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. 2016 Sep 13;7(37):59519-59534.
doi: 10.18632/oncotarget.11148.

The Human Box C/D snoRNAs U3 and U8 Are Required for pre-rRNA Processing and Tumorigenesis

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Free PMC article

The Human Box C/D snoRNAs U3 and U8 Are Required for pre-rRNA Processing and Tumorigenesis

Jean-Louis Langhendries et al. Oncotarget. .
Free PMC article

Abstract

Small nucleolar RNAs (snoRNAs) are emerging as a novel class of proto-oncogenes and tumor suppressors; their involvement in tumorigenesis remains unclear. The box C/D snoRNAs U3 and U8 are upregulated in breast cancers. Here we characterize the function of human U3 and U8 in ribosome biogenesis, nucleolar structure, and tumorigenesis. We show in breast (MCF-7) and lung (H1944) cancer cells that U3 and U8 are required for pre-rRNA processing reactions leading, respectively, to synthesis of the small and large ribosomal subunits. U3 or U8 depletion triggers a remarkably potent p53-dependent anti-tumor stress response involving the ribosomal proteins uL5 (RPL11) and uL18 (RPL5). Interestingly, the nucleolar structure is more sensitive to perturbations in lung cancer than in breast cancer cells. We reveal in a mouse xenograft model that the tumorigenic potential of cancer cells is reduced in the case of U3 suppression and totally abolished upon U8 depletion. Tumors derived from U3-knockdown cells displayed markedly lower metabolic volume and activity than tumors derived from aggressive control cancer cells. Unexpectedly, metabolic tracer uptake by U3-suppressed tumors appeared more heterogeneous, indicating distinctive tumor growth properties that may reflect non-conventional regulatory functions of U3 (or fragments derived from it) in mRNA metabolism.

