Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) Schematic depicting distal, proximal regulatory elements extending 3 kb on either side of CCL2 gene and the linkage disequilibrium (LD) between regulatory polymorphism rs1024611 and the transcribed polymorphism rs13900. rs1024611 is located 2578 base pairs upstream of the CCL2 translation start site and rs13900 is located in the CCL2 3′untranslated region (3′ UTR). ( B ) Allelic expression imbalance (AEI) in heterozygous donors is measured as a ratio of alternative allele (ALT) to reference allele (REF) in a transcribed polymorphism. ( C ) Representative chromatograms obtained following Sanger sequencing of PCR products obtained from genomic DNA (gDNA) and reverse transcription PCR of mRNA (cDNA) from three individuals heterozygous for rs13900. gDNA and mRNA were obtained from peripheral blood mononuclear cell (PBMC) treated with lipopolysaccharide (LPS) for 3 hr as previously described. The allelic ratios shown were determined by PeakPicker analysis. PeakPicker calculates allelic ratios by dividing the peak height of the alternate allele (rs13900 T allele) by that of reference allele (rs13900 C allele). The gDNA peaks were used for normalization. ( D ) Allelic ratio for cDNA and gDNA in six individuals heterozygous for rs13900 after treatment with LPS for 3 hr. Statistical significance for the difference in the level of expression between the alleles was determined using Student’s t test (p<0.003). Figure 1—source data 1. Numerical data used to generate .

Article Snippet: Commercial assay or kit , TaqMan SNP Genotyping Assay , Thermo Fisher Scientific , Thermo Fisher Scientific: 4351379 , Assay id: C___7449810_10.

Techniques: Expressing, Sequencing, Reverse Transcription

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: PBMCs were treated with 1 µg lipopolysaccharide (LPS) for 1, 3, or 6 hr. CCL2 mRNA expression was measured by quantitative real-time PCR and normalized to 18S rRNA. Data are presented as mean ± SD of triplicate samples.

Article Snippet: Commercial assay or kit , TaqMan SNP Genotyping Assay , Thermo Fisher Scientific , Thermo Fisher Scientific: 4351379 , Assay id: C___7449810_10.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) CCL2 mRNA expression in peripheral monocytes of heterozygous individuals (N=6) after treatment with lipopolysaccharide (LPS) for 3 hr and then incubated with 5 µg actinomycin D (Act D) for indicated times. mRNA was detected by RT-PCR. Results, normalized to 18S rRNA levels, are expressed as fold-increase over unstimulated cells (CNT). Levels shown in the bar graph represent mean ± SEM of result at time 0 (*p=0.019) versus unstimulated cells. ( B ) CCL2 mRNA half-life, calculated for each condition as the time (in hours) required for the transcript to decrease to 50% of its initial abundance (t 1/2 =ln (0.5)/slope). ( C ) Nascent RNA was isolated from treated monocytes from three individuals in the presence and absence of ActD. Allelic ratio was determined after 4 hr of incubation with or without ActD. Expression of the rs13900 T allele was much higher in ActD-treated samples. The difference between the groups was assessed by ANOVA with Fisher’s least significant difference (LSD) method (*p<0.05, **p<0.005). Figure 2—source data 1. Numerical data used to generate .

Article Snippet: Commercial assay or kit , TaqMan SNP Genotyping Assay , Thermo Fisher Scientific , Thermo Fisher Scientific: 4351379 , Assay id: C___7449810_10.

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) Validation of RBP-binding sites and polymorphism located on the 3′ untranslated region (3′ UTR) of CCL2 transcript from the Atlas of UTR Regulatory Activity and analysis of ENCODE genome-wide datasets detected specific enrichment of HuR (ELAVL1) at the region that contains the rs13900. ( B ) Predicted changes in the secondary structure using ViennaRNA Package 2.0; the black arrow indicates the changes in the secondary structure due to the rs13900 T allele. ( C ) Sequence logo of HuR-binding site as determined by HOMER. ( D ) Relative structural preference of HuR across different nucleotide contexts: P denotes paired regions, L denotes hairpin loops, U denotes unstructured (or external) regions, and M denotes miscellaneous regions.

Article Snippet: Commercial assay or kit , TaqMan SNP Genotyping Assay , Thermo Fisher Scientific , Thermo Fisher Scientific: 4351379 , Assay id: C___7449810_10.

Techniques: Biomarker Discovery, Binding Assay, Activity Assay, Genome Wide, Sequencing

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) HuR enrichment in immunoprecipitated material from macrophages stimulated with lipopolysaccharide (LPS). The input sample could not be included due to limited availability of material. To ensure comparable protein recovery across samples, β-actin was used as a loading control. ( B ) CCL2 3′ untranslated region (3′ UTR) was detected at significant levels in the samples precipitated by anti-HuR antibody when compared to the control IgG. ( C ) CCL2 mRNA expression in anti-HuR antibody enriched immunoprecipitated material analyzed by real-time quantitative PCR (RT-qPCR) (N=4). Statistical significance was calculated using Student’s t test (*p<0.005). The error bars represent SEM. ( D ) Relative expression levels of rs13900 C and T alleles in the anti-HuR-enriched immunoprecipitated complexes obtained from macrophages stimulated with LPS (N=6). Statistical significance was calculated using Student’s t test (*p<0.005). Figure 5—source data 1. PDF file containing original western blots for supporting . Figure 5—source data 2. Original files for western blot analysis displayed in . Figure 5—source data 3. PDF file containing original uncropped gel for , indicating the relevant bands and treatment. Figure 5—source data 4. Original files for agarose gel displayed in . Figure 5—source data 5. Numerical data used to generate .

