PDAP1, PDGFA-associated protein 1 RT-qPCR, reverse transcription quantitative PCR siRNA, small interfering RNA. Right: Quantification of the mean intensity of the PDAP1 signal in individual cells. (D) Memory T lymphocytes were stimulated with plate-bound anti-CD3 and anti-CD28 for the indicated days, followed by immunofluorescence for PDAP1. (C) Memory T lymphocytes were stimulated with plate-bound anti-CD3 and anti-CD28 for the indicated days, followed by RT-qPCR analysis of PDAP1 expression. N = 3 to 8 independent experiments (each dot represents one donor). The extent of PDAP1 down-regulation was measured by RT-qPCR (left), while cell proliferation was measured by BrdU incorporation assay. (B) Memory T lymphocytes were transfected with siRNAs targeting either MYB or PDAP1 or with a miR-150 mimic oligonucleotide. A representative western blot is shown on the left, while the densitometric quantification of independent experiments in shown on the right. Twenty-four hours after transfection, expression of PDAP1 was assessed by western blot. (A) Memory T lymphocytes were transfected with either a miR-150 mimic or control oligonucleotide. A.U., arbitrary unit CDS, coding sequence FC, fold change miRNA, microRNA RT-qPCR, reverse transcription quantitative PCR UTR, untranslated region WT, wild-type. (E) Same as in (D), except that the 4 putative miR-150 binding sites identified in the PDAP1 3′ UTR were mutated by site-directed mutagenesis. Luciferase reads were normalized to the renilla ones. (D) The 3′ UTR of the indicated genes was cloned in a dual-luciferase reporter vector and transfected into HEK293 cells together with either a miR-150 mimic or a control oligonucleotide. Twenty-four hours or 48 hours after transfection, the expression of the indicated genes was measured by RT-qPCR. (C) Activated memory cells were transfected with miR-150 mimic or control oligonucleotide. Both the 3′ UTRs and the CDS were manually searched for the presence of at least a 6-mer miR-150 or miR-146a binding site. (B) The list of genes obtained from (A) was intersected with several prediction databases using miRWalk 2.0. Genes in red were considered significantly differentially expressed when log 2FC ≥ 0.6, −log 10 p-value ≥ 2. (A) Volcano plot of differentially expressed genes between the indicated miRNA mimic and control oligonucleotides. CFSE, carboxyfluorescein succinimidyl ester miRNA, microRNA RT-qPCR, reverse transcription quantitative PCR.
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Data in the bar graph were normalized to the overall baseline signal on day 0, prior to stimulation, to compensate from experimental differences in basal CFSE loading. The extent of cell proliferation was measured 3 days after activation. (C) Freshly isolated memory T lymphocytes were loaded with CFSE, transfected with either a miR-150 mimic or a control oligonucleotide, and activated with anti-CD3 and anti-CD28 antibodies. MiRNA expression was measured by RT-qPCR, and data are expressed as 2 −ΔCt.
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(B) Total RNA was extracted from the indicated T-cell subsets freshly isolated from peripheral blood. The most highly expressed miRNAs are shown, and data are expressed as percentage of normalized counts over the total. (A) Total RNA was extracted from freshly isolated CD4 + naive, T CM, and T EM T-cell subsets, and miRNA expression was measured by NanoString SPRINT profiling.
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Overall, using an integrated approach involving quantitative analysis, unbiased genomics, and genome editing, we identified RFX factors, miR-150, and the PDAP1 RBP as the components of a regulatory axis that restrains proliferation of primary human T lymphocytes. PDAP1 acted as an RNA-binding protein (RBP), and its CRISPR/Cas-9-mediated deletion revealed that it prominently contributed to the regulation of T-cell proliferation. By performing miRNA pull-down and sequencing experiments, we identified PDGFA-associated protein 1 (PDAP1) as one main target of miR-150 in human T lymphocytes. Constitutive MIR150 gene expression required the RFX family of transcription factors, and its activation-induced down-regulation was linked to their reduced expression. We found that in primary human memory T helper lymphocytes, miR-150 was the most abundantly expressed miRNA, and its expression decreased drastically upon activation, suggesting regulatory roles. However, what are the mRNA targets regulated by miRNAs and how miRNAs are transcriptionally regulated themselves remain for the most part unknown.
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Within the immune system, microRNAs (miRNAs) exert key regulatory functions.