Keywords: cancer; nucleolus; ribosome; snoRNA; tumorigenesis.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. The box C/D snoRNAs U3 and U8 are required for human ribosomal subunit biogenesis
(A) Polysomes analysis. H1944 and MCF-7 cells were depleted of U3 or U8 for two days and then treated with cycloheximide, to freeze the polysomes. Total extracts were analyzed by sucrose gradient centrifugation. The reduction of polysomes upon snoRNA depletion is obvious (red arrows). U3 depletion leads to a marked subunit imbalance due to a deficit in 40S, while U8 depletion affects primarily 60S accumulation. (B) Mature rRNA accumulation. H1944 and MCF-7 cells were depleted of U3 or U8, for 1, 2, or 3 days. Total RNA was extracted, resolved on denaturing gels, and mature rRNAs were visualized by ethidium bromide staining. 7SL, detected by Northern blotting, was used as a loading control. (C) Densitometric quantification of the signals shown in panel B. rRNA levels in cells depleted for U3 or U8 normalized with respect to the levels observed in cells treated with a non-targeting silencer (SCR). Note that after 72 h of U8 depletion, in addition to inhibition of 28S rRNA synthesis, the 18S and other rRNAs, were also reduced; this reflects the important reduction in cell viability observed at this time-point (Figure S2D).
Figure 2
Figure 2. U3 and U8 are required for pre-rRNA processing in human cells
Total RNA extracted from H1944 or MCF-7 cells depleted of U3 or U8 for 1, 2, or 3 days was resolved on denaturing gels and analyzed by Northern blotting with specific probes (see Materials and Methods). As a control, cells were treated with a non-targeting silencer (SCR). Blots were probed with oligonucleotide LD1844 (panels I and II), LD2612 (panel III), LD1827 (panel IV), and LD1828 (panel V). The pre-rRNA species detected are indicated and represented as schematics with the probes used highlighted. A detailed pre-rRNA processing pathway and a description of all the RNA species detected are provided in Figure S1. Note that several abundant truncated forms of the 36S RNA are detected (highlighted with stars in panel V). We suggest that in the absence of ITS2/3′-ETS processing after U8 depletion, large subunit precursors are targeted for degradation, and that this is manifested by activation of cryptic cleavage sites within the 28S-coding part of the precursor, leading to production of these abnormal dead-end intermediates.
Figure 3
Figure 3. The depletion of U3 or U8 elicits a p53-dependent nucleolar antitumor surveillance pathway
(A) U3 and U8 depletion lead to an increased p53 steady-state level. Total protein extracted from H1944 or MCF-7 cells depleted of U3 or U8 for 1, 2, or 3 days was resolved on SDS-polyacrylamide gels and analyzed by Western blotting with specific antibodies (see Materials and Methods). As a control, cells were treated with a non-targeting silencer (SCR). In H1944 cells, at the late time points of depletion (48 and 72 h), p21 was detected as a doublet, suggesting it is post-translationally modified. The signals were quantitated with a ChemiDoc and normalized with respect to SCR-treated cells. As loading control, we probed the blots for β-actin. (B) The increase in p53 steady-state level observed upon U3 or U8 depletion depends on the presence of uL5 and uL18. H1944 and MCF-7 cells depleted of U3 or U8, or treated with a non-targeting control silencer (SCR), were codepleted of uL5 or uL18. Total protein was extracted and analyzed as in panel A. uL5 appears as a doublet, suggesting it is post-translationally modified.
Figure 4
Figure 4. Effects of U3 and U8 depletion on cell-cycle progression and apoptosis
(A) Cell-cycle analysis. The fractions of cells in the different phases of the cell-cycle (G0/G1, G2/M, or S) in populations of cells depleted of U3 or U8 for 1, 2, or 3 days were determined by staining nuclear DNA with propidium iodide and counting the fluorescence with a Muse (see Materials and Methods). As a control, cells were treated with a non-targeting silencer (SCR). (B) Apoptosis analysis. The fraction of apoptotic cells in populations of cells depleted of U3 or U8 or treated with the non-targeting control silencer (SCR) for 1, 2, or 3 days was determined by performing an Annexin V assay and counting the fluorescence with a Muse (see Materials and Methods). (C) Timeline of the appearance of phenotypes.
Figure 5
Figure 5. Effects of U3 and U8 depletion on nucleolar structure
(A) H1944 and MCF-7 cells depleted for 2 days of U3, U8, p53, or a combination of p53 and U3 or U8 were processed for immunofluorescence and decorated with antibodies specific to the nucleolar proteins fibrillarin (FBL) or PES1 and imaged by spinning-disc confocal microscopy. As a control, cells were treated with a non-targeting silencer (SCR). The inset (panel f’) is showing additional cells treated in the same conditions as those shown in panel f. (B) The mean number of nucleoli was established by PES1 staining of 100 cells for each condition and plotted. In H1944 cells, upon U8 depletion, the number of nucleoli could not be determined (ND) owing to nucleolar disruption (see f, f’, and l, in panel A).
Figure 6
Figure 6. The box C/D snoRNAs U3 and U8 are required for in vitro and in vivo tumorigenesis
(A) Soft-agar colony formation assay. H1944 and MCF-7 cells depleted of U3 or U8 for 3 days, and cells treated with a non-targeting control silencer (SCR), were layered on soft-agarose and incubated for 4-weeks, the colonies were stained with crystal blue and counted. Each data point represents a mean value of three independent experiments with SD. (B–E) Nude mouse xenograft experiment. Twelve nude mice were injected symmetrically in the upper body flanks with, on the right side of the animal, 5 million H1944 cells depleted of U3 or U8 for 3 days and, on the left side, 5 million H1944 cells treated with the non-targeting silencer control. (B) Percentages of mice bearing a tumor as monitored over a period of 64 days. The data show a one-month lag in tumor formation after injection of U3-suppressed cells, and no tumor formation after injection of U8-suppressed cells. (C) Tumor size estimated by caliper measurement. The data show that the tumors developed from U3-suppressed cells are substantially smaller and that no tumors developed from U8-suppressed cells. (D) One representative mouse (day 64) is shown for U3 depletion and one for U8 depletion. Large tumors are visible only in the flanks injected with control cancer cells (left side of the animals, white arrows). Left, digital photographic recording. Right, PET-CT tomograms. For simplicity, the 18F-FDG signals for the heart and bladder are not shown. The scale depicts the SUV (standard uptake values, see Materials and Methods) expressed in Bq/ml. Insets show resected tumors. Scale bar, 10 mm. (E) Tumor metabolic volume, tumor metabolic activity, and tumor heterogeneity were extracted and computed from the tomograms, according to refs [53, 75]. Tumor metabolic volume, tumor metabolic activity (through SUV peak), and tumor heterogeneity (through CSH-AUC) were compared between the U3-suppressed group and the control (scrambled depletion) group by means of an unpaired two-tailed t test, assuming equal variance between groups. The results, reported as p-values, were considered significant at p < 0.05. (*p ≤ 0.05; **p ≤ 0.01).

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