(A) Schematic diagram of the chromosome 4p16.3 tiling oligonucleotide microarray, spanning 430 Kb across the t(4;14) breakpoint region (breakpoints are indicated by arrows). Annotated genes are indicated with exon structures. (B) Tiling microarray expression data of representative t(4;14)-positive and -negative patients with MM. (C) Heat map of t(4;14)-positive (Y; n = 4) and -negative (N; n = 12) patient samples. (B and C) Numbers indicate base pairs on chromosome 4 (human genome assembly GRCh37). (D) Real-time PCR of ACA11 in samples from patients with MM. The inset shows that ACA11 is significantly associated with t(4;14) status. Normal, normal plasma cells (n = 10); t(4;14)⁺, t(4;14)-positive patient samples (n = 6); t(4;14)^–, t(4;14)-negative patient samples (n = 57). (E) Northern blot of ACA11 expression in human MM cell lines. t(4;14)-positive cell lines included KMS-11, OPM-2, LP-1, and H929 cells; t(4;14)-negative cell lines included U266, RPMI8226, and MM.1S cells; and non-MM cell lines included K562, HeLa, A375, and U1A cells. Loading controls were 28S and 18S rRNA. (F) ACA11 expression by real-time PCR is significantly associated with t(4;14) in MM cell lines. qRT-PCR data in D and F were normalized to the mean of 3 reference genes (GAPDH, UBC, YWHAZ). Data are presented as mean ± SD, with a box-whisker plot of minimum-to-maximum values (inset) (n = 3). The line within the box is the median value. The box represents the first and third quartiles. The whiskers show the largest and smallest events within at least 1.5 times the size of the box from the nearest edge.

(A–C) ACA11 is overexpressed in samples from patients with (A) bladder, (B) colon, and (C) esophageal cancer. Heat maps of RNA expression at the ACA11 locus were measured using our custom chromosome 4 tiling arrays. Each bar represents the normalized mean expression value obtained from quadruplicate probes representing each physical location. Numbers indicate base pairs on chromosome 4 (human genome assembly GRCh37). (D–F) Graphs of data shown in A–C. ACA11 levels in (D) bladder, (E) colon, and (F) esophagus samples were plotted using raw expression values of 4 probes. Intron, intron 18–19 of WHSC1 upstream of ACA11. Data in D–F are presented as mean ± SD (n = 4). M, malignant tumor samples. N, normal control samples.

(A) Predicted secondary structure of ACA11. Red brackets indicate H box, ACA box, and CAB motifs. Black lines indicate ASOs. (B) Endogenous ACA11 is not significantly localized to Cajal bodies (coilin, red) in MM cells. ACA11 or β-actin is in green; DNA (DAPI) is in blue. Scale bar: 5 μm. (C) Localization of ACA11 to nucleoli. ACA11 or β-actin is shown in red; NPM1 is shown in green; DNA (DAPI) is in blue. Scale bar: 5 μm. (D) Northern blot (top 2 panels) for ACA11 in cytoplasmic (CP), nucleoplasmic (NP), and nucleolar (NS) fractions. Western blot (bottom 3 panels) of cytoplasmic (SOD), nuclear (NUP62), and nucleolar (NPM1) markers.

(A) RNA EMSA confirming ACA11 binding to proteins from H929 cell nuclear extracts. Competitor, unlabeled ACA11 RNA. (B) Coomassie-stained SDS-PAGE gel of ACA11 pull-downs used for identification of proteins binding to ACA11 by MS. Representative proteins with the most abundant, unique peptides in each indicated band are marked with numbers that correspond to those in Table 2. M, protein marker. (C) Confirmation of ACA11-binding proteins by Western blot. The ACA11-protein complex was purified as in B and subjected to Western blot with antibodies to proteins identified by MS. The asterisk indicates nonspecific binding. Hsp90 was used as a negative control. (D) Confirmation of ACA11 presence in a DHX9/ILF3 ribonucleoprotein complex by CLIP followed by RT-PCR using H929 cells. H₂O was used as a water template for RT-PCR. GAPDH was used as an internal control. M, DNA marker. (E) Physical interaction of ACA11 ribonucleoprotein complex proteins NCL, ILF3, ADAR, and HNRNPU with DHX9 in H929 cells was confirmed by immunoprecipitation.

