Mechanistic and functional characterization of Makorin 1 mRNPs

Makorin(Mkrn)蛋白是一个进化高度保守的RNA结合蛋白家族，具有E3泛素连接酶功能。本项目旨在揭示Mkrn1mRNPs(信使核糖核蛋白复合体)在基因表达的转录后调控中的功能。我们专注于两个互补的系统，果蝇作为一个成熟的胚胎发育模式生物，以及用于复杂调控场景的人类细胞系，与人类生理学潜在的相关性。在第一个资助期，我们将发育研究与最先进的核糖体组学和蛋白质组学方法相结合，研究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中，Mkrn1与Poly(A)结合蛋白相互作用，并与Poly(A)序列上游的mRNAs结合。在一系列实验中，我们可以证明人类MKRN1在核糖体相关的质量控制中发挥作用，而果蝇中的Mkrn1结合特异性地调节Oskar mRNA的翻译，以控制胚胎模式。在第二个资助期，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 mRNPs的组成，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平行方法将使我们能够从不同的分子和进化角度阐明Mkrn1的功能。

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.