Fig 2: miR-150 restrains T-cell proliferation through MYB and PDAP1.(A) Primary memory T cells were transfected with Cas-9 RNPs to delete either MYB (left) or PDAP1 (right), followed by single-cell cloning. Individual clones were selected based on the presence of a large genomic deletion in the gene of interest, and proliferation was measured by BrdU incorporation assay. For the MYB gene, N = 18 control clones and N = 12 MYB-edited clones, from 2 independent donors. For the PDAP1 gene, N = 18 control clones and N = 25 PDAP1-edited clones, from 2 independent donors. Mean ± SD. Mann–Whitney test. (B) Memory T cells were transfected with Cas-9 RNP complexes containing 2 different sgRNAs targeting the MIR150 gene. Individual clones were selected based on the presence of a genomic deletion overlapping the MIR150 sequence, and proliferation was measured by BrdU incorporation assay. N = 31 control clones and N = 22 MIR150-edited clones, from 2 independent donors. Mean ± SD. Welch t test, 2 tailed. Underlying data can be found in S1 Data. sgRNA, single-guide RNA; KO, knockout; PDAP1, PDGFA-associated protein 1; RNP, ribonucleoprotein.

Fig 3: Identification of miR-150 targets.(A) Volcano plot of differentially expressed genes between the indicated miRNA mimic and control oligonucleotides. Genes in red were considered significantly differentially expressed when log2FC ≥ 0.6, −log10 p-value ≥ 2. (B) The list of genes obtained from (A) was intersected with several prediction databases using miRWalk 2.0. 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. (C) Activated memory cells were transfected with miR-150 mimic or control oligonucleotide. Twenty-four hours or 48 hours after transfection, the expression of the indicated genes was measured by RT-qPCR. N = 3 to 5 independent donors. Mean ± SD. Student t test, 2 tailed, paired. (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. Luciferase reads were normalized to the renilla ones. N = 3 to 4 independent experiments. Mean ± SEM. Student t test, 2 tailed, paired. (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. N = 3 independent experiments. Mean ± SEM. Student t test, 2 tailed, paired. Underlying data can be found in S1 Data. A.U., arbitrary unit; CDS, coding sequence; FC, fold change; miRNA, microRNA; RT-qPCR, reverse transcription quantitative PCR; UTR, untranslated region; WT, wild-type.

Fig 4: PDAP1 acts an RBP in primary human T lymphocytes.(A) Venn diagram showing the extent of overlap between RBPs identified in the indicated studies and cell types. (B) Activated memory T lymphocytes were UV-irradiated and lysed prior to poly-A mRNA pull-down and western blot. Nonirradiated samples were also used. Representative of N = 2 independent experiments. (C) Scatterplot of RIP-seq data (N = 3 independent donors). Blue dots indicate transcripts significantly enriched (FDR < 0.001) in RIP samples compared to input controls, with log2FC ≥ 2, as determined by DESeq2. All other mRNAs are indicated as gray dots. (D) Schematic representation of the experimental workflow to generate primary T lymphocytes KO for a protein of interest. (E) Primary human CD4+ memory T lymphocytes were transfected with CRISPR/Cas-9 RNPs to delete PDAP1, followed by single-cell cloning and screening. Clones showing a prominent genomic deletion in the PDAP1 gene were used to measure the expression of PDAP1 itself and of other transcripts bound by PDAP1. Each dot represents one individual clone (at least N = 8). Statistical test is unpaired t test or Mann–Whitney, depending on the distribution of the clones. Mean ± SD. Underlying data can be found in S1 Data. FDR, false discovery rate; FC, fold change; gDNA, genomic DNA; gRNA, guide RNA; KO, knockout; PBMC, peripheral blood mononuclear cell; PDAP1, PDGFA-associated protein 1; RBP, RNA-binding protein; RIP-seq, RNA immunoprecipitation sequencing; RNP, ribonucleoprotein.