Investigating the nucleolar stress response and targets that modulate p53 activation

Abstract

The nucleolus is a multifunctional organelle known as the central hub for ribosome biogenesis. Now, it is widely accepted that nucleolus also serves non-canonical role as a stress sensor, responding to numerous internal and environmental stressors. This response is termed the nucleolar stress response (NSR), which can be p53 dependent/independent. One of the cellular insults that activates NSR is perturbation of ribosome biogenesis. In the p53-dependent response, ribosome-free ribosomal proteins L5 and L11 (RPL5 and RPL11) bind to Murine double minute 2 (MDM2), stabilising the p53 protein, leading to cell arrest, apoptosis and/or senescene. However, the exact mechanism by which this process becomes activated remains unclear, and much detail is lacking. In human diseases, ribosomal protein (RP) mutations were frequently observed in Diamond Blackfan Anaemia (DBA) patients, with 20-25% cases of RPS19 mutation. Numerous bodies of evidence suggest that the NSR is the key mechanism of DBA pathogenesis. As such, we hypothesised that a functional genome-wide loss of function (RNAi) screen, performed under the condition of nucleolar stress (due to RPS19 depletion), would identify key proteins that regulate the NSR. Moreover, the candidates identified from this screen would provide new avenues for the treatment of diseases that are caused by abnormal ribosome biogenesis (e.g. ribosomopathies such as DBA and cancer).

Prior to the commencement of this study, a genome-wide RNAi screen using Dharmacon human siGENOME SMARTpool siRNAs (4 siRNA duplexes that target the expression of a single gene), was carried out to identify candidates that, when depleted, modulate the p53-mediated NSR caused by RPS19 knockdown. Approximately 400 top candidates that either enhancing or reducing p53 protein expression were selected for secondary screening using the Dharmacon siGENOME deconvoluted SMARTpool siRNA library, assaying each of the individual siRNAs which constituted the original SMARTpool siRNA screened in the primary screen. The 400 candidates were then categorised into 3 categories: high confidence (3/4 or 4/4 duplex hits- if the candidate had 3 or more individual siRNAs that demonstrate the same phenotype as what was observed in the primary screen), medium confidence (2/4 duplex hits) and low confidence (1/4 duplex hits). High and medium confidence candidates identified from the screens included RPL5, RPL11, p53, HEAT Repeat Containing 3 (HEATR3), Cirrhosis autosomal recessive 1A (CIRH1A), Retinoid X receptor alpha (RXRA), Insulin like growth factor 1 receptor (IGF1R), Nucleophosmin (NPM) and Phosphatase and tensin homolog (PTEN).

As part of these thesis studies, the high and medium confidence candidates identified in the screen were further validated using an in vitro candidate-based approach. The knockdown of candidates in A549 human lung cancer cells was first confirmed at the mRNA level, followed by measuring the resulting p53 expression via immunofluorescence and western blot. This was further complemented by examining how the cell cycle was affected, using flow cytometry. Apart from NPM, all the candidates were confirmed preventing p53 expression induced by RPS19 knockdown, demonstrating their important role underlying NSR. One of these candidates, HEATR3, was further evaluated to garner some insights into its normal role within cells, and how it influences the NSR.

Due to its novelty and previous literature demonstrating its role in ribosome 60S subunit biogenesis in yeast (Syo1), HEATR3 was further investigated to determine its specific role in the p53-mediated NSR and its normal cell functioning. Due to lack of robust tools (e.g. specific antibody) to study the endogenous function of HEATR3, a cell line stably expressing myc-tagged HEATR3 (A549-MT-HEATR3) was generated and used in most of the studies in this thesis. We aimed to investigate the subcellular localisation of HEATR3 by nuclear-cytoplasmic fractionation and immunofluorescence analysis. With these assays, the endogenous HEATR3 and MT-HEATR3 were found to be located in the nucleus. Furthermore, HEATR3 depletion prevented p53 activation/stabilisation after challenging cells with multiple RP depletion (knockdown of RPS6, RPS14 and RPL26). In contrast, HEATR3 depletion was unable to prevent the NSR induced by certain drugs/chemicals, including Doxorubicin, Camptothecin, Etoposide, CX-5461 and MG-132. In particular, HEATR3 depletion only rescued the p53 response with RP depletion/Leptomycin B treatment, suggesting specificity of HEATR3 involvement with these specific stresses. Overexpressed HEATR3 (MT-HEATR3) binds to overexpressed RPL5 and RPL11 (FLAG-tagged RPL5 an FLAG-tagged RPL11) evidenced in co-immunoprecipitation (CoIP) analysis, while its binding to endogenous RPL5 is specific, but not endogenous RPL11. We aimed to determine other binding partners of MT-HEATR3 using mass spectrometry; data analysis was prohibited due to sample variability.

Furthermore, we sought to determine whether HEATR3 depletion would alter the localisation of total RPL5/RPL11 (endogenous proteins), however, due to non-specificity of the antibodies, interpretation of the data was prevented. In addition, subcellular fractionation analysis suggested that HEATR3 depletion might not alter abundance of ribosome-free endogenous RPL5 and RPL11. While MT-HEATR3 may bind to 5S rRNA (statistically not significant), our ‘typical’ and ‘high salt’ polysome profiling analysis showed that HEATR3 is required for 60S ribosome subunit biogenesis and translation, which HEATR3 depletion reduced 60S ribosome subunit abundance and polysomes abundance. Particularly, HEATR3 knockdown alter translation of certain subsets of mRNA (decrease in some RPs mRNA translation and increase of Bcl-XL mRNA translation).

Overall, studies in this thesis have demonstrated the strength of functional genome-wide RNAi screens in identifying novel modifier genes of the p53-mediated NSR. We have also demonstrated the importance of HEATR3 in p53-mediated NSR and its normal function in ribosome biogenesis for the first time, and have begun to characterise the HEATR3 protein. The findings in this thesis will facilitate further studies in elucidating the impact of HEATR3 in human diseases and its potential role as a therapeutic target in diseases where the NSR is activated.