An RNA stem-loop structure and uORFs repress translation of the CD20 V1 isoform. (A) Diagram depicting the uORFs and the stem-loop in the 5′-UTR of the CD20 V1 isoform. The stem loop was predicted by the RNAfold web server. (B) Diagrams of mRNA products corresponding to various derivatives of V1-Puro. All AUG start codons found in the 5′-UTR of V1-Puro were mutated to AUC in the V1AUC-Puro and V1AUCDel-Stem-Puro constructs. Stem and Del-Stem refer to mutations stabilizing and destabilizing the predicted secondary structure, respectively. (C-F) Expression of V1- and V3-Puro and the V1-Puro mutants in stably transduced HEK293T cells. Pan-isoform CD20 RNA levels were quantified using RT-qPCR. The MFI of the total cellular CD20 protein was measured using flow cytometry. Representative histograms are shown on the right. Each bar or dot represents the average of repeated experiments (N = 2). All values are expressed as log₂ fold change relative to the Ctrl-Puro sample. (G) Diagram depicting putative Sam68 binding sites found within exons 2 and 3 of the V1. (H-I) Sam68 and CD20 expression measurements in Raji cells electroporated with a mixture of 2 Cas9-gRNA ribonucleoproteins targeting the CDS of Sam68. Shown in panel H is immunoblotting analysis of Sam68 protein expression, with ACTB serving as the loading control. Shown on the left side of panel I are Sam68 and CD20 mRNA levels, as quantified using RT-qPCR. Two PCR primer pairs measured nonoverlapping regions within the Sam68 transcript. The second primer pair is denoted with an asterisk (∗). Shown on the right side of panel I are MFI of total CD20 protein, as determined using flow cytometry. Representative histograms are shown on the far right. In both panels H and I, each row in the immunoblotting image and the heat map represent an independent experiment (N = 4).