TUMOR SUPPRESSOR p53
Translational regulation of p53 mRNA by trans-acting factors and cis-acting elementsTumor suppressor protein p53 is popularly considered as “guardian of the genome”. It is a master transcription regulator that is indispensable for controlling several cellular pathways. Its prime role is to adapt gene expression programs in order to maintain cellular homeostasis and genome integrity especially during the stress conditions. Most, if not all, tumors harbor mutations in the TP53 gene itself or acquire genetic and/or epigenetic alterations that compromise the p53 response. The regulation of p53 expression and activity is multi-layered. It is intricately controlled at the levels of transcription, RNA splicing, RNA stability, post-transcriptional gene-silencing, translation, post-translational modifications, protein stability and degradation. Extensive research has been carried out on regulation of p53 activation and function at the protein level, but translation of p53 mRNA is the focus of current research activities as this is one of the few steps in the regulation of any gene where response to any stimulus can be swiftly graded up or down in terms of the protein output.
Work done in our laboratory demonstrated that p53 mRNA has a dual Internal Ribosome Entry Site (IRES) structure that regulates translation of full-length p53 and its N-terminally truncated isoform ΔN-p53. The two IRESs show distinct cell-cycle phase-dependent activity with the IRES for full-length p53 being active at the G2–M transition and the IRES for ΔN-p53 shows highest activity at the G1–S transition (Ray et al, EMBO Reports, 2006). Translation from p53 IRESs depend upon IRES trans-acting factors (ITAFs) as well as cis-acting structural elements. We have demonstrated stress-dependent modulation of p53 IRESs by ITAFs. Polypyrimidine tract-binding protein (PTB) binds specifically to both the p53 IRESs but with differential affinity. During doxorubicin treatment, PTB protein translocates from nucleus to the cytoplasm, suggesting that the relative cytoplasmic abundance of PTB protein, under DNA-damaging conditions, might contribute to regulating the coordinated expression of the p53 isoforms, owing to the differential affinity of PTB binding to the two p53 IRESs (Grover et al, Cell Cycle. 2008). Further, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as p53 ITAFs. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53 mRNA appears to play a crucial role in IRES function (Sharathchandra et al, RNA Biology, 2012). In a collaborative project with Prof Adi Kimchi’s lab (Weizmann Institute of Science) we also found that an auxiliary translation initiation factor, eIF4G homolog Death Associated Protein 5Jan 30;33(5):611-8.promotes IRES-driven translation of p53 mRNA. Functional analysis indicated that DAP5 preferentially promotes translation from the second module of IRES residing in the coding sequence (Weingarten-Gabbay S and Khan et al, Oncogene, 2014).
To correlate p53 IRES structure with activity, we have mapped the putative secondary structure of p53-IRES RNA using information from chemical probing and nuclease mapping experiments. Compared to wild-type RNA, our results also indicate weaker and compromised IRES activities of the p53 IRESs bearing cancer-derived silent mutations in the 5’UTR or in the coding region of the IRES. Further, it was observed that few cytoplasmic trans-acting factors such as PTB and hnRNP C1/C2, critical for enhancing IRES function, were unable to bind these mutant IRESs as efficiently as in wild-type. In all these cases, knockdown of ITAFs or mutations in p53 IRESs show consequent changes in p53 dependent transactivation of target genes (Sharathchandra et al, 2012, RNA Biology. Vol. 9 (12), p1429-1439 ; Khan et al. 2012, Oncogene Vol.10, p1-12). Thus IRES-dependent translational control of p53 mRNA is uniquely indispensable in the complex scheme of p53 isoforms activation and function. We are also investigating the effect of p53 ITAFs in regulating cross-talk between 5’ UTR and 3’UTR of p53 mRNA and the role of miRNA and protein interactions at the 3’UTR in regulating p53 and ΔN-p53 expression from p53 mRNA.
Representation of proposed regulation of p53 IRES mediated translation. During stress and physiological conditions IRES trans-acting factors (green: DAP5, brown: hnRNPC1/C2, purple: PTB, yellow: ANXA2, and red: RPL26) come out from nucleus to cytoplasm to enhance IRES activity (Sharathchandra et al, Wiley Interdisciplinary Reviews: RNA, 2014)
The overarching aim of this “p53 project” is to identify novel factors that lead to differential expression of p53 isoforms.
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