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

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被称为microrna的小RNA分子，它可以抑制特定基因的表达。这些microrna有很多种，它们需要与一种叫做Argonautes的蛋白质结合才能正常工作。Argonaute蛋白需要招募其他能够抑制基因转化为蛋白质的蛋白质-翻译是发生在所有细胞中的基本过程。Argonaute与这些抑制蛋白的结合被称为microrna诱导沉默复合物，简称miRISC。人类细胞表达四种不同的argonaute，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。我们认为这些可以结合Argonautes的蛋白调节了不同miRISCs在调节基因表达方面的不同功能。为了研究这一点，我们将对细胞进行基因工程改造，将HaloTag附着在细胞中的AGOs上。HaloTag允许有效的纯化标记蛋白和先进的成像技术。在用HaloTag进行Argonaute纯化后，我们将对相关的microrna及其调控的基因进行测序。我们将根据细胞中LIMD1的存在来寻找microrna的模式和它们调节的基因类型。我们还将了解每种Argonaute蛋白在其结合的microrna和其调节的基因方面的功能差异。从大量的microRNA、基因表达和蛋白数据中，我们将利用计算方法构建LIMD1家族蛋白如何影响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Argonautes和microrna在细胞中的行为，以及LIMD1家族蛋白如何影响这种行为。总的来说，我们提出的研究将揭示一个新的miRISC调控层，细胞必须确保基因的适当表达。

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.