CD20 mRNA isoforms V3 and V4, but not V1 and V2, are efficiently translated into protein. (A) Polysome profiling of OCI-Ly8 and Raji cells. The top panel shows the ribosomal content measured at an absorbance of 254 nm, whereas the bottom panels show the relative distribution of specific transcripts across sucrose gradient fractions, as measured by RT-qPCR (supplemental Methods). (B) CD20 mRNA and protein measurements in HEK293T cells transiently transfected to express V1, V2, V3, or V4 mRNA isoforms. Pan-isoform CD20 transcripts were measured using RT-qPCR. Total CD20 protein was measured by flow cytometry and expressed as delta median fluorescence intensity (ΔMFI) (supplemental Methods). Representative scatter plots (bottom). ∗∗∗P < .001 per 1 way analysis of variance test (pairwise comparison [pwc]: Bonferroni). (C) Immunoblotting analysis of CD20 protein levels in cells from the previous panel. ACTB served as a loading control. Data from 2 independent experiments are shown. (D) CD20 mRNA and protein measurements in HEK293T stably expressing the empty vector (Ctrl-Puro) or the 5′-UTR and CDSs of V1, V2, or V3 (V1-, V2-, or V3-Puro). The kz-Puro Ctrl has the GCCACC Kozak consensus as its sole 5′-UTR element. The levels of CD20 mRNA were quantified by RT-qPCR. The MFI of total cellular PE-CD20 were determined by flow cytometry. The representative histograms are shown. (E) The same experiment was performed using OCI-Ly8 cells. (F) CD20 mRNA and protein measurements in OCI-Ly8 cells stably expressing the V1 or V3 5′-UTR sequences followed by a green fluorescence reporter (GFP) reporter (V1- and V3-GFP). The kz-GFP Ctrl has the GCCACC Kozak consensus as its sole 5’-UTR element. The GFP mRNA levels were measured using RT-qPCR. The MFI of GFP was measured using flow cytometry. Representative histograms are shown. In panels B,D-F, RNA levels are normalized to the reference gene RPL27. Each bar or dot represents the average of repeated experiments (N ≥ 2). RNA and MFI values are expressed as log₂ fold change relative to the Ctrl or Ctrl-Puro samples, as indicated. LTR, lentiviral tong terminal repeats.