Oncogene (2014) 33, 474–483 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Ribosomal protein S6 kinase activity controls the ribosome biogenesis transcriptional program C Chauvin1,2, V Koka1,2, A Nouschi1,2, V Mieulet1,2, C Hoareau-Aveilla3, A Dreazen4, N Cagnard2, W Carpentier5, T Kiss3, O Meyuhas4 and M Pende1,2 S6 kinases (S6Ks) are mechanistic target of rapamycin substrates that participate in cell growth control. S6Ks phosphorylate ribosomal protein S6 (rpS6) and additional proteins involved in the translational machinery, although the functional roles of these modifications remain elusive. Here we analyze the S6K-dependent transcriptional and translational regulation of gene expression by comparing whole-genome microarray of total and polysomal mouse liver RNA after feeding. We show that tissue lacking S6Ks 1 and 2 (S6K1 and S6K2), displays a defect in the ribosome biogenesis (RiBi) transcriptional program after feeding. Over 75% of RiBi factors are controlled by S6K, including Nop56, Nop14, Gar1, Rrp9, Rrp15, Rrp12 and Pwp2 nucleolar proteins. Importantly, the reduced activity of RiBi transcriptional promoters in S6K1;S6K2 À / À cells is also observed in rpS6 knock-in mutants that cannot be phosphorylated. As ribosomal protein synthesis is not affected by these mutations, our data reveal a distinct and specific aspect of RiBi under the control of rpS6 kinase activity, that is, the RiBi transcriptional program. Oncogene (2014) 33, 474–483; doi:10.1038/onc.2012.606; published online 14 January 2013 Keywords: signal transduction; ribosome biogenesis; mTOR INTRODUCTION that fully inhibit mTOR activity, impair mRNA translation to a much 16–18 Biochemical and genetic studies have unequivocally identified S6 greater degree than rapamycin. kinase 1 and 2 (S6K1 and S6K2) as the main in vivo kinases Here we address whether S6K activity controls expression of responsible for ribosomal protein S6 (rpS6) phosphorylation.1–4 specific mRNA classes at the transcriptional and translational levels The kinases sequentially phosphorylate rpS6 on five serine by combining microarray analysis on total and polysomal residues in the C-terminal tail of the protein.5,6 S6K1 and S6K2 fractions. Although we fail to uncover specific mRNAs under the are activated by the mammalian target Of rapamycin kinase translational control by S6Ks, our data reveal that S6K has a broad (mTOR, now officially named mechanistic target Of rapamycin),7 a influence on the transcription of the ribosome biogenesis (RiBi) master controller of growth integrating a large variety of program, providing the cell with nucleolar factors required for environmental cues.8 S6Ks participate in the mTOR-dependent ribosomal RNA (rRNA) synthesis, cleavage, post-transcriptional growth program, as outlined by the growth defect and perinatal modifications, assembly with ribosomal proteins (RPs) and lethality of loss-of-function mutant flies and mice.4,9,10 As rpS6 is a transport. As we demonstrate that the phosphorylation of rpS6 constitutive component of the 40S small ribosomal subunit and as participates in this regulatory event, our findings suggest the rpS6 phosphorylation is triggered by anabolic signals, the favored interesting possibility that the post-translation modification of a hypothesis is that S6K activity regulates growth by affecting RP affects RiBi through the synthesis of RiBi factors. protein synthesis, although direct experimental evidence is lacking. That S6Ks interact with the translational machinery is further RESULTS supported by the findings that additional substrates include Transcriptional versus translational control of gene expression by eukaryotic initiation factor 4B (eIF4B) and eukaryotic elongation S6Ks factor 2 kinase (eEF2K).11,12 Moreover, S6Ks are found in complex To uncover potential translational mRNAs targets of S6Ks, with the eukaryotic initiation factor 3.13 However, global protein ribonucleoprotein complexes were extracted from livers of wild- synthesis is not affected in S6K-deficient cells, as assessed by type and S6K1;S6K2 À / À mice after starvation or 4-h refeeding. The methionine incorporation and polysome profile analysis.14 liver was chosen for this in vivo analysis because the mTOR Consistently, the selective allosteric mTOR inhibitor rapamycin, pathways is readily regulated by nutrient status and the which abrogates S6K activity while having minor effects on the preservation of the ribonucleoprotein complexes is optimal. phosphorylation of other mTOR targets including the eukaryotic Starvation and refeeding affected the phosphorylation of the initiation factor 4E-binding proteins, has minimal effects on cap- mTORC1 substrates S6K1 and 4E-binding protein 1, and the dependent translation.15 In