When ribosome biogenesis defects occur, non-ribosomal 5S RNP accumulates in the nucleoplasm where it activates p53. However, the regulation of this 5S RNP-p53 signalling pathway, and whether other proteins are involved in this pathway remains unclear. It has been suggested that proteins involved in 5S RNP assembly, localization and incorporation
The 5S RNP assembly and its subsequent incorporation into ribosomes is a complicated process which has been shown to require the coordination from other protein co-factors. Some proteins interacting with RPL11 or RPL5 affect p53 induction, presumably via 5S RNP (Donati et al., 2013; Fregoso et al., 2013; Sloan et al., 2013a). Several proteins that are involved in either ribosome production (Mybbp1a, NML and B23), mRNA transcription
(HEXIM1) or splicing (SRSF1) have recently been linked to regulating p53 activity (see below).
1.5.1 SFSF1
Serine/Arginine rich Splicing Factor 1 (SRSF1), known as Alternative Splicing Factor 1
(ASF/SF2) was first identified as an alternative splicing factor protein, which is also involved in post-splicing events such as translation initiation (Michlewski et al., 2008), nuclear export of mature mRNA and nonsense mediated decay (Long and Caceres, 2009). The SRSF1 gene is up regulated in breast cancer and its overexpression was shown to promote transformation of mammalian cells (Anczukow et al., 2012; Anczukow et al., 2015).
Recently it has been reported that SRSF1 interacts with RPL5 (Fregoso et al., 2013). Interestingly they also showed that in BJ-TT cells, SRSF1 depletion abolishes ActD-induced p53 activation. This suggests that SRSF1 is involved in the 5S RNP-p53 regulatory signalling pathway (Fregoso et al., 2013).
In addition, Loren Gibson, a former member of the Watkins lab, confirmed that SRSF1 not only interacts with RPL5, but also the 5S RNP in U2OS cells (unpublished data). Unpublished results further demonstrated that the overexpression of SRSF1 stabilises p53, whereas depletion of SRSF1 reduces p53 basal activity and counteracts ActD-induced p53 activation in U2OS cells. SRSF1 depletion was also shown to reduce 5S RNP recruitment into the ribosome (Loren Gibson, unpublished data). Taken together, it was proposed that in normal conditions, SRSF1 is involved in the 5S RNP integration into the large subunit. In response to nucleolar stress, SRSF1 assists the 5S RNP in inhibiting MDM2.
Moreover, SRSF1 is highly phosphorylated and its function and cellular localization is regulated by its phosphorylation (Ma et al., 2008). One of the kinases involved is the
serine/arginine-rich protein-specific kinase (SRPK). SRPK phosphorylates specifically multiple serine residues located in the arginine/serine rich region (also known as the RS domain)
(Giannakouros et al., 2011; Gammons et al., 2013). Therefore, SRSF1 could be an important player in both ribosome biogenesis and p53 signalling.
1.5.2 HEXIM1
HEXIM1 protein has been well characterized as a positive transcription elongation factor b (P-TEFb) inhibitor which associates with RNA Pol II (Yik et al., 2003; Michels et al., 2004). Lew
et al. recently reported that HEXIM1 positively regulates p53. They first identified the
interaction between p53 and HEXIM1. They further reported evidence showing that overexpression of HEXIM1 leads to stabilised and more active p53, whereas depleting
HEXIM1 inhibits p53 activation in response to DNA damage and translation blockage stress in MCF7 cells (Lew et al., 2012). Since HEXIM1 is a substrate for ubiquitination by MDM2 (Lau
et al., 2009), it was proposed that HEXIM1 stabilises p53 by antagonizing the MDM2
ubiquitination function upon p53 (Lew et al., 2012; Lew et al., 2013).
Lew et al. also showed that ActD treatment, which blocks ribosome biogenesis, increased association between HEXIM1 and p53 in MCF7 cells (Lew et al., 2012), suggesting HEXIM1 could be a potential protein factor involved in the 5S RNP-p53 regulatory signalling pathway. Nevertheless, how HEXIM1 is related to ribosomal stress-inducing p53 activation remains unclear.
