Healthy and malignant B cells express 4 CD20 transcript variants (V1-V4) with distinct 5′-UTRs. (A) Oxford Nanopore long-read direct RNA sequencing of mRNA from Raji cells. Sequence alignments corresponding to full-length, cap-to-poly(A) CD20 mRNAs with 4 5′-UTR splice variants and 2 alternative 3′-UTRs are shown. Alignments were extracted and visualized using an Integrative Genomics Viewer. (B) Diagram depicting the CD20 pre-mRNA and 4 distinct 5′-UTR splice variants, from V1 to V4. (C-D) Relative abundance of the V1 (red), V2 (green), V3 (blue), and V4 (yellow) isoforms in OCI-Ly8, Raji, and MEC-1 cells. Panel C is based on RNA-seq data, with the stack plot showing the ratio of sequencing reads mapped to the unique exon junctions found in each 5′-UTR variant of CD20. Here and below, these variants are color-coded as shown in the Reads spanning panel. Here and below, pan-isoform reads mapping to any exon of CD20 are shown on the right for comparison as TPM. Panel D shows RT-qPCR-mediated quantification of pan- and 5′-UTR variant–specific CD20 levels in OCI-Ly8, Raji, and MEC-1 cells. RNA levels are normalized to the reference gene RPL27. Each bar represents the average from each repeated experiment (N = 4). (E-I) Relative abundance of the V1 (red), V2 (green), V3 (blue), and V4 (yellow) isoforms in primary samples corresponding to healthy and malignant B cells. Each bar represents data from a single donor. The corrected TPM (cTPM) values at the top of panel H are the TPM values corrected for potential batch effects between the different data sets. Healthy B-cell subsets were fluorescence-activated cell sorting–enriched from the human bone marrow and tonsils (E) and peripheral blood (F). In panel E, CD19⁺IgM⁺IgD⁺ naïve tonsillar B cells were treated with anti-IgM or an isotype control. Western blots of phosphorylated SYK are displayed at the bottom right corner as a marker of B-cell activation, with actin serving as a loading control.