Article Snippet: Commercial assay or kit , TaqMan SNP Genotyping Assay , Thermo Fisher Scientific , Thermo Fisher Scientific: 4351379 , Assay id: C___7449810_10.

Techniques: Immunoprecipitation, Control, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Agarose Gel Electrophoresis

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) Schematic representation of the luciferase reporter vectors containing CCL2 3′untranslated region (3′ UTR) with either rs13900 C or T allele. ( B ) HEK-293 cells were transfected with the equal quantities of CCL2 3′ UTR reporter vectors, and luciferase activity was measured 48 hr later. The relative luciferase activities of the 3′ UTR reporter plasmids were expressed as a percentage reduction in the luminescence when compared to the control vector that was set to 100% after normalizing for the protein content of the lysates. The error bars indicate the standard error of mean, and statistical significance was calculated using two-tailed Student’s t test (*p<0.05) (N=3). ( C ) HEK-293 cells were transfected with either pCMV6-HuR (0.5 μg) or pCMV-Entry (0.5 μg), and after 72 hr they were co-transfected with the two plasmid constructs (0.5 μg). Twenty-four hours after transfection, the relative change in luciferase activity was determined (N=3). ( D ) Cells were co-transfected with 125 pmol HuR siRNA or control siRNA and with the two plasmid constructs (0.5 μg). Twenty-four hours after transfection, the relative change in luciferase activity was determined, normalized to total protein concentration, data from three independent experiments (mean ± SEM; N=3). Statistical analyses were performed using Fisher’s least significant difference (LSD) method (*p<0.05). Figure 6—source data 1. Numerical data used to generate .

Article Snippet: Commercial assay or kit , TaqMan SNP Genotyping Assay , Thermo Fisher Scientific , Thermo Fisher Scientific: 4351379 , Assay id: C___7449810_10.

Techniques: Luciferase, Transfection, Activity Assay, Control, Plasmid Preparation, Two Tailed Test, Construct, Protein Concentration

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) Polysome profile obtained by sucrose gradient centrifugation from macrophages before and after stimulation with LPS (1 µg/mL) for 3 hr. The polysome profile shows a shift from monosomal fractions to heavier polysomal fractions upon LPS stimulation, indicating active translation. ( B ) The percentage of CCL2 mRNA loading on the polysome fraction was calculated using the ΔCT method (mean ± SEM, N=4). *p<0.025.

Article Snippet: Commercial assay or kit , TaqMan SNP Genotyping Assay , Thermo Fisher Scientific , Thermo Fisher Scientific: 4351379 , Assay id: C___7449810_10.

Techniques: Gradient Centrifugation

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) HEK-293 cells were transfected by nucleofection with control and CCL2 3′ untranslated region (3′ UTR) reporter constructs with rs13900 C and T alleles. The nucleofected cells were plated separately and harvested for total RNA isolation or lysed for mRNA level or protein level expression of luciferase, respectively, after 24 hr. The reporter mRNA levels from the transfected 293T cells were quantified by real-time quantitative PCR (RT-qPCR), and 18S rRNA was used for normalization (N=6). ( B ) The relative luciferase activities of the 3′ UTR reporter plasmids were expressed as a percentage reduction in the luminescence when compared to the control vector that was set to 100% after normalizing for the protein content of the lysates (N=4). ( C ) mRNA translatability was calculated as luciferase activity normalized by the reporter luciferase mRNA level. The error bars indicate the standard error of mean from four independent experiments (N=4), and statistical significance was calculated using ANOVA and post hoc contrast with Fisher’s least significant difference (LSD) method. *p<0.01, **p<0.005. Figure 7—source data 1. Numerical data used to generate .

Article Snippet: Commercial assay or kit , TaqMan SNP Genotyping Assay , Thermo Fisher Scientific , Thermo Fisher Scientific: 4351379 , Assay id: C___7449810_10.

Techniques: Transfection, Control, Construct, Isolation, Expressing, Luciferase, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Activity Assay

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) HuR expression in primary human macrophages following lentiviral transduction. Macrophages obtained from four individuals who are homozygous for either rs13900 C or rs13900 T allele were transduced with either CMV-null or HuR expressing lentiviral particles (pCMV6-HuR) for 72 hr followed by lipopolysaccharide (LPS) stimulation for 3 hr. ( B ) CCL2 expression determined by real-time quantitative PCR (RT-qPCR) (N=4). Error bars represent SEM. Statistical analyses were performed using Student’s t test (*p<0.005). Figure 8—source data 1. Numerical data used to generate .

Article Snippet: Commercial assay or kit , TaqMan SNP Genotyping Assay , Thermo Fisher Scientific , Thermo Fisher Scientific: 4351379 , Assay id: C___7449810_10.

Techniques: Expressing, Transduction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR