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Molecular basis of IGF2BPs-mediated lncRNAs degradation

Final Report Summary - LNCRNATURNOVER (Molecular basis of IGF2BPs-mediated lncRNAs degradation)

IGF2 mRNA binding protein 1 (IMP/IGF2BP1) is an oncofetal protein produced at high levels in the human embryo that is important for the development of our nervous system. A faulty protein or a reduced production of IGF2BP1 results in subnormal embryonic development and in a smaller cerebral cortex, the section of the brain where cognitive functions are located. When looking at the level of individual cells, IGF2BP1 has been shown to be important for changes in the way cells divide, but also how they are shaped and importantly how they move. In the embryonic brain, IGF2BP1 regulates the wiring of the nascent neurons to create the enormous complexity of connections between neurons that is necessary to process the information the brain will receive. It also help strengthening the connections themselves, a process essential for memory a general thinking. In adults, IGF2BP1 expression is normally low, but the protein is expressed at high levels in some cancers. This has been correlated with both the growth of a tumour and its capability to create metastasis and therefore the final outcome of that cancer.
At the molecular level IGF2BP1 performs several functions. A key function, which we explore in this project, is to regulate the stability of specific molecules of RNA. Recently, it has been reported that IGF2BP1 interacts with HULC long non-coding RNA (lncRNA) in hepatocellular carcinoma cells and mediates its degradation through recruitment of CCR4-NOT deadenylation complex. IGF2BP1 acts as an adaptor for a multi-protein complexes that regulate RNA degradation, that is it recruits this complexes to the HULC lncRNA targets. Therefore to perform its function in RNA degradation IGF2BP1 must first recognise specifically the RNA to be degraded. To do this, IGF2BP1 can count on six potentially RNA-binding units (or domains) organized in three two-domain units. Also, IGF2BP1 needs to interact with a multi-protein complex degrading RNA and recruit it to the RNA molecule. Importantly, such RNA degradation mechanism is likely not limited to the HULC lncRNA but it may function in the degradation of other targets of IGF2BP1. The project investigates how RNAs are recognised by IGF2BP1 and regulated via this mechanism. The outcome will help understand if and how this mechanism represent a promising target to counteract the action of IGF2P1 in cancer.
A key question in understanding the biology of this system and in establishing its potential as a therapeutic target is how the different domains - or interaction units - of the protein can be used in a combinatorial fashion to recognize a sets of targets and to recruit them to a core RNA regulation complex in the cell. In order to answer this question, we need both to understand how the recognition of the RNA targets takes place and how the IGF2BP1 binds to the multi-protein complex required for degradation. As far as RNA recognition is concerned, we have established a novel procedure that assess the contribution of individual domain to the recognition of the whole ensemble of cellular targets. This is done by eliminating the RNA binding capability of individual domains using mutations and assessing RNA binding to the cellular RNA using iCLIP (individual Nucleotide Crosslinking and ImmunoPrecipitation). Overall our results indicate that IGF2BP1 uses different combinations of domains to recognise different targets. Such a combinatorial RNA recognition implies that a potential exist for disrupting the interaction between IGF2BP1 and groups of RNA targets by targeting one specific domains with a small molecule (i.e. a drug). Indeed the RNA binding surface of this type of domains is a relative small and deep hydrophobic grove that, in other proteins, has been shown to be blocked by amino glycosides.
As far as the recruitment of the multi-protein complex mediating RNA degradation is concerned, we show that the protein interact with this complex directly. We also show that the interaction takes place in different cells, and is likely to represent a general mechanism for RNA degradation by IGF2BP1. Overall, these results point to a role of IGF2BP1 as a finely tuned regulator of specific gene expression programs, identify the molecular element for specific interactions and establish a more general role in the recruitment of the CCR4-NOT machinery for RNA de-adenylation and decay. Our work show that, in principle, it may be possible to develop compounds that block the interaction of the protein with its targets in a selective, target-dependent way.