RESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Secuencia Conservada/genética , Neoplasias Pulmonares/genética , Precursores del ARN/genética , ARN Ribosómico/genética , ARN Nucleolar Pequeño/genética , Animales , Carcinogénesis , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Procesamiento Postranscripcional del ARN , ARN Interferente Pequeño/genética , Proteínas Ribosómicas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The function of RNA is subtly modulated by post-transcriptional modifications. Here, we report an important crosstalk in the covalent modification of two classes of RNAs. We demonstrate that yeast Kre33 and human NAT10 are RNA cytosine acetyltransferases with, surprisingly, specificity toward both 18S rRNA and tRNAs. tRNA acetylation requires the intervention of a specific and conserved adaptor: yeast Tan1/human THUMPD1. In budding and fission yeasts, and in human cells, we found two acetylated cytosines on 18S rRNA, one in helix 34 important for translation accuracy and another in helix 45 near the decoding site. Efficient 18S rRNA acetylation in helix 45 involves, in human cells, the vertebrate-specific box C/D snoRNA U13, which, we suggest, exposes the substrate cytosine to modification through Watson-Crick base pairing with 18S rRNA precursors during small subunit biogenesis. Finally, while Kre33 and NAT10 are essential for pre-rRNA processing reactions leading to 18S rRNA synthesis, we demonstrate that rRNA acetylation is dispensable to yeast cells growth. The inactivation of NAT10 was suggested to suppress nuclear morphological defects observed in laminopathic patient cells through loss of microtubules modification and cytoskeleton reorganization. We rather propose the effects of NAT10 on laminopathic cells are due to reduced ribosome biogenesis or function.
Asunto(s)
Acetiltransferasas/metabolismo , Acetiltransferasa E N-Terminal/metabolismo , ARN Ribosómico 18S/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilación , Acetiltransferasas/química , Secuencia de Aminoácidos , Línea Celular , Secuencia Conservada , Citosina/metabolismo , Humanos , Acetiltransferasa E N-Terminal/química , Acetiltransferasas N-Terminal , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN de Planta/química , ARN de Planta/metabolismo , ARN Ribosómico 18S/química , ARN Nucleolar Pequeño/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Mature ribosomal RNAs (rRNAs) are produced from polycistronic precursors following complex processing. Precursor (pre)-rRNA processing has been extensively characterized in yeast and was assumed to be conserved in humans. We functionally characterized 625 nucleolar proteins in HeLa cells and identified 286 required for processing, including 74 without a yeast homolog. For selected candidates, we demonstrated that pre-rRNA processing defects are conserved in different cell types (including primary cells), defects are not due to activation of a p53-dependent nucleolar tumor surveillance pathway, and they precede cell-cycle arrest and apoptosis. We also investigated the exosome's role in processing internal transcribed spacers (ITSs) and report that 3' end maturation of 18S rRNA involves EXOSC10/Rrp6, a yeast ITS2 processing factor. We conclude that human cells adopt unique strategies and recruit distinct trans-acting factors to carry out essential processing steps, posing fundamental implications for understanding ribosomopathies at the molecular level and developing effective therapeutic agents.