Computational and Experimental Modelling of Post-transcriptional Gene Regulatory Networks Involved in Cancer

Das Mandal, Sukhen (2022) Computational and Experimental Modelling of Post-transcriptional Gene Regulatory Networks Involved in Cancer. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Sukhen Das Mandal (15RS045)) 15RS045.pdf - Submitted Version Restricted to Repository staff only Download (6MB)

Abstract

Interplay between RNA-binding proteins (RBPs) and microRNAs (miRNAs) in post-transcriptional gene regulatory networks regulates the translation/turnover of manymRNAs involved in cancer development and progression. Expression of the tumor suppressor protein p53 is regulated at the post-transcriptional level by crosstalk between the RBP HuR and the miRNA miR-125b. HuR antagonizes miR-125b-mediated repression of p53 mRNA translation in response to UV irradiation. We have combined computational systems modelling with experimental validation to elucidate the translation regulatory network that controls p53 expression under UV-induced DNA damage condition. The simulated dynamics fitted with the experimentally observed pulse of p53 and HuR when hypothetical inhibitors of synthesis and degradation of HuR were incorporated into the known p53 translation regulatory network. Experimentation showed that miR-125b inhibited HuR synthesis by repressing translation of HuR mRNA and HuR protein was proteasomally degraded by the E3-ubiquitin ligase tripartite motif-containing 21 (TRIM21). The integrated regulation by miR-125b and TRIM21 gives rise to an intricate regulatory system that controls pulsatile expression of HuR and p53 in response to DNA damage. Furthermore, we have investigated the role of a new player, RNA modification, in the interplay between miRNAs and RBPs in post-transcriptional gene regulatory networks. N⁶-methyladenosine (m⁶A), the most prevalent epitranscriptomic modification in eukaryotes, is enriched in 3′-untranslated regions (3′ UTRs) of mRNAs and regulates mRNA metabolism at multiple levels. m6A has been shown to influence both RBP and miRNA interactions with mRNAs and may therefore play a determining role in the interplay between RBP and miRNA binding to target mRNAs. Using transcriptome-wide computational analysis, we have found a strong positive correlation between the number of m⁶A sites, miRNAs and RBPs binding to mRNAs, suggesting that m6A-modified mRNAs are more targeted by miRNAs and RBPs. m⁶A sites are also located proximally to miRNA target sites and binding sites of RBPs independent of cell type. Further, miRNA target sites and RBP-binding sites located close to each other are also proximally located to m⁶A sites indicating three-way interplay between m⁶A, microRNA and RBP binding. From the subset of mRNAs which show three-way proximity between m⁶A sites and target sites of miR-125b and HuR, we have selected COX7A2L, which encodes a component of the mitochondrial respiratory chain and is upregulated in estrogen-treated breast cancer cells. We have found that expression of COX7A2L is regulated by both miR-125b and HuR. We also found that m⁶A modification facilitates the binding of miR-125b to COX7A2L mRNA 3’UTR and HuR antagonizes the miR-125b mediated repression COX7A2L expression. This thesis therefore uses combined computational and experimental approaches to discover new mediators and interactions which influence the HuR- and miR-125b-driven post-transcriptional gene regulatory network of p53 and further introduces and validates the role of m⁶A RNA modification in modulating post-transcriptional regulation by HuR and miR-125b.

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