(A) Experimental systems for phenotypic analysis. Raji cells (N = 2) with BIRC3 protein knock-out (KO). Controls: parental Raji cells (WT) and Raji clone after CRISPR procedure that retains BIRC3 protein (ctrl KO). Raji cells with stable expression of shRNAs against the long BIRC3 3′UTR isoform (LU KD, N = 2) are compared to cells expressing a control shRNA (ctrl KD). Arrow indicates transcriptional start and pA indicates polyadenylation signal.
(B) Viability at day 2 after Fludarabine treatment for biological replicates of ctrl Raji (N = 4), LU KD (N = 4), and BIRC3 KO cells (N = 2). T-test for independent samples, *, p < 0.039.
(C) B cell migration after 3.5 hours in media with 50 ng/ml CXCL12 as mean ± SD from biological replicates. Mann Whitney test, *, p = 0.02.
(D) FACS analysis of endogenous surface CXCR4 expression in ctrl and KO cells as mean fluorescence intensity (MFI) ± SD of biological replicates. Mann Whitney test, *, p = 0.03. Representative example in Figure S2J.
(E) As in (D), but for ctrl and LU KD cells. Mann Whitney test, **, p = E-5. Representative example in Figure S2K.
(F) FACS analysis of endogenous surface CXCR4 levels in KO3 cells after expression of constructs from Figure 1E. GFP expression in left panel and mean MFI of surface CXCR4 ± SD from biological replicates in right panel. Mann Whitney test, **, p = 0.003. Representative example in Figure S2L.
(G) FACS analysis of endogenous surface CXCR4 levels from Figure S2M. Shown is mean MFI ± SD from biological replicates. Mann Whitney test, *, p = 0.03.
(H)
CXCR4 mRNA level of NB (N = 4) and CLL B (N = 10) as mean 3′-seq TPM ± SD. Mann Whitney test, p = NS.
(I) FACS analysis of endogenous surface CXCR4 shown as MFI ± SD for normal (N = 2) and CLL B (N = 8) cells. Mann Whitney test, *, p = 0.037.
(J) 3′UTR-dependent trafficking regulation by BIRC3 is important for CLL biology. Similar mRNA level of BIRC3 and CXCR4, but increased expression of the long BIRC3 3′UTR in CLL which correlates with increased surface CXCR4 protein, allowing CLL cell migration towards the CXCL12 cytokine expressed in bone marrow niches.