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

miRISC 词典：解构 miRNA 诱导沉默复合物的功能复杂性

• 批准号: BB/V009567/1
• 负责人: Tyson Sharp
• 金额: $ 67.22万
• 依托单位: Queen Mary University of London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV009567%2F1
• 关键词: Lexicon; miRISC; Deconstructing; functional; complexity

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.

在细胞中，基因表达受到非常严格的调控，其中一种机制依赖于称为microRNAs的小RNA分子，它可以抑制特定基因的表达。这些microRNAs种类繁多，它们需要被称为ArgAertes的蛋白质结合，才能正常发挥作用。ArgAerte蛋白质需要招募其他蛋白质来抑制基因到蛋白质的翻译-翻译是所有细胞中发生的一个基本过程。精氨酸与这些抑制蛋白结合后被称为microRNA诱导沉默复合体，简称miRISC。人类细胞表达四种不同的精氨酸，但它们之间的功能差异尚不清楚。我们的实验室研究的是LIMD1蛋白家族，其中有三个成员-LIMD1、Ajuba和WTIP。我们认为这些蛋白可以结合ArgAertes，调节不同miRISCs在调控基因表达方面的不同功能。为了研究这一点，我们将通过基因工程使细胞将HaloTag连接到细胞中的agos上。HaloTag允许高效纯化标记的蛋白质和先进的成像技术。在用HaloTag对ArgAerte进行纯化后，我们将对相关的microRNAs及其调控的基因进行测序。我们将寻找microRNAs中的模式及其调控的基因类型，这取决于细胞中LIMD1的存在。我们还将了解每种ArgAerte蛋白在其结合的microRNAs和它调节的基因方面是如何在功能上有所不同的。从大量的microRNA、基因表达和蛋白质数据中，我们将使用计算方法来构建LIMD1家族蛋白质如何影响miRISC功能的数据库和预测模型。我们还将使用先进的显微镜技术来观察ArgAertes和microRNAs在细胞中的行为，以及LIMD1家族蛋白如何影响这种行为。总体而言，我们拟议的研究将揭示一层新的miRISC调控，细胞必须确保基因得到适当表达。