addition, recent data using ribosome downstream S6K substrates rpS6 and eIF4B, as expected profiling and methionine incorporation, demonstrate that (Figure 1a).4,11 After sucrose gradient fractionation, actively Adenosine triphosphate (ATP)-competitive inhibitors of mTOR translating polyribosomes (polysomes) were separated from 1INSERM, U845, Paris, France; 2Universite´ Paris Descartes, Sorbonne Paris Cite´, Faculte´ de Me´decine, UMRS-845, Paris, France; 3Laboratoire de Biologie Mole´culaire Eucaryote, Universite´ de Toulouse-UPS and Centre National de La Recherche Scientifique, Toulouse, France; 4Department of Biochemistry and Molecular Biology, IMRIC, Hebrew University- Hadassah Medical School, Jerusalem, Israel and 5Plateforme Post-Ge´nomique Pitie´-Salpe´trie`re, Groupe Hospitalier Pitie´-Salpe´trie`re, Universite´ Pierre et Marie Curie, Paris, France. Correspondence: Dr M Pende, U845, Inserm, 156 rue de Vaugirard, Paris, F-75015, France. E-mail: [email protected] Received 23 July 2012; revised 21 September 2012; accepted 4 November 2012; published online 14 January 2013 S6 kinases control RiBi program C Chauvin et al 475 monosomes (80S), large and small ribosomal subunits (60S and These factors may associate with the box C/D class of small 40S), free mRNA and proteins (Figure 1b). After measuring the nucleolar ribonucleoproteins (Nop56 and Rrp9), or with the box relative abundance of peaks corresponding to 40S-60S-80S and H/ACA class (Gar1). As the majority of these mRNAs was also found polysomes, there was an increase in the area under the curve of downregulated in the polysomal fractions (Figure 1c and polysomes after refeeding as compared with starved controls. Supplementary Figure 2), it is likely that S6K deletion also However, we could not detect a significant difference between decreases RiBi expression at the protein level. Immunoblot wild-type and S6K1;S6K2 À / À liver profiles, both in starved and analysis with commercially available antibodies to Nop56 con- refed conditions. These data were consistent with previous firmed a 50% reduction of protein amount, whereas fibrillarin observations in other cell types and experimental conditions,14 levels were not affected (Figure 1e and Supplementary Figure 3). suggesting that S6Ks do not affect global translation. To address Thus, S6Ks regulate the abundance of factors that are implicated whether specific mRNA classes were regulated at the translational in a variety of nucleolar functions. level, whole-genome microarray analysis was performed on To gain further insights on the physiological regulation of the mRNAs associated with polysomes and compared with the RiBi program by S6Ks, mice underwent different nutritional, results from the whole-cell extract. A total of 456 mRNAs were pharmacological and genetic manipulations that affected S6K downregulated in the whole-cell extract from S6K1;S6K2 À / À livers activity (Figure 1a). We focused this analysis on Nop56, Gar1 and after refeeding (Supplementary Table 1). To specifically assess Pwp2 mRNA expression that displayed a high degree of regulation control of gene expression at the translational level, the ratio of by S6Ks. As shown in Figure 2a, 4-h refeeding after overnight signal intensities from the microarray analysis on the whole-cell starvation led to 2.5- to 3-fold increase of gene expression in wild- extracts and polysomal fractions was calculated, as previously type mice. A kinetic study from 2- to 8-h refeeding indicated a performed in distinct experimental settings.19 Strikingly, only similar response throughout the entire time period (Figure 2b). five mRNAs displayed a decreased polysomal/whole-cell ratio Strikingly, S6K deletion abrogated the nutrition-induced gene in S6K1;S6K2 À / À extracts as compared with wild type expression, as Nop56, Gar1 and Pwp2 mRNA levels in S6K1; (Supplementary Table 1). Two of these mRNAs were not S6K2 À / À livers after refeeding were equivalent to the starved annotated. The remaining encoded for myeloid/lymphoid or mice of both S6K1;S6K2 À / À and wild-type genotypes (Figure 2a). mixed-lineage leukemia 5, polycomb group ring finger 5 and A transient inhibition of mTORC1/S6K activities by the rapamycin retinal outer segment membrane protein 1 mRNAs. The signal derivative Temsirolimus in wild-type mice mimicked the effect of intensities for these mRNAs on the microarray chip were close to S6K deletion on RiBi gene expression. Similarly, in primary cultures the low threshold of detection. In addition, when real-time of hepatocytes, serum stimulation increased Nop56 and Pwp2 quantitative PCR (RT–QPCR) analysis was attempted on a larger gene expression in wild-type cells in a rapamycin-sensitive number of mice, we could not confirm a statistical significant