream because the open reading frame (ORF) is less structured. The more relaxed structure allows a faster elongation rate resulting in a longer window of the AUG proximity effect. In addition, the brief ORF translation also allows efficient translation re-initiation to occur at codon 32. This re-initiation diminishes the levels of residual exon junction complexes on the mRNA, thus contributing to the overall repression of NMD.
Figure Legend Snippet: A model for the effect of an AUG-proximal premature termination codon (PTC or stop codon). ( A ) The effect in the human β-globin transcript. During cap-mediated translation initiation, cytoplasmic poly(A) binding protein 1 (PABPC1) interacts with the eukaryotic initiation factor 4G (eIF4G). This interaction indirectly tethers PABPC1 to the 40S ribosomal subunit via the interaction of eIF4G with eIF3, which interacts with the 40S. The resulting configuration brings PABPC1 into the vicinity of the AUG initiation codon as a consequence of 43S scanning. The maintenance of the PABPC1-eIF4G-eIF3 interactions with the 40S during the first steps of translation elongation brings PABPC1 into close proximity with the termination complex at an AUG-proximal PTC. This proximity allows PABPC1 to interact with the release factor eRF3, thus impairing the UPF1-eRF3 interaction, resulting in efficient translation termination and inhibition of NMD—this was called the ‘AUG-proximity effect’. ( B ) In the human α-globin transcript, the AUG-proximity effect is observed for PTCs located further downstream because the open reading frame (ORF) is less structured. The more relaxed structure allows a faster elongation rate resulting in a longer window of the AUG proximity effect. In addition, the brief ORF translation also allows efficient translation re-initiation to occur at codon 32. This re-initiation diminishes the levels of residual exon junction complexes on the mRNA, thus contributing to the overall repression of NMD.

Techniques Used: Binding Assay, Inhibition

Related Articles

Sequencing:

Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity
Article Snippet: .. The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template. .. The βWT-(CAA)25 and β39-(CAA)25 gene variants were obtained by replacing the first 25 codons of the native β-globin ORF with 25 consecutive ‘CAA’ repeats, by overlap-extension PCR with overlapping primers #86 and #87, and the βWT or β39 genes as DNA templates.

Polymerase Chain Reaction:

Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity
Article Snippet: .. The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template. .. The βWT-(CAA)25 and β39-(CAA)25 gene variants were obtained by replacing the first 25 codons of the native β-globin ORF with 25 consecutive ‘CAA’ repeats, by overlap-extension PCR with overlapping primers #86 and #87, and the βWT or β39 genes as DNA templates.

Mutagenesis:

Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity
Article Snippet: .. The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template. .. The βWT-(CAA)25 and β39-(CAA)25 gene variants were obtained by replacing the first 25 codons of the native β-globin ORF with 25 consecutive ‘CAA’ repeats, by overlap-extension PCR with overlapping primers #86 and #87, and the βWT or β39 genes as DNA templates.

Construct:

Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity
Article Snippet: .. The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template. .. The βWT-(CAA)25 and β39-(CAA)25 gene variants were obtained by replacing the first 25 codons of the native β-globin ORF with 25 consecutive ‘CAA’ repeats, by overlap-extension PCR with overlapping primers #86 and #87, and the βWT or β39 genes as DNA templates.

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    Stratagene native β globin orf
    The sequence of the <t>ORF</t> determines the extension of the AUG-proximity effect. ( A ) Physical maps of the α-globin and the hybrid β/α- and α/β/α-globin mRNAs studied. Dark-gray rectangles represent α-globin exons and light-gray rectangles represent <t>β-globin</t> sequences. Translation initiation codons (AUG) and native termination codons (UAA) or premature translation termination codons (PTCs) are depicted by heavy black vertical lines. The name of each mRNA is indicated to its right. ( B ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments). mRNA levels are normalized to the expression level of the wild-type mRNA (αWT). A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. P -values were estimated using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied β-globin and hybrid α/β- and β/α/β-globin mRNAs, using the same depiction code as for (A) (see above). ( D ) Illustrative RPA of RNA from HeLa cells transiently transfected with the constructs identified beneath each lane. Identification of the β-globin and puro R protected fragments is indicated to the left of the autoradiograph. Average levels (and standard deviations from three independent experiments) of β-globin mRNA (plotted above each respective lane) were measured relatively to the puro R mRNA as in (B) (see above). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control.
    Native β Globin Orf, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/native β globin orf/product/Stratagene
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    The sequence of the ORF determines the extension of the AUG-proximity effect. ( A ) Physical maps of the α-globin and the hybrid β/α- and α/β/α-globin mRNAs studied. Dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin sequences. Translation initiation codons (AUG) and native termination codons (UAA) or premature translation termination codons (PTCs) are depicted by heavy black vertical lines. The name of each mRNA is indicated to its right. ( B ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments). mRNA levels are normalized to the expression level of the wild-type mRNA (αWT). A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. P -values were estimated using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied β-globin and hybrid α/β- and β/α/β-globin mRNAs, using the same depiction code as for (A) (see above). ( D ) Illustrative RPA of RNA from HeLa cells transiently transfected with the constructs identified beneath each lane. Identification of the β-globin and puro R protected fragments is indicated to the left of the autoradiograph. Average levels (and standard deviations from three independent experiments) of β-globin mRNA (plotted above each respective lane) were measured relatively to the puro R mRNA as in (B) (see above). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control.

