Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: Protein-protein interaction of NRT1.5 and AHA2. (A) THY.AP4 cells were transformed with NRT1.5 or NRT1.5 G209E fused to Cub and AHA2 fused to Nub. To detect protein-protein-interaction, 12 µl yeast cell culture with an OD 600 nm =1 and OD 600 nm =2, respectively, were dropped on YNB-Leu-Trp-His-Ade medium supplemented with 20 mM 3-amino-1,2,4-triazole (3-AT) and on YNB-Leu-Trp medium as growth control. The addition of 3-AT reduces non-specific yeast growth not based on protein-protein interactions. Plates were incubated at 30°C for three days and then photographed. (B) Confirmation of the interaction of NRT1.5 with AHA2 by bimolecular complementation assay. Shown are confocal microscopy images of N. benthamiana epidermis cells two days post-infiltration co-expressing non-fused nYFP, cYFP-NRT1.5 and RFP (top row), nYFP-AHA2, cYFP-NRT1.5 and RFP (center row), and nYFP-AHA2, cYFP-NRT1.5 G209E and RFP (bottom row). Column YFP: YFP fluorescence in yellow indicates interaction between proteins. Column RFP, RFP fluorescence in red visualizes the cytoplasm and the lumen of the nucleus as expression control. Column Chl, autofluorescence of chlorophyll (represented in violet). Column BF, bright field image. Column overlay: overlay of all 4 channels. Scale bar = 20 µm.

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Transformation Assay, Cell Culture, Incubation, Confocal Microscopy, Expressing, Fluorescence

Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: Phenotyping of wild-type and mutant plants under low (LK) and high nutrient (HK) conditions. (A) RT-PCR analysis of NTR1.5 and AHA2 transcripts in Col-0 and the nrt1.5/aha2 double mutant. GAPC1 (At3g04120) was used as reference housekeeping gene (B) Rosette phenotypes, (C) Leaf fresh weight, and (D) Fraction of chlorotic leaves (%) of Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 plants that were pre-germinated in fertilized soil for seven days and transferred to unfertilized soil type for another 30 days while watered with HK or LK medium, respectively. Shown are the means ± SD of the fraction of chlorotic leaves in 20 individual plants. Different letters above columns indicate statistically significant differences (Tukey’s test; P <0.05).

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: Elemental analysis by ICP-OES of Arabidopsis Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 plants grown under low (LK) and high (HK) nutrient conditions. (A) Macronutrient and (B) micronutrient concentration in shoots of 30 days-old Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 plants grown in LK and HK medium. Different letters above columns indicate statistically significant differences (Tukey’s test; P <0.05) between mutants and Col-0 (means ± SD, n =8).

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Concentration Assay

Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: Phenotypes of Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 seedlings at 0K or HK supply. (A) Root phenotypes. Seedlings were pre-germinated in 0.5 × MS medium for five days and then transferred to 0K or HK media for fourteen days further. White arrows show lateral root formation. Dotted lines represent the maximum primary root growth of Col-0 in each condition. (B) Quantification of the density of lateral roots from (A) The number of lateral roots formed/main length of the primary root (cm) was plotted as lateral root density. Different letters indicate statistically significant differences (Tukey’s test) between mutants and Col-0 with P < 0.05, (means ± SD, n ≥15). (C) Root hair phenotypes of seedlings in (A) . (D) Root hair density was determined as the number of hair roots in a 15 mm section from the starting point of the root tip under a light stereomicroscope (SZX12, Olympus) and quantified using Image J software. Results were expressed as the mean ± standard error. Different letters indicate statistically significant differences (Tukey’s test) between mutants and Col-0 with P < 0.05, (means ± SD, n =15). (E) Shoot phenotypes of seedlings in A after a week of growth in the conditions indicated.

