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: Translational Psychiatry

Article Title: Immunoglobulin G modulation of the melanocortin 4 receptor signaling in obesity and eating disorders

doi: 10.1038/s41398-019-0422-9

Figure Lengend Snippet: a Representative images of hMC4R GFP+ HEK 293 cells (green) 30 min after application of DyLight 550®-labeled (red) α-MSH affinity-purified IgG from eating disorder (anorexia nervosa, n = 9; bulimia nervosa, n = 7; binge eating disorder, n = 7), obese ( n = 10), and Ctrl ( n = 9) preincubated or not with α-MSH. Quantification of DyLight 550®-positive spots in hMC4R+ HEK 293 cells ( n = 50/group): b at the membrane; c intracellularly (cytosolic), and d ratios of cytosolic/membrane staining. Affinity kinetics properties of α-MSH/IgG IC for hMC4R + HEK 293 cells including e dissociation equilibrium constant (KD); f association rate (ka), and g dissociation rate (kd). Data are means ± s.e.m. Kruskal–Wallis test with Dunns’ post-tests ( b – d , f , g ) or analysis of variance with Tukey’s post-test ( e ), *** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: MC4R-expressing HEK 293 cells (AMS Biotechnology, Abingdon, UK) were cultured in glass bottom Petri dishes (MatTek, ≈250,000 cells/dish).

Techniques: Labeling, Affinity Purification, Staining

Journal: Translational Psychiatry

Article Title: Immunoglobulin G modulation of the melanocortin 4 receptor signaling in obesity and eating disorders

doi: 10.1038/s41398-019-0422-9

Figure Lengend Snippet: a cAMP dose–response curves to α-MSH alone or α-MSH/IgG IC formed by IgG pooled in patents and control groups and adjusted to α-MSH-reactive IgG plasma levels of controls. cAMP dose–response curves to α-MSH preincubated or not with individual total IgG and corresponding EC50 ( b ) and maximal cAMP production ( c ). d Control experiments including cAMP dose–response curves to α-MSH by MC4R-expressing and non-expressing control HEK 293 cells and to α-MSH 1–4 peptide by MC4R-expressing cells ( n = 4). e cAMP dose-response curves to α-MSH and IgG from patients and controls without their overnight pre-incubation. f , cAMP dose–response curves to α-MSH alone or α-MSH/IgG IC co-administered (solid line) with agouti-related protein (AgRP; 100 nM) or added after AgRP preincubation (dotted line— n = 2/group) as well as in g cAMP maximal response. h , i cAMP dose–response curves of α-MSH preincubated with h purified total IgG from patients and controls depleted for α-MSH-reactive IgG ( n = 3/group) and i affinity-purified α-MSH-reactive IgG ( n = 6/group); j EC 50 and k maximal cAMP production at the plateau. Data are means ± s.e.m. Analysis of variance with Tukey’s post-test ( b , c , g , j ) or Kruskal–Wallis test with Dunns’ post-tests ( m ), *** p < 0.001, ** p < 0.01, * p < 0.05; Mann–Whitney test, $ p < 0.05. a α-MSH ( n = 9), Ctrl, anorexia nervosa (AN), bulimia nervosa (BN), and binge eating disorder (BED; n = 6), obese (OB; n = 4); d HEK 293-hMC4R+ ( n = 5), HEK 293-CTRL ( n = 6); e α-MSH ( n = 3), Ctrl, BN, and BED ( n = 2), AN and OB ( n = 3)

Article Snippet: MC4R-expressing HEK 293 cells (AMS Biotechnology, Abingdon, UK) were cultured in glass bottom Petri dishes (MatTek, ≈250,000 cells/dish).

Techniques: Expressing, Incubation, Purification, Affinity Purification, MANN-WHITNEY

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of the preparatory and analytical workflows. (A) POMC derived ligands and their downstream signaling cascades. (B) Schematic overview of the thermal proteome profiling (TPP) workflow. MC3R-expressing HEK293 cells were treated with ACTH, α-MSH, or γ-MSH at concentrations of 20, 100, and 500 nM or with DMSO as a vehicle-only negative control. (C) Schematic overview of the TPP data analysis workflow. Protein identification and relative quantification were achieved by direct analysis of the raw LC–MS data, after which various bioinformatics tools were used to infer changes in transcription factor (TF) activity, perform enriched pathway analysis, and identify thermally affected proteins.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Derivative Assay, Expressing, Negative Control, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of identified proteins and thermally stabilized or destabilized proteins. (A) Venn diagrams showing the numbers of proteins exhibiting altered melting points, associations with enriched pathways, and phosphorylation in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH. (B) Venn diagrams showing the numbers of stabilized, destabilized, and phosphorylated proteins after incubation with ACTH, α-MSH, and γ-MSH. (C) Upset plot representing individual numbers of stabilized and destabilized proteins for each ligand and those common between various combinations of ligands.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Phospho-proteomics, Expressing, Incubation

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Characterization of transcription factors. (A) Heat map showing the relative abundance (compared to vehicle-only controls) of the transcription factors CCAR2, HMGB2, DDX21, SRSF7, and TET2 in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH at different ligand concentrations and temperatures. (B) Phosphorylation of tryptic peptides derived from the thermally stabilized and destabilized transcription factors shown in panel A whose activity was inferred to change following stimulation with ACTH, α-MSH, or γ-MSH. Phosphorylation sites are indicated by asterisks next to the modified amino acid (shown in parentheses when the exact amino acid is unknown). (C) Transcription factor activities and relational networks inferred from differential expression data using BITFAM. The heatmap shows fold changes in transcription factor activities (relative to vehicle-only treatments) in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, or γ-MSH. (D) Network showing the interconnectivity of the transcription factors identified within our experimental LC–MS data set.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Expressing, Incubation, Phospho-proteomics, Derivative Assay, Activity Assay, Modification, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy