A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly

Abstract

TP53(tumour protein 53) is one of the most frequently mutated genes in human cancer and its role during cellular transformation has been studied extensively. However, the homeostatic functions of p53 are less well understood. Here, we explore the molecular dependency network of TP53 through an RNAi-mediated synthetic interaction screen employing two HCT116 isogenic cell lines and a genome-scale endoribonuclease-prepared short interfering RNA library. We identify a variety of TP53 synthetic interactions unmasking the complex connections of p53 to cellular physiology and growth control. Molecular dissection of the TP53 synthetic interaction with UNRIP indicates an enhanced dependency of TP53-negative cells on small nucleolar ribonucleoprotein (snoRNP) assembly. This dependency is mediated by the snoRNP chaperone gene NOLC1 (also known as NOPP140 ), which we identify as a physiological p53 target gene. This unanticipated function of TP53 in snoRNP assembly highlights the potential of RNAi-mediated synthetic interaction screens to dissect molecular pathways of tumour suppressor genes.More than 30 years of intense research on the tumour suppressor gene TP53 has revealed its relevance in many aspects of tumour biology (reviewed in ref. 1). It is now well established that p53 (the protein product of TP53) activation leads to the execution of a complex genetic program that limits cellular proliferation and leads to senescence or apoptosis 2,3 . Several lines of evidence have pointed to an under-appreciated homeostasis function of p53 in normal, unstressed cells 4 . As a transcription factor, p53 can mediate the expression levels of its target genes, thereby adjusting cellular homeostasis of basic metabolism 5 , balance of reactive oxygen species 6,7 , stem cell identity and reprogramming 8 and animal reproduction 9 . These data establish an important role for p53 in cellular homeostasis and indicate that p53-loss-of-function cells become genetically rewired in a way that discriminates them from wild-type cells, a premise that could be explored for the identification of possible p53 genetic dependencies.Synthetic interaction screens have led the way in identifying new and unanticipated functional interactions between seemingly unrelated protein complexes in yeast 10,11 . In mammalian cells, RNA interference (RNAi)-based synthetic interaction screens are a promising approach to dissect genetic dependencies and the first studies on mammalian synthetic interactions of oncogenes have demonstrated the power of this approach 12,13 . Furthermore, synthetic lethal screens have identified genes that sensitize cells to different chemical drugs 14,15 . Several synthetic interactions with tumour suppressor genes have also been published 11,16 , but to our knowledge a genome-scale survey for synthetic interactions of a tumour suppressor gene has not been reported. Here, we present the results of a systematic investigation of TP53 genetic dependencies and characterize in molecular detail a homeostatic function of p53 in snoRNP biogen...

Plk2 regulates mitotic spindle orientation and mammary gland development

“…RNA interference (RNAi) has been used successfully in high-throughput screens to identify potential drug targets (Gargiulo et al, 2013;Liu et al, 2013). In particular, RNAi has been used to identify putative tumor suppressors in forward genetic screens (Westbrook et al, 2005;Krastev et al, 2011;Iorns et al, 2012). Recently, our laboratory performed RNAi screens designed to specifically target kinases and phosphatases, characterizing PTPN12 as a tumor suppressor (Sun et al, 2011).…”

Polycomb group ring finger 1 cooperates with Runx1 in regulating differentiation and self-renewal of hematopoietic cells

“…Only with the development of RNA interference have systematic large-scale genetic interaction screens become feasible in human cancer cell lines. 17,18 However, despite the artificial cellular background that cultured cancer cells provide, such screens have been rarely carried out in primary cells. In the present study, we report a pooled genome-wide, shRNA-based Runx1 genetic interaction screen in primary mouse hematopoietic cells.…”

Ribosome biogenesis and p53: Who is regulating whom?

“…cells without effecting the p53-negative cells. 5 This finding suggests that the identified genes present specific checkpoints of the ribosome assembly pathway that are monitored by p53. When the amount of these proteins ceases in the cell (in this case because of their knockdown), p53 is activated, which, in turn, stops proliferation and ultimately leads to cell death.…”

“…It turns out that NOLC1 levels are dependent on basal p53 activity, thus revealing a homeostatic function of p53 to support ribosome biogenesis in unstressed cells. 5 Furthermore, when cells experience stress (e.g., DNA damage or treatment with p53 agonist Nutlin-3a), NOLC1 levels are downregulated, showing that this homeostatic function can be turned into a stress response. In consequence, both UNRIP and NOLC1 converge in snoRNP assembly, explaining the selective dependency of p53-negative cells on UNRIP expression (Fig.…”