1.5.3 Mybbp1a
Myb-binding protein 1a (Mybbp1a) was originally identified as a binding protein for the proto-oncogene c-Myb and since then, increasing evidence connects its involvement in rRNA production and in p53 acetylation and regulation, suggesting its association with ribosome biogenesis and p53 regulation.
Mybbp1a is a nucleolar protein that is associated with RNA Pol I in HeLa cells, and the expression of Mybbp1a represses rRNA transcription (Hochstatter et al., 2012). They also showed that Mybbp1a is linked to rRNA processing. Furthermore, they demonstrated that
tetramerization of p53 (Kuroda et al., 2011; Ono et al., 2013). However, more intriguingly, ActD treatment promotes p53-K382 acetylation and increases protein levels of p21 in MCF7 cells, whereas depleting Mybbp1a counteracts such effects caused by the ActD treatment (Kuroda et al., 2011).
1.5.4 NML
Nucleomethylin (NML; also known as RRP8) was identified as a component of the energy- dependent nucleolar silencing complex (eNoSC). This complex includes two other protein components, sirtuin 1 (SIRT1) and Histone-lysine N-methyltransferase (SUV39H1). NML helps to recruit SIRT1 and SUV39H1, resulting in methylation and deacetylation of histone H3K9 which inhibits rDNA transcription when cells are in low glucose conditions (Vaquero et al., 2007; Murayama et al., 2008; Yang et al., 2013).
NML is an RNA binding protein associating with 5S, 5.8S and 28S rRNA. The NML-SIRT1 protein interaction is inhibited by the rRNA binding. During nutrient starvation, reduced rRNA production which promotes eNoSC recruitment via NML, results in a positive feedback loop further inhibiting the production of rRNAs. It was proposed that the NML-SIRT1
interaction couples nutrient levels to ribosome biogenesis (Yang et al., 2013).
NML is also a methyltransferase which is essential for the N1-methyladenosine (m1A) modification in the human and mouse 28S rRNA (Waku et al., 2016). In addition, Oie et al. reported evidence showing NML protein is also linked to fatty acid metabolism in a mouse model. NML-null mice showed reduced lipid accumulation in liver cells and after feeding these mice with a high fat diet, their weight gain and fat accumulation were lower than their wild type counterparts fed with the same diet (Oie et al., 2014). Kumazawa et al. provided evidence showing that in response to the glucose starvation Mybbp1a translocates to the nucleoplasm where it promotes p53 acetylation and activation. They also showed that Mybbp1a depletion reduces p53 activation in response to glucose starvation (Kumazawa et
al., 2011).
1.5.5 B23
Nucleophosmin, also known as phosphoprotein B23, NPM1 or numatrin, is a multifunctional protein which is involved in multiple stages of ribosome biogenesis (Lindström, 2011). Wild
type B23 is predominantly localized in the nucleolus, whereas the NPMc+, a mutated form of B23 caused by chromosomal aberrations, is found mislocated in the cytoplasm in acute myeloid leukaemia (AML) cells (Wang et al., 1993). There are approximately 35% of AML patients that have NPMc+ mutations (Federici and Falini, 2013).
The interaction between B23 and MDM2 was confirmed biochemically in SaOS-2 cells and UV radiation increases B23/MDM2 complex formation (Kurki et al., 2004). Knockdown of B23 protein reduces p53 stability and activity in response to radiation treatment (Colombo
et al., 2002; Kurki et al., 2004). It was proposed that B23 inactivates MDM2 and
subsequently stabilising p53 in response to DNA damage stress.
Furthermore, evidence also suggests that B23 itself is able to associate with p53 (Kurki et al., 2004; Lambert and Buckle, 2006). In addition, B23 interacts with both RPL5 and the 5S rRNA (Yu et al., 2006). B23 translocating from nucleoli to the nucleoplasm was shown to be caused by ActD treatment (Huang et al., 2005; Yao et al., 2010; Brodská et al., 2016) and UV