Mechanistic and functional characterization of Makorin 1 mRNPs

Makorin (Mkrn) 蛋白是一个进化上高度保守的 RNA 结合蛋白 (RBP) 家族，具有 E3 泛素连接酶功能。该项目旨在揭示 Mkrn1 mRNP（mRNA 核糖核蛋白复合物）在基因表达转录后调控中的功能。我们专注于两个互补的系统，果蝇作为胚胎发育的成熟模型生物，以及人类细胞系，用于与人类生理学潜在相关的复杂调控场景。在第一个资助期间，我们将发育研究与最先进的核糖组学和蛋白质组学方法相结合，以解决 Mkrn1 在果蝇卵子发生和人类翻译中的作用。有趣的是，我们发现在这两种生物体中，Mkrn1 与 Poly(A) 结合蛋白相互作用，并与 Poly(A) 序列上游的 mRNA 特异性结合。在一系列实验中，我们可以证明人类 MKRN1 起到核糖体相关质量控制的作用，而果蝇中的 Mkrn1 结合特异性调节 oskar mRNA 的翻译以控制胚胎模式。在第二期资助期间，我们将继续将细胞生物学、生物化学和高通量方法与计算生物学相结合，研究Mkrn1功能的机制原理。我们将描述 Mkrn1 mRNP 的组成部分，剖析它们的功能相互作用并追踪它们与 mRNA 修饰的联系。我们在果蝇和人类细胞中的并行方法将使我们能够从不同的分子和进化角度阐明 Mkrn1 功能。

The Makorin (Mkrn) proteins are an evolutionarily highly conserved family of RNA-binding proteins (RBPs) with an E3 ubiquitin ligase function. This project aims to unravel the function of Mkrn1 mRNPs (mRNA ribonucleoprotein complexes) in the posttranscriptional regulation of gene expression. We focus on two complementary systems, Drosophila as a well-established model organism for embryonic development, as well as human cell lines for complex regulatory scenarios with potential relevance in human physiology.In the first funding period, we have combined developmental studies with state-of-the-art ribonomics and proteomics approaches to address the roles of Mkrn1 in Drosophila oogenesis and human translation. Intriguingly, we have found that in both organisms, Mkrn1 interacts with the poly(A)-binding protein and binds to mRNAs specifically upstream of poly(A) sequences. In a line of experiments, we could show that human MKRN1 serves in ribosome-associated quality control, while Mkrn1 binding in Drosophila specifically regulates the translation of oskar mRNA to control embryonic patterning. In the second funding period, we will continue to integrate cell biology, biochemistry and high-throughput approach with computational biology to study the mechanistic principles of Mkrn1 function. We will characterize the components of the Mkrn1 mRNPs, dissect their functional interactions and follow their link to mRNA modifications. Our parallel approaches in Drosophila and human cells will enable us to illuminate Mkrn1 function from different molecular and evolutionary perspectives.