    Journal: Nucleic Acids Research

    Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

    doi: 10.1093/nar/gkv588

    Figure Lengend Snippet: The sequence of the ORF determines the extension of the AUG-proximity effect. ( A ) Physical maps of the α-globin and the hybrid β/α- and α/β/α-globin mRNAs studied. Dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin sequences. Translation initiation codons (AUG) and native termination codons (UAA) or premature translation termination codons (PTCs) are depicted by heavy black vertical lines. The name of each mRNA is indicated to its right. ( B ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments). mRNA levels are normalized to the expression level of the wild-type mRNA (αWT). A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. P -values were estimated using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied β-globin and hybrid α/β- and β/α/β-globin mRNAs, using the same depiction code as for (A) (see above). ( D ) Illustrative RPA of RNA from HeLa cells transiently transfected with the constructs identified beneath each lane. Identification of the β-globin and puro R protected fragments is indicated to the left of the autoradiograph. Average levels (and standard deviations from three independent experiments) of β-globin mRNA (plotted above each respective lane) were measured relatively to the puro R mRNA as in (B) (see above). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control.

    Article Snippet: The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template.

    Techniques: Sequencing, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing

    The boundary of the AUG-proximity effect for NMD resistance is determined by the mRNA secondary structure. ( A ) Schematic representation of the studied human β-globin mRNAs. Heavy black vertical lines indicate the positions of translation initiation codons (AUG) and natural (UAA) or premature termination codons (PTCs). Diagonally-striped rectangles represent 25 consecutive ‘CAA’ triplets [(CAA) 25 ]. The identification of each transcript is indicated to the right. ( B ) Representative RPA of RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of β-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of β-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments) normalized to the expression level of βWT mRNA. A 2-fold RNA sample (2x βWT) from HeLa cells transfected with the βWT gene was also analyzed to ascertain that the RPA was carried out in probe excess. P -values were determined using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied human α-globin mRNAs, using the same depiction code as for (A) (see above). Loop shapes in dark rectangles represent pseudoknot (pk) structures. The alteration of the two potential re-initiating AUGs, at codons 32 and 76, to ACG triplets is indicated by short and thick vertical lines (32–76Met→Thr). The identification of each mRNA is indicated to the right of the diagram. ( D ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The positions of α-globin and the puromycin-resistance (puro R ) protected fragments are indicated to the right of the autoradiograph. Resulting levels of α-globin mRNA were quantified as in (B). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( E ) Luminometry assay to test translation re-initiation after translation of a highly structured (pseudoknot; pk) or unstructured [(CAA) 26 ] ORF. The 5′-part of α-globin gene upstream of codon 32 (AUG), was fused with the firefly luciferase ORF at its translation initiation codon. Volume-shaped rectangles represent luciferase exons, dark-gray rectangles represent α-globin sequences. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (UAG)] codons, are represented as black vertical lines. A control construct was created, in which the α-globin translation initiation codon was mutated to AUA allowing to control for the maximum of luciferase activity. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids and protein levels were analyzed as above for Figure 2C .

