BIC RNA and miR-155 in cell lines. (A) Total RNAs of different cell lines were analyzed by semiquantitative RT-PCR for BIC RNA and β-actin (as a normalization control) by using the primer pair A/C (Fig. 6) and the primers described in Methods, respectively. PCR products obtained after 30 cycles (BIC) and 24 cycles (β-actin) of reaction were analyzed by gel electrophoresis. BL, Burkitt lymphoma; LBL, EBV-immortalized lymphoblastoid cell line; MM, multiple myeloma; ALL, acute lymphoblastic leukemia; ATLL, adult T cell leukemia/lymphoma (human T cell leukemia virus type-1-positive). (B) Northern blot of total RNA isolated from different cell lines was probed with a [³²P]5′ end-labeled oligonucleotide with sequence complementary to the sequence of human miR-155. tRNAs served as a loading control. The 22-nt miR-155 is indicated. The predicted ≈60-nt pre-miR-155 could be detected as a weak band in some samples. (C) Northern analysis of miR-155 in HEK-293T cells and HEK-293T transfected with the vector pcDNA3.BIC expressing BIC exon 3 sequences (see Fig. 6).

Intracellular localization of BIC RNA and miR-155. (A) Nuclear (N) and cytoplasmic (C) RNAs isolated from OCI-Ly3 and L1236 cells were reverse-transcribed and amplified with primers specific for spliced or unspliced BIC RNA (primer pairs A/C and B/C, producing 476- and 467-bp products, respectively) (see Fig. 6). The reactions were performed with and without reverse transcriptase (+RT and -RT) and in the absence of template (0). PCR was performed for the indicated number of cycles. (B) Northern analysis was performed on total RNA isolated from the nuclear and cytoplasmic fractions of OCI-Ly3 and L1236. The 22-nt miR-155 and the ≈60-nt predicted precursor are marked.

Numbers of molecules (copies per cell) of BIC RNA and miR-155 in lymphoma cells. Quantification was by miRNA and mRNA Invader assays designed to detect miR-155 and BIC RNA, respectively (see Methods). Samples were tested in triplicate. (A, C, and E) Orange bars and aqua bars are miR-155 and BIC RNA levels, respectively. (A–D) Samples tested in A and C are indicated on the x axes of B and D, respectively, and abbreviations are as described for Fig. 1. (A) miR-155 and BIC RNA levels in several cultured lymphoma cell lines. Note the expanded scale for BIC RNA (right axis). Error bars represent one standard deviation. (B) Ratio of copy number per cell of miR-155 and BIC RNA in cultured lymphoma cells shown in A. (C) miR-155 and BIC RNA levels in cells of clinical isolates of lymphomas. Scales and error bars are as described for A. Samples S4 and S5, normal circulating B cells, are taken as normal controls. (D) Ratio of copy numbers per cell of miR-155 and BIC RNA in the samples shown in C. (E) Comparison of the copy numbers per cell in DLBCL cells exhibiting the GC vs. ABC phenotypes for miR-155 and BIC RNA. A total of 23 cases were tested (11 shown in C and D and reported in Table 2 plus 12 reported in Table 3). The indicated P values are calculated from a t test between the GC phenotype (n = 4) and the ABC phenotype (n = 19). Error bars represent one standard error. (F) Lack of correlation between amounts of miR-155 and BIC RNA in clinical isolates of lymphomas. The graph represents the 23 cases shown in E. Linear regression (R² values are indicated) was performed with and without sample D6 (filled circle).

Quantification of miR-15a, miR-16, and let-7a miRNAs in the normal B cells and clinical isolates of lymphoma cells analyzed in Fig. 3C by using Invader miRNA assays. For a summary of the quantification of miRNAs in cultured lymphoma cell lines analyzed in Fig. 3A, see Fig. 10, which is published as supporting information on the PNAS web site. Samples were measured in triplicate (see Table 2 for values), and error bars represent one standard deviation.

Pathways for generation of pre-miR-155. Pre-miR-155 may be generated by the nuclear Drosha-containing microprocessor complex acting on the primary BIC gene transcript or on spliced and polyadenylated BIC RNA that has not yet been exported from the nucleus. Cytoplasmic BIC RNA does not have access to this complex and, thus, cannot be used to make pre-miR-155.