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

Reference	BB/V009567/1
Principal Investigator / Supervisor	Professor Tyson Sharp
Co-Investigators / Co-Supervisors	Professor Viji Draviam, Dr Paul Grevitt, Dr Faraz Mardakheh, Dr Jun Wang
Institution	Queen Mary University of London
Department	Barts Cancer Institute
Funding type	Research
Value (£)	527,571
Status	Current
Type	Research Grant
Start date	01/01/2022
End date	31/12/2024
Duration	36 months

Abstract

The microRNA-induced silencing complex (miRISC) is critical for post-transcriptional regulation. miRISCs consist of Argonaute proteins, which bear miRNAs, TNRC6 proteins which are critical for translational repression and a host of other effectors which destabilise and degrade mRNAs. Multiple isoforms of AGOs and TNRC6 proteins exist. Our previous work has shown that the LIMD1 family of proteins (LIMD1, Ajuba and WTIP, collectively termed LAW) interact with AGOs and TNRC6 proteins. We propose LAW proteins modify canonical cytoplasmic AGO function in human cells and the range of AGO, TNRC6 and LAW isoforms can assemble into multiple different miRISCs (miRISCLIMD1, miRISCAjuba, miRISCWTIP or combinations thereof miRISCLAW), which differ significantly in function. Our proposal focuses on an overarching hypothesis: "LAW proteins drive the formation specialised miRISCs with distinct localisation patterns and functions". To address this, we have the following aims: Aim 1: Determine functions of different miRISCs on the silencing landscape for a predictive model of miRISC-LAW association and regulatory output. Aim 2: To image functional miRISCs, RNA and P-bodies with regard to LAW protein association To address our Aims, we will utilise the HaloTag system in an endogenous context to tag AGOs. This will facilitate AGO purification for RNA-seq studies on AGO-bound miRNAs and mRNAs. Such experiments will be performed in LIMD1+/+ and -/- cells in parallel so the effect of LIMD1 (and other LAW proteins once they are ablated) on miRISC function and gene expression will be ascertained. To complement, proteomic data on these cells will also be obtained. Ultimately, these datasets will feed into machine learning programmes which will identify structural and functional patterns to LAW-dependent miRNA silencing. We will use the HaloTag and microinjection of labelled RNAs for single molecule imaging techniques to visualise the localisation and function of specialised miRISCs.

Summary

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 thatcells have to ensure genes are expressed appropriately.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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