    Journal: Nucleic Acids Research

    Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

    doi: 10.1093/nar/gkv588

    Figure Lengend Snippet: The boundary of the AUG-proximity effect for NMD resistance is determined by the mRNA secondary structure. ( A ) Schematic representation of the studied human β-globin mRNAs. Heavy black vertical lines indicate the positions of translation initiation codons (AUG) and natural (UAA) or premature termination codons (PTCs). Diagonally-striped rectangles represent 25 consecutive ‘CAA’ triplets [(CAA) 25 ]. The identification of each transcript is indicated to the right. ( B ) Representative RPA of RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of β-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of β-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments) normalized to the expression level of βWT mRNA. A 2-fold RNA sample (2x βWT) from HeLa cells transfected with the βWT gene was also analyzed to ascertain that the RPA was carried out in probe excess. P -values were determined using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied human α-globin mRNAs, using the same depiction code as for (A) (see above). Loop shapes in dark rectangles represent pseudoknot (pk) structures. The alteration of the two potential re-initiating AUGs, at codons 32 and 76, to ACG triplets is indicated by short and thick vertical lines (32–76Met→Thr). The identification of each mRNA is indicated to the right of the diagram. ( D ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The positions of α-globin and the puromycin-resistance (puro R ) protected fragments are indicated to the right of the autoradiograph. Resulting levels of α-globin mRNA were quantified as in (B). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( E ) Luminometry assay to test translation re-initiation after translation of a highly structured (pseudoknot; pk) or unstructured [(CAA) 26 ] ORF. The 5′-part of α-globin gene upstream of codon 32 (AUG), was fused with the firefly luciferase ORF at its translation initiation codon. Volume-shaped rectangles represent luciferase exons, dark-gray rectangles represent α-globin sequences. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (UAG)] codons, are represented as black vertical lines. A control construct was created, in which the α-globin translation initiation codon was mutated to AUA allowing to control for the maximum of luciferase activity. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids and protein levels were analyzed as above for Figure 2C .

    Article Snippet: The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template.

    Techniques: Cellular Antioxidant Activity Assay, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing, Luciferase, Activity Assay

    Contrary to what occurs in β-globin mRNA, α-globin mRNAs allow for efficient translation re-initiation, but only partially contributes to NMD inhibition. ( A ) Schematic representation of the studied human α-globin mRNAs. Vertical lines represent translation initiation (AUG) or termination [native (UAA) or premature (PTC)] codons. The conversion of the two potential re-initiation AUGs, at codons 32 and 76, to ACG codons is indicated by short and thick vertical lines. The name of each transcript is specified on the right. ( B ) A representative RPA of RNA isolated from HeLa cells transiently transfected with each gene construct is shown. Identification of the α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the right of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations) normalized to the expression level of the αWT gene. A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. For each case, three independent experiments were performed. P -values were determined using a student's t -test and refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) The 5′-part of α-globin gene upstream of codon 32 (AUG), or upstream of codon 55 (AUG) of β-globin gene, was fused with the firefly luciferase open reading frame (ORF). Volume-shaped rectangles represent luciferase ORFs, dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin exons. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (PTC)] codons, are represented as black vertical lines. A positive control construct was created for each hybrid gene, in which the translation initiation codon for α- or β-globin gene was mutated to AUA. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids. Luciferase activity was analyzed 16 h after transfection. In parallel, both firefly and Renilla luciferase mRNA levels were determined by semi-quantitative RT-PCR. The activities of firefly and Renilla luciferase were calculated relative to their respective mRNA levels (Relative mRNA Levels, left bars), and then the resulting values for firefly luciferase activity/mRNA were quantified relative to those of Renilla luciferase activity/mRNA. The obtained luciferase relative activities from hybrid mRNAs were normalized to the relative activity of the uncoupled firefly luciferase (Luc). The average values and standard deviations from three independent experiments corresponding to three independent sets of transfections are shown.