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Software

Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: pH and plasma membrane potential are influenced by NRT1.5 and AHA2 expression. (A) Root growth phenotypes of Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 in response to 5 µg/ml HygB. 5-day-old seedlings were transferred to 0.5 × MS medium pH 5.5 supplemented with or without 5 µg/ml HygB for fourteen more days. The white bar represents 1 cm. (B) Extracellular pH measurements in Col-0, nrt1.5 , aha2, nrt1.5/aha2 roots under HK and 0K nutrition supply. Ten days-old seedlings were incubated with 0.25 x MS liquid medium supplemented with 30 µg/ml of FITC-dextran for 16 h. Media pH was determined by a fluorescence standard curve with a pH range from 4.5 to 7.5. Different letters indicate statistically significant differences (Tukey’s test) between mutants and Col-0 with P <0.05, (means ± SD, n =12). (C) BY4741 wild-type and BYT12 mutant yeast strains were transformed with empty expression vectors (p425-TEF and p426-TEF) or expression constructs for the indicated proteins. Twelve 20 µl drops of untransformed (negative control) or transformed yeast cell suspension (OD 600 nm =1) were spotted along the HygB gradient (from 0 to 0.5 g/l) on SD-leu-ura pH 6.0, 0.1 M KCl plates and incubated at 30°C for two days. p425-TEF and p426-TEF are the empty expression vectors.

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Membrane, Expressing, Incubation, Fluorescence, Mutagenesis, Transformation Assay, Construct, Negative Control, Suspension

Journal: Cell reports

Article Title: Stem-loop-induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control

doi: 10.1016/j.celrep.2024.113976

Figure Lengend Snippet: Revisiting translational control of human ATF4: Reporters and experimental setup (A) Schematic showing the 5′ end of the mRNA encoding human activating transcription factor 4 ( ATF4 ; NM_182810.2, transcript variant 2), featuring the color-coded Start-stop (St-st) element (yellow), REI-permissive (green) 3-codon uORF1, and inhibitory uORF2 (red) overlapping with the beginning of the main ATF4 ORF, frames 0 and 1. Distances are given in nucleotides. (B) Schematic of the WT CMV-driven ATF4-HA-tagged construct; the HA tag was placed immediately upstream of the ATF4 stop codon. (C) Experimental workflow described in the form of a timeline diagram. PN, Jess protein normalization detection module. (D) All HEK293T cell lysates were subjected to protein separation and immunodetection using the Jess system. The signal, detected in the capillary, is represented as an electropherogram (a single peak of the ATF4-HA tag full-length protein, size of 53 kDa, left) and was automatically quantified (right). Expression of the WT construct under 3 h of Tg stress conditions 8 h post transfection, detected by anti-mouse HA tag antibodies, is shown. (E) Stress-induced upregulation of ATF4-HA protein expression under Tg stress (blue) compared with non-stress conditions (green). Quantified “fold induction” data were plotted (n = 17, right). The differences between experimental groups were tested by a t test. Variables are presented as mean ± SD, and p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (F) Stress-induced upregulation of the ATF4-HA protein expression from the WT reporter under tunicamycin stress compared with non-stress conditions (set to 1). Quantified “fold induction” data were plotted (n = 3) and analyzed as in (E). (G) Experimental setup illustrating determination of fold change values when comparing (1) each mutant reporter construct vs. the WT construct separately under non-stress (top horizontal arrow) and Tg stress conditions (bottom horizontal arrow) and (2) the fold induction expression of a mutant under stress with the same mutant under non-stress (right vertical arrow) or the WT under stress vs. no stress (blue left vertical arrow). The red diagonal arrow indicates calculated fold induction changes of a given mutant under stress vs. WT under non-stress conditions; i.e., how much each mutant increases or decreases the ~5.2-fold induction of the WT reporter.

