Mapping mRNP-NUP interactions during nuclear export with a unique mRNA-trap

真核mRNA在细胞核中转录，需要穿过核孔到达细胞质进行翻译。这一通道对mRNA功能至关重要，而失败与一系列人类疾病有关。核mRNA输出的研究是具有挑战性的，因为它是非常快的，mRNA和核孔蛋白之间的相互作用是短暂的。我们最近发现锥虫缺乏mRNA输出检查点，通常阻止未成熟的mRNA开始核输出。因此，mRNA与其附着的蛋白质（mRNP，信使核糖核蛋白颗粒）可以被捕获，而被输出，简单地通过阻断转录或剪接。在这里，我们计划利用锥虫的这种独特功能作为mRNA陷阱来研究mRNP和核孔蛋白之间的相互作用。我们将使用新开发的蛋白质和RNA的邻近标记技术的组合，辅以经典的生化纯化方法。我们的综合数据将创建一个具有高空间分辨率的核孔蛋白和运输中的mRNP之间相互作用的地图。尽管进化的距离，核孔的基本结构是保守的锥虫，它是可能的发现是可转移到后突，在那里的“mRNA陷阱”是不可能的。相反，我们的方法将阐明锥虫和哺乳动物系统之间mRNA输出和输出控制的差异：这些研究的综合结果将确定寄生虫特有的机制，推进我们对mRNA输出复杂途径如何演变的认识。此外，可以利用那些代表脆弱进程的进程来开发药物，以防治被忽视的热带疾病，如非洲昏睡病、恰加斯病和利什曼病。

Eukaryotic mRNAs are transcribed in the nucleus and need to pass through the nuclear pores to reach the cytoplasm for translation. This passage is essential for mRNA function and failures are linked to a range of human diseases. The study of nuclear mRNA export is challenging, because it is very fast and interactions between mRNAs and nuclear pore proteins are transient. We recently found that trypanosomes lack the mRNA export checkpoint that usually prevents immature mRNAs from starting nuclear export. As a consequence, mRNAs with their attached proteins (mRNPs, messenger ribonucleoprotein particles) can be trapped while being exported, simply by blocking transcription or splicing. Here, we plan to employ this unique feature of trypanosomes as an mRNA trap to study interactions between mRNPs and nucleoporins. We will use a combination of newly developed proximity labeling techniques for proteins and RNA, complemented by classical biochemical purification approaches. Our combined data will create a map of interactions between the nuclear pore proteins and the mRNPs in transit with high spatial resolution. Despite the evolutionary distance, the basic structure of the nuclear pore is conserved in trypanosomes and it is likely that findings are transferrable to opisthokonts, where an ‘mRNA trap’ is not possible. Conversely, our approach will elucidate differences in mRNA export and export control between trypanosomatids and mammalian systems: The combined outcome from these studies will identify mechanisms that are unique to the parasite, advancing our knowledge on how the complex pathway of mRNA export has evolved. Moreover, those that represent vulnerable processes can be leveraged for drug development against the neglected tropical diseases African Sleeping Sickness, Chagas disease and Leishmaniasis.