Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Steady-state mRNA levels of firefly luciferase variants. Shown is a northern blot, probing for the common 3’UTR within different firefly luciferase reporter mRNAs, which vary by optimal codon content, and U6 snRNA loading control. Quantification of Firefly luciferase mRNA (relative to 0% optimal construct) is shown at bottom (error bars denote standard deviation; n=3). (B) More optimal luciferase mRNA variants are more stable. Transcriptional shut-off experiments were performed in Tet-Off HEK293 cells, and firefly luciferase mRNA levels were determined by northern blots. Timepoints correspond to the time after the addition of doxycycline, which shut-offs transcription of the reporter. t½ corresponds to the half-life (min) ± standard deviation (n=3). See for loading control. (C) More optimal MECP2 mRNA variants are more stable. As in B, expect for MECP2 . See for loading control. (D) More optimal CFTR ΔF508 mRNA variants are more stable. As in B, except for CFTR ΔF508. See for loading control. See also and Tables S1 and S2.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Luciferase, Northern Blot, Construct, Standard Deviation

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Schematic of the ORFeome workflow. The ORFeome collection contains ~16,000 full-length coding sequences corresponding to 14,000 genes in a lentiviral background. Each ORF derived from the ORFeome is flanked by invariant UTRs, and also contains a C-terminal V5 tag. Lentiviral pools containing ~3000 ORFeome clones were used to infect HEK293T and generate stable cell lines. Metabolic labeling was used to determine stabilities of endogenous and ORFeome-derived mRNAs in these stable lines. (B) Changing coding regions changes mRNA stability. Plotted are boxplots of half-lives for endogenous HEK293T (End.) and ORFeome-derived mRNAs (in blue and gray, respectively). The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (C) ORFeome mRNAs show as much variation in stability as endogenous mRNAs. Plotted are density destributions of median-centered half-lives for endogenous HEK293T and ORFeome-derived mRNAs (in blue and gray, respectively). (D) Treatment with 4EGI-1 inhibits translation. Shown are A 254 traces from sucrose density gradients of cell lysates from HEK293T cells treated with the translation inhibitor 4EGI-1 (orange) or DMSO (grey). (E) Transaltion inhibition destabilizes endogenous mRNAs. Plotted are boxplots of half-lives for endogenous HEK293T mRNAs with DMSO or 4EGI-1 treatment (in grey and orange, respectively). The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (F) Translation inhibition has a minor effect on the variation in stability for endogenous mRNAs. Plotted are density destributions of median-centered half-lives for endogenous HEK293T in cells treated with DMSO or 4EGI-1 (in gray and orange, respectively). (G) Inhibition of translation destabilizes ORFeome-derived mRNAs and reduces the variation in stability. As in E, except for ORFeome mRNAs. DMSO, in blue; 4EGI-1, in orange. (H) Translation inhibition reduces the variation in stability for ORFeome-derived mRNAs. As in F, except for ORFeome mRNAs. DMSO, in blue; 4EGI-1, in orange. See also and Table S2.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Derivative Assay, Clone Assay, Stable Transfection, Labeling, Inhibition