Article Snippet: WT ATF4-HA Tag was created by inserting Hind III -Not I digested h ATF4 -wt-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector. dSt-st was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -St-st_AGG-F and h ATF4 -St-st_AGG-R using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d1 was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -uORF1_AGG-F and h ATF4- uORF1_AGG-R primers using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d2 was created by inserting the Pst I -EcoR V digested fusion PCR product obtained with primers h ATF4 -uORF2_AGG and SW ATF4 d120 SphI R using wt ATF4-HA Tag as a template into Pst I -EcoR V digested wt ATF4-HA Tag. d-all was created by inserting Hind III -Not I digested h ATF4 -d -all-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector.

Techniques: Variant Assay, Construct, Immunodetection, Expressing, Transfection, Mutagenesis

Journal: Cell reports

Article Title: Stem-loop-induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control

doi: 10.1016/j.celrep.2024.113976

Figure Lengend Snippet: Sequence analysis of the 5′ UTR of the human ATF4 mRNA and ribosome queuing model expanding the mode of ATF4 translational control (A) Schematic of previously unknown, bioinformatically predicted, potential regulatory features within the 5′ UTR of the ATF4 mRNA and beginning of the ATF4 main ORF; point mutations are depicted. For details, please see the main text. (B) Model of the ribosome queuing mechanism under non-stress vs. stress condition employing SL3 and near-cognate CUG as an additional layer of ATF4 translational control. For details, please see the main text. Created with BioRender.

Article Snippet: WT ATF4-HA Tag was created by inserting Hind III -Not I digested h ATF4 -wt-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector. dSt-st was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -St-st_AGG-F and h ATF4 -St-st_AGG-R using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d1 was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -uORF1_AGG-F and h ATF4- uORF1_AGG-R primers using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d2 was created by inserting the Pst I -EcoR V digested fusion PCR product obtained with primers h ATF4 -uORF2_AGG and SW ATF4 d120 SphI R using wt ATF4-HA Tag as a template into Pst I -EcoR V digested wt ATF4-HA Tag. d-all was created by inserting Hind III -Not I digested h ATF4 -d -all-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector.

Techniques: Sequencing

Journal: Cell reports

Article Title: Stem-loop-induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control

doi: 10.1016/j.celrep.2024.113976

Figure Lengend Snippet: SL3 delays the flow of ribosomes in the uORF2/ATF4 overlap (A) Same as for better comparison. (B) Same as except that the SL3 ATF4 mutant constructs depicted at the top of the corresponding panels were subjected to Jess analyses. Relative ATF4-HA protein expression levels were plotted as ratios of values obtained with an indicated mutant construct vs. the WT set to 1 under “non-stress” (row 1) and “Tg stress” (row 2) conditions; “fold-induction” plots (row 3) depict ratios of Tg stress vs. non-stress values obtained with a given mutant construct. The differences between experimental groups were tested by the t test, except wt-SL3 Mut−1 Tg stress and d2-SL3 Mut−1 non-stress, where a Mann-Whitney test was used (n ≥ 3).

Article Snippet: WT ATF4-HA Tag was created by inserting Hind III -Not I digested h ATF4 -wt-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector. dSt-st was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -St-st_AGG-F and h ATF4 -St-st_AGG-R using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d1 was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -uORF1_AGG-F and h ATF4- uORF1_AGG-R primers using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d2 was created by inserting the Pst I -EcoR V digested fusion PCR product obtained with primers h ATF4 -uORF2_AGG and SW ATF4 d120 SphI R using wt ATF4-HA Tag as a template into Pst I -EcoR V digested wt ATF4-HA Tag. d-all was created by inserting Hind III -Not I digested h ATF4 -d -all-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector.

Techniques: Comparison, Mutagenesis, Construct, Expressing, MANN-WHITNEY

Journal: Cell reports

Article Title: Stem-loop-induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control

doi: 10.1016/j.celrep.2024.113976

Figure Lengend Snippet: SL3 genetically interacts with the upstream CUG near-cognate codon (A) Same as for better comparison. (B) Same as except that the CUG to CUA and SL3 Mut−1 ATF4 mutant constructs depicted at the top of the corresponding panels were subjected to Jess analyses (n ≥ 3).

