The human box C/D snoRNAs U3 and U8 are required for pre-rRNA processing and tumorigenesis

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.

The cellular landscape of mid‐size noncoding RNA

“…More recently, mncRNAs with less established functions including canonical and noncanonical snoRNAs are being increasingly associated with human disease. For example, several snoRNAs were either upregulated or downregulated in different types of cancer including breast (Langhendries, Nicolas, Doumont, Goldman, & Lafontaine, 2016), lung (Langhendries et al, 2016;Liao et al, 2010;Mannoor, Shen, Liao, Liu, & Jiang, 2014;Mei et al, 2012;Su et al, 2016), brain (Dong et al, 2009), pancreatic (Cui et al, 2017;Kitagawa et al, 2019), and prostate (Martens-Uzunova et al, 2015). These cancer-associated snoRNAs have been shown to both induce and suppress tumor development (Mannoor et al, 2014).…”

Novel intramolecular base-pairing of the U8 snoRNA underlies a Mendelian form of cerebral small vessel disease

“…In cell culture models, tp53 is required for activation of cell cycle arrest within 24 hours from the onset of ribosomal stress before, over time, impaired ribosome function becomes rate limiting for cellular growth and division (11,(32)(33)(34). Depletion of U8 has recently been shown to result in potent induction of TP53 in human cells (13,16), and tp53 signaling in the U8-3 mutant would be predicted to induce an earlier and more complete inhibition of the cell cycle. In keeping with this, the angiogenic sprouting defect observed in the U8-3 mutant at 24hpf was largely, but not completely, rescued by genetic inactivation of tp53; with the partial rescue probably explained by a preferential reduction of 28S in U8-3 mutants indicating that ribosomal dysfunction is already manifested by this time.…”

“…U8 is required for removal of the 3´ external transcribed spacer (3´-ETS) sequence, and in particular the biogenesis of 28S and 5.8S rRNAs (14)(15)(16). Tapestation analysis indicated that U8-3 mutants exhibit a preferential reduction in 28S biogenesis compared to 18S (Fig.…”

“…Total RNA with integrity values in excess of 9 were selected for 28S:18S quantitation ASO-knockdown of U8 and rRNA processing assays in fibroblasts. ASO-mediated depletion of U8 in control cells (HCT116, colon carcinoma) was performed as described previously (16).…”

“…RNA extraction and pre-rRNA processing analysis of U8 depleted cells and patient-derived fibroblasts (LCC) was performed as described previously (16,43). The ATTCC fibroblast control PCS-201-012 (primary dermal fibroblasts normal human adult) was used.…”