(A–F) Graphical representation of sequencing peaks mapping to the 6 targets of ACA11 snRNP. The horizontal gray bars indicate the locations of snoRNAs visualized in the UCSC genome browser. Arrows represent primers used for RT-PCR in H. (G) The same data as in E shown with lower vertical scale demonstrate smaller peaks of RPL10 exons. (H) RT-PCR confirms the presence of the indicated snoRNA intermediates resident within RP genes in the ILF3-, SF3B1-, or control IgG-precipitated complexes from H929 cells. H₂O was used as a water template. GAPDH was used as control.

WHSC1 is upregulated in all 4p16 patients (arrow), and the nearby FGFR3 gene (arrow) is upregulated in most but not all 4p16 patients. Previously defined patient subgroup clusters (see Methods) are indicated by orange, dark green, red, dark blue, light green, and light blue. Highly significant downregulation (**P = 4.8 × 10^–20) of RP genes was found in the 4p16 (i.e., WHSC1, orange) subgroup.

(A) Overexpression of ACA11 confirmed by Northern blot analysis in Arf^–/– MEFs stably transduced with ACA11 lentiviruses. β-Actin was used as loading control. (B) Overexpression of ACA11 in Arf^–/– MEFs as measured by qRT-PCR. (C) RP mRNA expression in Arf^–/– MEFs overexpressing ACA11 was analyzed by qRT-PCR. (D) Expression of RPs in Arf^–/– MEFs overexpressing ACA11 demonstrated by Western blot. γ-Tubulin was used as loading control. (E) Overexpression of ACA11 confirmed by Northern blot analysis in MM.1S cells stably transduced with ACA11 lentiviruses. β-Actin was used as loading control. (F) qRT-PCR showing the expression of ACA11 in MM.1S cells in E. (G) RP mRNA expression in MM.1S cells overexpressing ACA11 was analyzed by qRT-PCR. (H) Downregulation of RPs in MM.1S cells overexpressing ACA11 demonstrated by Western blot. γ-Tubulin was used as loading control. (I) Small RNAs were isolated from MM.1S cells overexpressing ACA11. snoRNA expression was analyzed by qRT-PCR. U43, snoRNA located in RPL3; U64, snoRNA located in RPS2; U32A, U33, and U35A, snoRNAs located in RPL13A. qRT-PCR data were normalized to the average of 3 reference genes, (B, C, F, and G) GAPDH, UBC, and YWHAZ or (I) U6, U44, and U48, and are shown as fold change relative to that of mock-treated cells. Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01.

(A and B) ROS levels, quantified by DCFH-DA (DCF) labeling, are suppressed by ACA11 expression in (A) Arf^–/– MEFs or (B) MM.1S cells at baseline and upon challenge with hydrogen peroxide (H₂O₂). (C and D) ACA11 expression increased growth and proliferation of MM.1S cells as measured by (C) cell counting and (D) BrdU-ELISA relative to mock-treated cells. (E) ACA11 increased resistance of MM.1S cells to cytotoxic chemotherapy as measured by MTT viability assay. (F and G) Northern blot and qRT-PCR analyses of ACA11 knockdown in H929 cells 4 days after transfection with ASOs. β-Actin was used as loading control. U23, unrelated snoRNA. αA2 and αA3 are ASOs that target ACA11. (H–K) Knockdown of ACA11 in H929 cells (H) increased ROS levels, (I) decreased cell growth, (J) decreased cell proliferation, and (K) decreased resistance to cytotoxic chemotherapy. (L and N) Growth of (L) KMS-11 and (N) RPMI8226 cells in immunocompromised mice is reduced by ACA11 knockdown. (M and O) qRT-PCR showing the expression of ACA11 in (M) KMS-11 and (O) RPMI8226 xenograft tumors. Cells were nucleofected with 600 pmol ASOs per 10⁶ cells and cultured for (I) 24 hours or (H, J–L, and N) 48 hours before further analysis. qRT-PCR data in G, M, and O were normalized to the average of 3 reference genes (GAPDH, UBC, YWHAZ) and are shown as fold change relative to that of mock-treated cells. All data shown represent mean ± SD (n = 3 [A–E and G–K], n = 5 [L–O]). *P < 0.05, **P < 0.01.