    Journal: Nucleic Acids Research

    Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

    doi: 10.1093/nar/gkv588

    Figure Lengend Snippet: Contrary to what occurs in β-globin mRNA, α-globin mRNAs allow for efficient translation re-initiation, but only partially contributes to NMD inhibition. ( A ) Schematic representation of the studied human α-globin mRNAs. Vertical lines represent translation initiation (AUG) or termination [native (UAA) or premature (PTC)] codons. The conversion of the two potential re-initiation AUGs, at codons 32 and 76, to ACG codons is indicated by short and thick vertical lines. The name of each transcript is specified on the right. ( B ) A representative RPA of RNA isolated from HeLa cells transiently transfected with each gene construct is shown. Identification of the α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the right of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations) normalized to the expression level of the αWT gene. A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. For each case, three independent experiments were performed. P -values were determined using a student's t -test and refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) The 5′-part of α-globin gene upstream of codon 32 (AUG), or upstream of codon 55 (AUG) of β-globin gene, was fused with the firefly luciferase open reading frame (ORF). Volume-shaped rectangles represent luciferase ORFs, dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin exons. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (PTC)] codons, are represented as black vertical lines. A positive control construct was created for each hybrid gene, in which the translation initiation codon for α- or β-globin gene was mutated to AUA. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids. Luciferase activity was analyzed 16 h after transfection. In parallel, both firefly and Renilla luciferase mRNA levels were determined by semi-quantitative RT-PCR. The activities of firefly and Renilla luciferase were calculated relative to their respective mRNA levels (Relative mRNA Levels, left bars), and then the resulting values for firefly luciferase activity/mRNA were quantified relative to those of Renilla luciferase activity/mRNA. The obtained luciferase relative activities from hybrid mRNAs were normalized to the relative activity of the uncoupled firefly luciferase (Luc). The average values and standard deviations from three independent experiments corresponding to three independent sets of transfections are shown.

    Article Snippet: The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template.

    Techniques: Inhibition, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing, Luciferase, Positive Control, Activity Assay, Quantitative RT-PCR

    A model for the effect of an AUG-proximal premature termination codon (PTC or stop codon). ( A ) The effect in the human β-globin transcript. During cap-mediated translation initiation, cytoplasmic poly(A) binding protein 1 (PABPC1) interacts with the eukaryotic initiation factor 4G (eIF4G). This interaction indirectly tethers PABPC1 to the 40S ribosomal subunit via the interaction of eIF4G with eIF3, which interacts with the 40S. The resulting configuration brings PABPC1 into the vicinity of the AUG initiation codon as a consequence of 43S scanning. The maintenance of the PABPC1-eIF4G-eIF3 interactions with the 40S during the first steps of translation elongation brings PABPC1 into close proximity with the termination complex at an AUG-proximal PTC. This proximity allows PABPC1 to interact with the release factor eRF3, thus impairing the UPF1-eRF3 interaction, resulting in efficient translation termination and inhibition of NMD—this was called the ‘AUG-proximity effect’. ( B ) In the human α-globin transcript, the AUG-proximity effect is observed for PTCs located further downstream because the open reading frame (ORF) is less structured. The more relaxed structure allows a faster elongation rate resulting in a longer window of the AUG proximity effect. In addition, the brief ORF translation also allows efficient translation re-initiation to occur at codon 32. This re-initiation diminishes the levels of residual exon junction complexes on the mRNA, thus contributing to the overall repression of NMD.

    Journal: Nucleic Acids Research

    Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

    doi: 10.1093/nar/gkv588

    Figure Lengend Snippet: A model for the effect of an AUG-proximal premature termination codon (PTC or stop codon). ( A ) The effect in the human β-globin transcript. During cap-mediated translation initiation, cytoplasmic poly(A) binding protein 1 (PABPC1) interacts with the eukaryotic initiation factor 4G (eIF4G). This interaction indirectly tethers PABPC1 to the 40S ribosomal subunit via the interaction of eIF4G with eIF3, which interacts with the 40S. The resulting configuration brings PABPC1 into the vicinity of the AUG initiation codon as a consequence of 43S scanning. The maintenance of the PABPC1-eIF4G-eIF3 interactions with the 40S during the first steps of translation elongation brings PABPC1 into close proximity with the termination complex at an AUG-proximal PTC. This proximity allows PABPC1 to interact with the release factor eRF3, thus impairing the UPF1-eRF3 interaction, resulting in efficient translation termination and inhibition of NMD—this was called the ‘AUG-proximity effect’. ( B ) In the human α-globin transcript, the AUG-proximity effect is observed for PTCs located further downstream because the open reading frame (ORF) is less structured. The more relaxed structure allows a faster elongation rate resulting in a longer window of the AUG proximity effect. In addition, the brief ORF translation also allows efficient translation re-initiation to occur at codon 32. This re-initiation diminishes the levels of residual exon junction complexes on the mRNA, thus contributing to the overall repression of NMD.

    Article Snippet: The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template.

    Techniques: Binding Assay, Inhibition