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) ORFeome complexity was maintained through stable cell line generation. Shown is a western blot probing lysates from the pooled ORFeome stable lines with V5 (the common C-terminal tag in the ORFeome collection). WT, parental HEK293T line; S, supernatant; P, pellet. (B) ORFeome-derived mRNAs are expressed in the stable cell lines. Shown is a scatter plot comparing steady-state RNA-seq reads (with a +1 pseudocount) for each gene between the two pooled lines used in this study. In black, genes in neither pool; in blue, genes in pool 1; in orange, genes in pool 4; in green, genes in both pools. Red dashed lines represents y = 3X and y = X/3, which were used as cut-offs to classify genes as ORFeome-expressed. ORFeome genes that did not pass threshold were not used for subsequent analysis (see Methods for more details). Numbers refer to the total number of genes in each pool and the number passing the 3-fold threshold. (C) ORFeome mRNAs are expressed in a pool-dependent fashion. Shown are boxplots of normalized read counts (with a +1 pseudocount) for ORFeome-derived mRNAs (split into pool 1 and pool 4) in the two pooled stable cell lines. Abundance in cell line 1 is shown in blue; in cell line 4, in orange. Note that the ORFeome pools are expressed in the appropriate cell line. (D) 4EGI-1 substantially reduces translation. Cells were treated with DMSO, 4EGI-1, or cyclohexamide (as a positive control) for the indicated times and then briefly treated with puromycin, which is incorporated into nascent peptides. Lysates were separated by gel electrophoresiss and probed for puromycin (top) or tubulin (bottom). (E) DMSO treatment does not substantially affect mRNA stability. Shown are scatterplots comparing half-lives for endogenous genes (averaged from both pools) from the original experiment and DMSO-treated cells. Red dashed line represents x = y. (F) 4EGI-1 treatment affects mRNA stability. As in E, except comparing half-lives from the original experiment and 4EGI-1-treated cells.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Stable Transfection, Western Blot, Derivative Assay, RNA Sequencing Assay, Positive Control

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Endogenous mRNA stability negatively correlates with length. Shown are boxplots for half-lives of endogenous HEK293T mRNAs binned into quartiles by ORF length. (B) ORFeome mRNA stability does not correlate with length. As in A, except for ORFeome mRNAs. (C) Endogenous mRNA stability weakly correlates with local secondary structure. For each ORF, the folding energy in 100 bp sliding windows was calculated, and the minimum value taken. Shown are boxplots for half-lives of endogenous HEK293T binned into quartiles by folding energy (with increased secondary structure on the right). (D) ORFeome mRNA stability does not correlate with local secondary structure. As in B, except for ORFeome mRNAs. (E) microRNA-mediated regulation cannot explain the variation in ORFeome stability. ORFs were classified as containing or lacking seed-matched sites for the top five expressed mRNAs (site ORFs [orange] and no site ORFs [blue], respectively). Shown are boxplots for their half-lives. Significance was calculated by the Kolmogorov-Smirnov test. (F) AU-rich elements cannot explain the variation in ORFeome stability. As in E, except for AU-rich elements. (G) AU-rich elements in ORFs destabilize mRNAs upon translational repression. As in F, except for half-lives determined in the presence of 4EGI-1.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques:

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Codons are differentially associated with stability. Shown are spearman correlations, for each codon, of their frequency with mRNA stability (codon stability coefficient; CSC) for endogenous HeLa mRNAs, endogenous HEK293T mRNAs, and ORFeome mRNAs. The 15 codons most associated with stability (as defined by the ORFeome collection) were designated as “optimal” (blue), while 15 codons most associated with instability were designated as “non-optimal” (orange). (B) HeLa and HEK293T cells have similar codon stability coefficients (CSCs). Plotted are the CSC values for endogenous HEK293T mRNAs compared to endogenous HeLa mRNAs (C) As in B, except comparing endogenous HEK293T and ORFeome-derived CSC values. (D) ORFeome mRNAs with more optimal codons are more stable. Shown are boxplots of mRNA half-lives for ORFeome mRNAs, binned into quartiles by the frequency of optimal codons. The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (E) As in D, except for endogenous HEK293T mRNAs. (F) Endogenous HEK293T CSCs weakly correspond with pause scores. Using HeLa ribosome profiling, pause scores were calculated for each codon in the A site, and then codons were divided into three groups (slow in orange; neutral in green; fast in blue). Shown are boxplots for the corresponding CSC values as determined by endogenous HEK293T mRNAs. (G) As in F, except for ORFeome-derived CSCs. See also .

