Interplay Between microRNA-21 and RNA-binding Proteins in the Regulation of Translation of the Pro-inflammatory Gene Programmed Cell Death 4 (PDCD4)

Poria, Dipak Kumar (2016) Interplay Between microRNA-21 and RNA-binding Proteins in the Regulation of Translation of the Pro-inflammatory Gene Programmed Cell Death 4 (PDCD4). PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Inflammation is the primary protective response of the body to infection and injury; however unresolved and chronic inflammation can exacerbate tissue damage and contribute to multiple disease conditions, including a transition to cancer. To limit the undesirable consequences of excessive and chronic inflammation, many stimuli that trigger the inflammatory response simultaneously trigger a regulatory programme that actively resolves inflammation. Translational control of pro-inflammatory genes plays an important role in regulating the inflammatory response and preventing chronic inflammation. Translation regulation is mediated by the signal-dependent binding of regulatory RNA-binding proteins (RBPs) or microRNAs (miRNAs) to specific sequence/structural elements in the 3’ and 5’ untranslated regions (UTRs) of mRNAs. Therefore the interactions of specific regulatory proteins with sequence/structural elements in the 3’UTR of an mRNA can modulate specific miRNA-mRNA interactions, and vice versa, resulting in coordinated and fine-tuned control of translation. The pro-inflammatory tumor suppressor protein PDCD4 plays an important role in maintaining the balance between inflammation and tumorigenesis by inhibiting neoplastic transformation, tumour progression and metastasis. PDCD4 expression gets stimulated by inflammatory stimuli such as LPS, and it acts as a tumor suppressor by inhibiting the expression of multiple tumorigenic factors in later stages of inflammation. miR-21, an oncogenic miRNA upregulated in many cancers, inhibits the translation of PDCD4 mRNA. While a number of studies have shown the translation regulation of PDCD4 by miR-21, RBP-mediated regulation of PDCD4 has not been reported, and the proteins binding to the PDCD4 3’UTR are not known. In this thesis the interplay between microRNA-21 and RBPs in the regulation of PDCD4 translation has been investigated. In the first part of the thesis, a ubiquitously expressed RNA-binding protein HuR, belonging to the Hu/ELAV (embryonic lethal abnormal vision) family of RBPs, was found to interact with PDCD4 3’UTR. HuR was found to enhance PDCD4 expression and reverse miR-21-mediated translation repression of PDCD4 mRNA in MCF7 breast carcinoma cells. Interaction of HuR was found to dissociate miR-21-RISC from the PDCD4 mRNA to activate its translation. In unstimulated cells, HuR protein was predominantly localized in the nucleus but treatment with the inflammatory agonist, bacterial lipopolysaccharide (LPS) was found to cause an increase in cytoplasmic HuR level by its nuclear-cytoplasmic translocation. This resulted in increasing binding with PDCD4 mRNA and reversal of miR-21-mediated translation silencing. PDCD4 mRNA was found to be mostly present in the non-translating free mRNA pool in cells overexpressing miR-21, but was redistributed to translating heavy polysomes on overexpression of HuR or LPS treatment. The antagonistic interplay between miR-21 and HuR in regulating PDCD4 expression was reflected in alteration of cellular physiology. Cells overexpressing miR-21 showed higher rate of proliferation and reduced apoptosis, which was reversed by HuR expression. Together, these observations constitute the first report of an RBP that is involved in the regulation of PDCD4 expression by an antagonistic crosstalk with miR-21-mediated translation inhibition. In the second part of the thesis, we attempted to systematically decipher the mechanism of integration of these two modes of regulation of PDCD4 mRNA translation by miR-21 and HuR. Using a consecutive deletion-based approach, HuR was found to interact with two regions of the PDCD4 3'UTR in the vicinity of the miR-21 target site, and HuR binding to any of these sites was found to result in reversal of miR-21-mediated translation repression. However, remarkably, HuR was also found to be able to function in trans to rescue miR-21-mediated translation repression of PDCD4. This was found to be due to the ability of HuR to bind to miR-21 directly, and independently of PDCD4 mRNA, thereby sequestering it and preventing its interaction with the PDCD4 mRNA 3'UTR, leading to the rescue of translation. Therefore, HuR can act as an "miRNA sponge", sequestering free miRNA from the cytoplasm, together with binding to the PDCD4 mRNA, and thereby shifting the equilibrium of miRNA-binding to PDCD4 mRNA more towards free miRNA and reversing the translation repression. Bioinformatic analysis suggested that multiple miRNAs might be targets of HuR, and "miRNA sponging" might be a generalized mechanism by which HuR sequesters multiple miRNAs and modulates miRNA-mediated translation regulation, resulting in fine-tuned gene expression in complex regulatory environments. In the third part of the thesis, more regulatory molecules which might be involved in the interplay between RBPs and miRNA to regulate PDCD4 expression have been explored. An unbiased mass spectrometry-based method to find RBPs interacting with the PDCD4 3'UTR showed that the human Lupus antigen La, an RBP involved in IRES mediated translation and tRNA maturation, interacts with the PDCD4 3’UTR. The interaction with La was found to enhance PDCD4 expression in cells. In addition, La over expression was found to rescue miR-21-mediated repression of PDCD4 expression in the cells. Three La binding sites were found in the PDCD4 3’UTR among which the La binding site proximal to the miR-21 target site was found to be sufficient for rescuing miR-21-mediated translation inhibition. Interestingly, treatment with LPS increased cellular La protein level, which contributed to the LPS-mediated increase in PDCD4 expression. LPS treatment has previously been shown to cause cytoplasmic localization of HuR. HuR and La was found to facilitate each other's binding to the PDCD4 3'UTR resulting in a cooperative effect on PDCD4 mRNA translation and rescue of miR-21-mediated translation repression. Therefore, this thesis demonstrates that an inflammatory stimulus causes the activation of two different RBPs HuR and La, in different ways, to trigger an antagonistic interplay between these RBPs and miR-21, resulting in the reversal of miRNA-mediated repression of PDCD4 expression. This interplay involves both mRNA-binding and miRNA-binding by RBPs, suggesting a highly expanded regulon of RBPs which might be involved in regulating gene expression in the context of various stimuli and stress conditions.

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