Article Snippet: WT ATF4-HA Tag was created by inserting Hind III -Not I digested h ATF4 -wt-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector. dSt-st was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -St-st_AGG-F and h ATF4 -St-st_AGG-R using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d1 was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -uORF1_AGG-F and h ATF4- uORF1_AGG-R primers using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d2 was created by inserting the Pst I -EcoR V digested fusion PCR product obtained with primers h ATF4 -uORF2_AGG and SW ATF4 d120 SphI R using wt ATF4-HA Tag as a template into Pst I -EcoR V digested wt ATF4-HA Tag. d-all was created by inserting Hind III -Not I digested h ATF4 -d -all-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector.

Techniques: Comparison, Mutagenesis, Construct

Journal: Cell reports

Article Title: Stem-loop-induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control

doi: 10.1016/j.celrep.2024.113976

Figure Lengend Snippet: Ribosome queuing and substantial leaky scanning at AUG1 of ATF4 contributes to its overall translational control (A–D) Same as in except that the 63 c-Myc tag insertion in frame with ATF4 (A), combined with the SL3 Mut−1 mutation (B), in the otherwise WT construct (A and B) vs. the construct lacking uORF2 (C and D), all depicted at the top of the corresponding panels, were subjected to Jess analyses. The differences between experimental groups were tested by a t test, except d2_ins_SL3 Mut−1 Tg stress, where a Mann-Whitney test was used (n ≥ 4). (E) The first AUG of the ATF4 ORF is robustly leaky scanned. The electropherograms of the ATF4 construct bearing the 63 c-Myc tag in-frame insertion probed with the anti-c-Myc (left) and anti-HA (right) antibodies are shown. For details, see the main text.

Article Snippet: WT ATF4-HA Tag was created by inserting Hind III -Not I digested h ATF4 -wt-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector. dSt-st was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -St-st_AGG-F and h ATF4 -St-st_AGG-R using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d1 was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -uORF1_AGG-F and h ATF4- uORF1_AGG-R primers using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d2 was created by inserting the Pst I -EcoR V digested fusion PCR product obtained with primers h ATF4 -uORF2_AGG and SW ATF4 d120 SphI R using wt ATF4-HA Tag as a template into Pst I -EcoR V digested wt ATF4-HA Tag. d-all was created by inserting Hind III -Not I digested h ATF4 -d -all-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector.

Techniques: Mutagenesis, Construct, MANN-WHITNEY

Journal: Cell reports

Article Title: Stem-loop-induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control

doi: 10.1016/j.celrep.2024.113976

Figure Lengend Snippet: Ribosome protection assay demonstrating that SL3 pauses ribosomes and prompts their queuing under both non-stress and stress conditions (A) Schematic showing the ATF4 mRNA with the sequences of three primer pairs amplifying three different amplicons (A1–A3; also indicated in ): 1a-1b (the latter shown in the 3′ to 5′ direction for better illustrative purposes) for the putative queuing fragment and 2a-2b and 3a-3b for two control fragments downstream of SL3 used in the ribosome protection assay. (B) HEK293T cells were cross-linked with formaldehyde (HCHO) and then subjected to the ribosome protection assay as described in . qPCR product levels of the recovered putative queuing region (A1) are normalized to the region immediately downstream of SL3 (A2) as well as to the internal RNA isolation control (SPIKE) with the non-stress values set to 1. Results are representative of three independent replicates, and values are expressed as mean ± SD. Statistical significance was assessed using unpaired two-sided t test (*p < 0.01, **p < 0.001) with Bonferroni correction. (C) HEK293T cells were treated with cycloheximide (a non-cross-linking agent) and then subjected to the ribosome-protection assay as described in . Results from three independent replicates were analyzed as described in (B) with non-stress values set to 1 (*p < 0.01, **p < 0.001). (D) HEK293T cells were transiently transfected with plasmids carrying either WT or SL3-mutated (in SL3 Mut−1 ) ATF4 reporters and treated as described in (B). Results from three independent replicates were analyzed as described in (B) with the WT values set to 1 (*p < 0.01, **p < 0.001, ***p < 0.0001).

Article Snippet: WT ATF4-HA Tag was created by inserting Hind III -Not I digested h ATF4 -wt-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector. dSt-st was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -St-st_AGG-F and h ATF4 -St-st_AGG-R using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d1 was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -uORF1_AGG-F and h ATF4- uORF1_AGG-R primers using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d2 was created by inserting the Pst I -EcoR V digested fusion PCR product obtained with primers h ATF4 -uORF2_AGG and SW ATF4 d120 SphI R using wt ATF4-HA Tag as a template into Pst I -EcoR V digested wt ATF4-HA Tag. d-all was created by inserting Hind III -Not I digested h ATF4 -d -all-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector.

Techniques: Isolation, Transfection

Journal: Cell reports

Article Title: Stem-loop-induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control

doi: 10.1016/j.celrep.2024.113976

Figure Lengend Snippet: mRNA methylation further fine tunes ATF4 translation (A) Same as for better comparison. (B) Same as except that A235G (left) and A326G either alone (center) or in combination with SL3 Mut−1 (right) ATF4 mutant constructs depicted at the top of the corresponding panels were subjected to Jess analyses (n ≥ 3). (C) mRNA fragments prepared from either HEK293T (left) or HeLa (right) cells carrying the A235G and A326G mutations were subjected to the T3 ligation assay as described in the main text. The normalized percentage of unmodified A 235 or A 326 bases of the ATF4 mRNA expressed in mock-treated vs. Tg-treated cells, with the latter set to 100%, was plotted as shown (n ≥ 4).

Article Snippet: WT ATF4-HA Tag was created by inserting Hind III -Not I digested h ATF4 -wt-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector. dSt-st was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -St-st_AGG-F and h ATF4 -St-st_AGG-R using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d1 was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -uORF1_AGG-F and h ATF4- uORF1_AGG-R primers using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d2 was created by inserting the Pst I -EcoR V digested fusion PCR product obtained with primers h ATF4 -uORF2_AGG and SW ATF4 d120 SphI R using wt ATF4-HA Tag as a template into Pst I -EcoR V digested wt ATF4-HA Tag. d-all was created by inserting Hind III -Not I digested h ATF4 -d -all-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector.

Techniques: Methylation, Comparison, Mutagenesis, Construct, Ligation

Journal: Cell reports

Article Title: Stem-loop-induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control

doi: 10.1016/j.celrep.2024.113976

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: WT ATF4-HA Tag was created by inserting Hind III -Not I digested h ATF4 -wt-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector. dSt-st was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -St-st_AGG-F and h ATF4 -St-st_AGG-R using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d1 was created by inserting the Hind III -Pst I digested fusion PCR product obtained with primers h ATF4 -uORF1_AGG-F and h ATF4- uORF1_AGG-R primers using wt ATF4-HA Tag as a template into Hind III -Pst I digested wt ATF4-HA Tag. d2 was created by inserting the Pst I -EcoR V digested fusion PCR product obtained with primers h ATF4 -uORF2_AGG and SW ATF4 d120 SphI R using wt ATF4-HA Tag as a template into Pst I -EcoR V digested wt ATF4-HA Tag. d-all was created by inserting Hind III -Not I digested h ATF4 -d -all-HA-Tag (GeneArt String DNA Fragment; Invitrogen) into Hind III -Not I digested pCMV-EGFP-N2 high copy vector.

Techniques: Virus, Recombinant, Lysis, Ligation, Plasmid Preparation, Fluorescence, Software, Western Blot