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Codon stability coefficients (CSCs) have little relationship to tRNA abundance. Plotted are the normalized tRNA abundance in comparison to CSC values for endogenous HEK293T and and ORFeome mRNAs (left and right panels, respectively). (B) tRNA abundance has little impact on elongation speed. Codons were divided into thirds by their A-site pause scores (slow in orange; neutral, green; fast, blue). Shown are boxplots for the abundance of corresponding tRNAs. The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (C) Amino acids are differentially associated with stability. Shown are spearman correlations, for each amino acid, of their frequency with mRNA stability (amino acid stability coefficient or AASC) for endogenous HeLa mRNAs, endogenous HEK293T mRNAs, and ORFeome mRNAs. Polar amino acids (in pink) have charged or highly electronegative side chains; nonpolar amino acids (dark gray) have aliphatic and weakly electronegative side chains. (D) HeLa and HEK293T have similar AASCs. Plotted are the AASC values for endogenous HEK293T mRNAs compared to endogenous HeLa mRNAs (E) As in B, except comparing endogenous HEK293T and ORFeome-derived AASC values. (F) Hydrophobic amino acids are associated with stability. Plotted are the hydrophobicity scores for each amino acid compared to their stability coefficient for endogenous HEK293T mRNAs. (G) As in F, except for ORFeome-derived AASC values. (H) Schematic diagram of LSM8 reporter constructs. In the middle of the LSM8 coding region, five repeats of instability-associated amino acids (S, H and E) or stability-associated amino acids (V, I, and L) were inserted. (I) Insertion of instability-associated amino acids destabilizes the LSM8 reporter mRNA. Transcriptional shut-off experiments were performed in Tet-Off HEK293 cells, and LSM8 mRNA levels were determined by northern blots. Timepoints correspond to the time after the addition of doxycycline. t½ corresponds to the half-life (min) ± standard deviation (n=4). See for loading control. (J) The destabilized LSM8 reporter mRNA has shorter poly(A) tails. High resolution northern blotting was performed to measure poly(A)-tail lengths on the SHE and VIL LSM8 mRNAs. Arrow indicates deadenylated mRNA species; dT, oligo(dT)/RNase H treated mRNA control. (K) LSM8 reporter mRNAs are deadenylated. Transcription of the SHE and VIL LSM8 reporters was shut-off, as in I, and poly(A)-tail lengths were measured by high-resolution northern blotting. Timepoints represent time elapsed after transcription shutoff with 2 μg/mL doxycycline. Arrow indicates deadenylated mRNA species; dT, oligo(dT)/RNase H treated mRNA control. See also and Tables S1 and S2.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Derivative Assay, Construct, Northern Blot, Standard Deviation

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Valine frequency correlates with mRNA stability. Shown are boxplots of mRNA stabilities for HeLa, endogenous HEK293T, and ORFeome mRNAs binned into quartiles by valine frequency. (B) Serine frequency negatively correlates with mRNA stability. As in A, except for serine. (C) U6 snRNA northern analysis for transcription shut-off experiments for the LSM8 variants shown in . (D) U6 snRNA Northern analysis for LSM8 variable amino acid content transcription shutoff/Northern mRNA decay analyses shown in .

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Northern Blot

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Amino acids, when in the A-site, are translated at different rates. Plotted are the pause scores for each amino acid when in the predicted A-site (see methods for details). Nonpolar amino acids, grey; polar amino acids, pink. (B) Amino acid stability coefficients correlate with A-site pause scores. Shown are plots comparing A-site pause scores for each amino acid with its stability coefficient, as defined by endogenous HeLa, endogenous HEK293T, and ORFeome mRNAs (left, middle, and right, respectively). (C) As in A, except for the P site. (D) As in A, except for the E site. (E) A-site pause scores correlate poorly with P- and E-site pause scores. Plotted are the pairwise comparisons for A-, P-, and E-site pause scores. (F) ORFeome amino acid stability coefficients poorly correlate with P-site pause scores. Shown are plots comparing P-site pause scores for each amino acid with its stability coefficient, as defined by ORFeome mRNAs. (G) As in F, except for E-site pause scores.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques:

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: For each pair of codons, the absolute difference in the corresponding CSC values was calculated and then normalized to the maximal difference (to correct for differences in overall variance between organisms). Pairs of codons were binned into these encoding the same or different amino acid (n = 87, in grey, and n = 1742, in green, respectively). Shown are boxplots of those differences for S. pombe , trypanosomes, zebrafish, and endogenous HEK293T mRNAs. Significance determined by Wilcoxon rank sum test.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques:

