The Lexicon of miRISC: Deconstructing the functional complexity of the miRNA induced silencing complex

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In the cell, gene expression is regulated very tightly and one mechanism relies on small RNA molecules called microRNAs, which can repress the expression of specific genes. These microRNAs, of which there are many kinds, need to be bound by proteins called Argonautes for them to function correctly. Argonaute proteins are required to recruit other proteins that can repress the translation of genes into proteins - translation being a fundamental process occurring in all cells. Argonaute in combination with these repressive proteins is called the microRNA-induced silencing complex, or miRISC for short.Human cells express four different Argonautes but the functional differences between them are unclear. Our lab works on the LIMD1 family of proteins of which there are three members - LIMD1, Ajuba and WTIP. We think that these proteins, which can bind Argonautes, regulate the diverse functions of different miRISCs in regulating gene expression.To investigate this, we will genetically engineer cells to attach the HaloTag onto AGOs in the cell. The HaloTag permits efficient purification of tagged proteins and advanced imaging techniques. Following Argonaute purification with the HaloTag, we will sequence associated microRNAs and the genes they are regulating. We will look for patterns in the microRNAs and types of genes they regulate depending on the presence of LIMD1 in the cell. We will also get an idea of how each Argonaute protein is functionally distinct in terms of the microRNAs it binds and the genes it regulates. From the large amounts of microRNA, gene expression and protein data, we will use computational methods to construct a database and predictive model for how LIMD1 family proteins influence miRISC function. We will also use advanced microscopy techniques to see how the Argonautes and microRNAs behave in the cell and how LIMD1 family proteins are influencing this behaviour. Overall, our proposed research will reveal a new layer of miRISC regulation that cells have to ensure genes are expressed appropriately.