Fig 1: Development of LC–MS/MS method for the detection of hnRNP-A1 in human blood. (A) Chromatographic separation of a mixture of synthetic peptides corresponding to hnRNP-A1223-228 bearing unmethylated, mono-methylated, asymmetrically-dimethylated arginine 225, and the unmodified hnRNP-A1154-167 peptide. (B) Detection of chymotryptically-derived hnRNP-A1 peptides in human PBMC lysates, in which hnRNP-R225 is detected in its dimethylated form. (C) Determination of method accuracy for the detection of the DM-R225-hnRNP-A1 peptide employing a complete protease inhibitor cocktail (PIC red horizontal line), a PIC without serine protease inhibitors (yellow horizontal line) or no protease inhibitors (blue horizontal line) on chymotryptically-digested human PBMC lysates diluted 1:2 (filled circle), 1:4 (filled square) and 1:10 (filled triangle) with PBS. The levels of DM-R225-hnRNP-A1 in undiluted lysates were 988 ng/mL, 3153 ng/mL and 2800 ng/mL in presence of complete PIC, custom PIC and no PIC, respectively.
Fig 2: Reduction of ADMA on hnRNP-A1 across normal PBMCs and cancer cells. (A) AlphaLISA for candidate substrates identified in the MethylScan study using lysates from TCR-activated human PBMCs treated with either DMSO or 2 µM GSK3368712 for 72 h (two-tailed Student’s t test: **p < 0.01, ***p < 0.001; p-values not indicated if no statistically-significant difference was observed). (B) ADMA-hnRNP-A1 levels in lysates from non stimulated PBMCs and cancer cell lines, Jurkat and Toledo, treated with increasing concentrations of GSK3368712 for 72 and 48 h, respectively. Data are expressed as the mean ± standard deviation (SD).
Fig 3: Silencing of hnRNP‐A1 reduces MELOE‐1 internal ribosomal entry sequence (IRES) activity. (A) Efficacy of siRNA‐mediated depletion of hnRNP‐A1 on M113 assessed by RT‐qPCR 48 h postlipofection. (B) FLuc/RLuc ratio (*100) was measured in M113 melanoma cell lysate 48 h post‐transfection with pRF bicistronic vectors in which Renilla luciferase (RLuc) translation is cap‐dependent and Firefly luciferase (FLuc) translation is controlled either by MELOE‐1 IRES, encephalomyocarditis virus (EMCV) IRES, or nothing (no IRES). Where indicated, cells were cotransfected with hnRNP‐A1 siRNA (siRNA#1, 10 µm, Santa‐Cruz Biotechnologies, siRNA#2, 5 µm, Qiagen) or with a universal control siRNA (5–10 µm). Data are expressed as mean ± SD (n = 7 independent experiments). P‐values were calculated using repeated‐measure one‐way ANOVA followed by Holm–Sidak’s multiple comparison test.
Fig 4: Functional characterization of the GBP2 intronic variant c.1149+14T>C. (A) Minigene analysis of c.1149+14T>C. Illustration of the GBP2 minigene. The 5′ splice site of exon seven and the downstream sequence covering the c.1149+14T>C variation is enlarged (top). RT-PCR analysis of minigene splicing in HepG2 cells. The analysis shows that the minigene carrying the mutant sequence (MUT) has a higher degree of exon seven skipping than the minigene carrying the wild type sequence (WT) (bottom, left). Quantification of the exon seven inclusion and exclusion rates (blue and orange columns, respectively). The inclusion/exclusion is quantified on a Fragment Analyzer instrument and represent the intensity of the 544 bp or 263 bp bands over the total intensity in the lane (bottom, right). (B) SPRi measurements of hnRNP A1 (splicing silencer factor) binding to the WT (the left hand side) and MUT (the right hand side) oligonucleotides. Dots correspond to the raw SPRi measurements while black lines correspond to the model-implied fits. Protein (hnRNP A1) was injected in increasing 2-fold concentrations from 50 to 400 nM shown in color dots. (C) RT-PCR analysis of c.1149+14T>C from the mRNA of the mutation carrier (the father of P2; samples: c1 and c2) and three healthy controls (samples: a1, a2 and a3). (D) qRT-PCR analysis of total GBP2 mRNA from whole blood in the carrier (the father of P2) and three healthy controls. GBP2 transcript levels were normalized by RPL13A expression. The values presented are the medians of duplicate determinations ±SD.
Fig 5: RNA secondary structures upstream of MELOE‐1 ORF, redrawn from predictions by UNAFold (http://www.unafold.org), revealed typical stem‐loop elements and putative hnRNP‐A1 binding sites. (A) Schematic representation of the predicted RNA secondary structure of the 275 nt sequence upstream of MELOE‐1 ORF (AUG initiation codon in red). (B) Focus on the proximal regions of the wt, variant 1, and variant 2 internal ribosomal entry sequence (IRES), highlighting the putative hnRNP‐A1‐binding sites, circled in orange (5′CAG‐3') and blue (5′UAG‐3'). Nucleotide changes in variants 1 and 2, shown in red, are predicted not to change the rest of the IRES sequence.
Supplier Page from Abcam for Recombinant Human hnRNP A1 protein