Journal: Immunology and Cell Biology

Article Title: The vaccinia‐based Sementis Copenhagen Vector coronavirus disease 2019 vaccine induces broad and durable cellular and humoral immune responses

doi: 10.1111/imcb.12539

Figure Lengend Snippet: Spike‐specific antibody and SARS‐CoV‐2 neutralization responses following SCV‐S vaccination. (a) S1 and S2 subunit endpoint IgM and IgG ELISA titers determined from serum of female C57BL/6J mice ( n = 3) at the indicated times after a single vaccination with 10 7 PFU of SCV‐S, with (b) ratio of S1‐ and S2‐specific IgG2c to IgG1 endpoint ELISA titers determined 28 days after vaccination. (c) S1 IgG ELISA binding titers (left panel) and cPass neutralization titers (right panel) in outbred ARC(s) and inbred C57BL/6J female mice ( n = 5) 21 days after a single vaccination with 10 7 PFU of SCV‐S or vector control, with (d) durability of response in the outbred cohort shown by S1‐specific endpoint IgG ELISA titers (left panel) and neutralization titers (right panel) at the indicated times. (e) S1‐specific IgG ELISA (left panel) and neutralization titers (right panel) 50 days after a single‐dose (day 0) or prime‐boost (day 0 and 28) vaccination of female C57BL/6J mice ( n = 5) with 10 7 PFU SCV‐S or control vector, with (f) neutralizing activity in ACE2 and RBD blocking ELISA, and (g) neutralizing activity (IC 80 titers) against lenti‐SARS‐CoV‐2‐S pseudoviruses bearing spike protein from the Wuhan reference strain, the alpha or beta variant, shown for prime‐boost samples. (h) Neutralization titers in young (6–8 weeks old; n = 5) and middle‐aged (9–10 months old; n = 10) C57BL/6J mice at the indicated times after prime‐boost vaccination with 10 7 PFU SCV‐S. Results shown are representative of four independent experiments (indicated above) with binding and neutralizing antibody levels comparable at similar doses and time points across all experiments. Symbols represent individual mice and bars show the mean ± s.e.m. from independent experiments. Data were log transformed and statistical significance determined using Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ACE2, angiotensin‐converting enzyme‐2; IC 80 , 80% inhibitory concentration; Ig, immunoglobulin; ns, not significant; PFU, plaque‐forming units; RBD, receptor‐binding domain; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SCV‐S, Sementis Copenhagen Vector spike protein.

Article Snippet: ACE2‐HEK293 recombinant cell line (catalog number 79951; BPS Bioscience, San Diego, CA, USA) was maintained in Dulbecco’s Modified Eagle Medium supplemented (Sigma Aldrich, MO, USA) with 10% fetal bovine serum, 2 mM l ‐glutamine and penicillin–streptomycin.

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Binding Assay, Plasmid Preparation, Activity Assay, Blocking Assay, Variant Assay, Transformation Assay, Concentration Assay

Journal: Cancers

Article Title: Silibinin Overcomes EMT-Driven Lung Cancer Resistance to New-Generation ALK Inhibitors

doi: 10.3390/cancers14246101

Figure Lengend Snippet: Targeted effects of silibinin against the TGFβ/TGFβR/SMAD signaling pathway. ( A ) Expression levels of E-cadherin, SNAIL, vimentin, phospho-SMAD2/3, total SMAD2/3 were detected by immunoblotting in H2228/H2228CR and H3122/H3122CR cells using specific antibodies. The intensity of the bands was measured using the ImageJ software. Fold-change of each protein relative to parental cells was calculated using GAPDH as a loading control. The figure shows representative immunoblots of multiple ( n ≥ 5) independent experiments. E: Epithelial; M: Mesenchymal. ( B ) Top: Relative luciferase activity using SBE Reporter–HEK293 cells pre-incubated during 4–5 h with graded concentrations of SB525334 and silibinin before stimulation with TGFβ1. Bottom: Expression levels of phospho-SMAD2/3 and total SMAD2/3 were detected by immunoblotting in HEK293 cells stimulated with TGFβ1 (0, 6, and 24 h) in the absence/presence of either silibinin or SB431542 using specific antibodies. The intensity of the bands was measured using the ImageJ software. Fold-change of each protein relative to untreated samples was calculated using GAPDH as a loading control. The figure shows representative immunoblots of multiple ( n ≥ 3) independent experiments. ( C ) Volcano plots of the results from analyses of the Applied Biosystems TM TaqMan TM Array Human TGFβ Pathway in H2228/H2228CR cells cultured in the absence/presence of silibinin (100 μmol/L) for 48 h. Each dot represents a transcript with its corresponding mean Log2 fold-change (FC) ( x axis) and Benjamini–Hochberg corrected p -value (−log10, y axis). Colored dots illustrate differential lipid species, using a cutoff of p < 0.05 and log2FC > 1 or < 1. ( D ) The figure depicts the backbone of the overall crystal structure of TGFβR1 (5E8S) and TGFβR2 (5E8Y) with rainbow colors showing the best docked poses of silibinin A and silibinin B at the catalytic site. The uncropped western blot figures were presented in .

Article Snippet: The SBE Reporter–HEK293 cell line (Cat. #60653; BPS Bioscience, San Diego, CA, USA) was employed for monitoring the impact of silibinin on the activity of the TGFβ/SMAD signaling pathway.

Techniques: Expressing, Western Blot, Software, Luciferase, Activity Assay, Incubation, Cell Culture

Journal: Chemical biology & drug design

Article Title: 2-Aminothiophene derivatives as a new class of positive allosteric modulators of glucagon-like peptide 1 receptor

doi: 10.1111/cbdd.14039

Figure Lengend Snippet: Potential allosteric effect of S-1 and S-2 on human GLP-1R. (a) The effect of GLP-1 on HEK293 cells expressing human GLP-1R or empty vector in the presence or absence of S-1 (23.5 μM). (b) The effect of GLP-1 on HEK293 cells expressing human GLP-1R or empty vector in the presence or absence of S-2 (71 μM). GLP-1R activation was assessed as luminescence normalized to protein concentration and plotted as luminescence fold change with respect to vehicle control (0.5% DMSO). Data are average of three independent experiments with at least three technical replicates for each conditions, and error bars for each concentration were plotted as SEM (n = 3). Statistical analysis was done using 2-way ANOVA (****p < 0.0001; **p < 0.001; *p < 0.5). The comparison is done between the data points of the dose–response curve generated due to GLP-1’s effect on GLP-1R in the presence and absence of S-1 or S-2

Article Snippet: HEK293 cell stably expressing CRE/CREB luciferase reporter gene (BPS Bioscience #60515), RPMI medium (Corning #10-040), Krebs-Ringer Solution, HEPES-buffered (Alfa Aesar #J67795-K2), L-Glutamine (Gibco #25030-081), HEPES (Gibco #15630-080), Sodium Pyruvate (Gibco #11360-070), β-mercaptoethanol (MP #806444), D-Glucose (#G-7528), Fetal Bovine Serum (Fisher #03600511), penicillin/streptomycin (Corning #30-002-Cl), Hygromycin B (Alfa Aesar #J60681), Lipofectamine 2000 (Invitrogen #11668027), vasoactive intestinal peptide receptor (VIPR) peptide agonist (Sigma #V3628), VIPR peptide antagonist (Sigma #SCP0260), GLP-1R peptide agonist (Sigma #9416), 6-well cell culture plates (Ultra Cruz #sc-204443), 96-well cell culture plates (Sigma #CLS9102), Luciferase cell culture lysis reagent (Promega #E1531), Luciferase assay reagent (Promega #E1501), and Ultra-Sensitive Rat Insulin Kit (Crystal Chem #90060) were purchased from vendors.

Techniques: Expressing, Plasmid Preparation, Activation Assay, Protein Concentration, Control, Concentration Assay, Comparison, Generated